BSRG 2012-2013

2012-2013

Biennial Report

BSRG | Biological Sciences Research Group

12-13 20 12-13 Biennial Report 12-13

Contents

5 Executive Summary

6 Research Programs Highlights

43 Publications

49 Communications in International Conferences

51 Communications in National Conferences

53 Other Scientific Activities

55 BSRG Members

4

Biological Sciences Research Group

5

20 12-13 Biennial Report

12-13

The outcome of the research activities of the IBB Biologi-

cal Sciences Research Group (BSRG) during the bienni-

um 2012-2013 is detailed in this report, which we gladly

present to the readers. Key research outputs that have

contributed to the consolidation and international visibility

of the BSRG activities in the fields of Molecular and Cellu-

lar Microbiology, Functional and Comparative Genomics,

and Microbial Biotechnology are described. Interdiscipli-

nary programmes involving molecular biosciences across

disciplines, from molecules to systems, were developed to

understand how biological systems orchestrate multiple

functions, envisaging the exploitation/control of their activ-

ities in Industrial, Health, Environmental and Agro-Food

Biotechnology.

Over the past decade, the group has reinforced and gen-

eralized the use of Functional and Comparative Genomics

and Bioinformatics approaches, giving a Molecular Sys-

tems Microbiology dimension to the research programmes

active during the biennium; this is highlighted under the

general thematic line “Integrative (micro)biology: post-

genomic approaches to boost biosciences research”. An-

other wide-spanning thematic line dedicated to “Microbial

Response and Resistance to Environmental Challenges”

integrates most of our research lines and projects, as

highlighted in the next pages. This thematic area aims at

understanding the complexity of cellular responses to

environmental alterations and insults, one of the major

challenges in Biology and essential for successful Bio-

technological and Biomedical applications.

During the biennium a total number of 47 research papers

were published in international peer-reviewed journals

since 2012, of which 70% were published in the first quar-

tile of scientific journals from different subject areas, 9

book chapters were co-authored and 2 patents were is-

sued. The YEASTRACT database, at the forefront of bio-

informatics tools that support gene and genomic regula-

tion analysis and systems biology studies in yeast, was

updated and upgraded. The impact of our published work

among the international scientific community was also

significant. Around 1000 of the total number of citations

gathered by the group’s publications were obtained during

this biennium.

BSRG members also continued their essential activity in

the advanced training of human resources at Instituto

Superior Técnico (IST), Universidade de Lisboa, both at

the undergraduate and postgraduate levels in all the study

cycles offered by IST at the interface between Biological

Sciences and Engineering, having launched the ULisboa

inter-schools Master’s Programme in Microbiology. They

also served in evaluation panels (e.g. Portuguese Agency

for the Evaluation and Accreditation of Higher Education

(A3ES)) and in the governing boards of scientific societies

(Microbiology and Biotechnology). Internationalization was

pursued through relevant collaborations with several for-

eign institutions, the participation in a COST action and 2

EraNets (Industrial Biotechnology and Pathogenomics),

the exchange of students and scientists, the international

dissemination of results, the participation in editorial

boards of several international journals and as guest edi-

tors of special issues, the organization and scientific com-

mittees of international conferences, congresses, and

schools, and through the participation in international ad-

visory boards and as evaluators for international funding

agencies, (e.g. European Research Council). In summary,

the mission of the BSRG to create biosciences-based

solutions to societal problems, providing useful services to

society by combining R&D activities with advanced educa-

tion was fulfilled during 2012-2013.

Executive Summary

Isabel Sá-Correia

Head of the BSRG


6

Objectives

A wide-spanning thematic line dedicated to research

on the response and resistance to environmental

challenges integrates a number of research lines and

projects highlighted in the next pages. The indicated

bibliographic references are those listed as the BSRG

publications in the period 2012-2013.

This thematic line aims at understanding the

complexity of cellular responses to environmental

alterations and insults, which is one of the major

challenges in Biology and is also crucial for

successful biotechnological and biomedical

applications. In fact, the survival and performance of

living cells depends on their ability to sense

alterations in the environment and to appropriately

respond to the new stressing situations by

remodelling genomic expression. Also, cellular

resistance to multiple drugs/xenobiotics is implicated

in the failure of many therapeutic, food-preservation

and crop protection actions but may help to improve

the productivity of biotechnological processes.

Research Topics

1. Adaptation and resistance to environmental

stresses and antifungals in yeasts

During 2012-2013, we have been contributing to the

understanding of the toxic effects caused by ethanol

and the molecular mechanisms triggered by

Saccharomyces cerevisiae at a genomic scale to

cope with this stress [24] and successfully genetically

engineered yeast cells for increased ethanol tolerance

and productivity [45]. The impact of assimilable

nitrogen in S. cerevisiae glucose uptake kinetics

during alcoholic fermentation was also examined,

providing useful guidelines for the control of nitrogen-

limited alcoholic fermentations [33]. The genetic

engineering of the transcription factor Haa1 and of the

Haa1-regulon for improved yeast tolerance to acetic

acid is also on the focus of current research. The

mechanisms underlying the intrinsic resistance to

acetic acid in the acidic food spoilage yeast

Zygosaccharomyces bailii were also examined to

guide food preservation strategies [23,31,

Palma&Roque et al., submitted]. Resistance to

natural growth inhibitors and antifungal therapy in

pathogenic Candida yeast species has also been

studied [7-9], providing new directions for the

treatment of the increasing number of drug resistant

fungal infections.

2. Multidrug/multixenobiotic resistance transporters

Multidrug/multixenobiotic resistance (MDR/MXR) is

many times the result of the action of MDR/MXR

transporters found at the membranes of all living cells.

Our longstanding research has been dedicated to the

study of the biological role and regulation of drug/

xenobiotic -pumps of the Major Facilitator Superfamily

(MFS) or the ATP Binding Cassette (ABC)

Superfamily using the eukaryotic model yeast S.

cerevisiae. This knowledge is also being explored to

functionally characterize membrane transporters from

the model plant Arabidopsis thaliana and to

understand their role in resistance to abiotic stress.

Since abiotic stress represents the most pervasive

cause of loss of plant productivity worldwide under

agricultural conditions, this thematic line attempts to

uncover novel roles for MFS transporters in plant

responses to adverse environments, paving the way

for the development of efficient strategies to improve

crop tolerance to abiotic stress [38, 39]. The

extension of the current knowledge on yeast MDR to

pathogenic yeasts is another task of this thematic line.

Mechanisms of antifungal drug resistance acquisition

in clinical isolates of C. albicans and C. glabrata have

also been examined, in particular the biological role of

several C. glabrata MDR transporters in resistance to

antifungal drugs, acetic acid and polyamines [7-9].

3. Environmental toxicology and bioremediation

One of our goals is to predict toxicological outcomes

of exposure to environmental pollutants/pesticides,

with emphasis on microbial toxicogenomics and

bioremediation of environmental pollutants. In this

context, the catabolic ability of two soil s-triazine–

degrading bacterial strains, Pseudomonas sp. ADP

and Arthrobacter aurescens TC1 is being explored

and bioassays for the assessment of the toxicity of

xenobiotics (e.g. pesticides, synthetic dyes and

others) were developed [6,47,56], based on

experimental models (the yeast S. cerevisiae and the

Response and resistance to

environmental challenges BSR

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nematode Caenorhabditis elegans). The optimization

of bioremediation strategies to decontaminate soils

polluted with s-triazine-based herbicidal formulations

is expected to allow the prevention of adjacent

freshwater compartment contamination with the

herbicides and their toxic chlorinated metabolites [6].

4. Microbial pathogenesis and host-bacteria

interactions

Chronic pulmonary infections with Burkholderia

cepacia complex (Bcc) are largely associated with a

worse prognosis and increased risk of death among

cystic fibrosis (CF) patients (Coutinho et al., 2011,

Front Cell Infect Microbiol, 1: 12). Bcc species

possess remarkable genome plasticity, providing an

invaluable advantage for adaptation to the highly

stressful CF lung environment, where bacteria must

adapt to selecting pressures resulting from

challenges of the immune system, antimicrobial

therapy, nutrient availability, oxygen limitation, etc..

The identification of the mechanisms involved in this

adaptation is crucial to define new potential drug

targets and treatment strategies. During 2012-2013

we have continued to investigate this issue, and

found further evidences supporting the crucial role of

bacterial adaptation to the evolving host

environment, highlighting the importance of

metabolic reprograming in the adaptation and

increased pathogenic potential of Bcc bacteria

occurring alongside with disease progression [25].

We have also focused on the molecular

mechanisms that trigger mucoid morphotype

variation among Bcc isolates [41], on the proteins

directing exopolysaccharide (EPS) biosynthesis, and

on EPS-mediated interaction with hosts, either in

symbiosis or pathogenesis [19,43]. Small non-coding

RNAs are increasingly recognized as powerful tools

used by bacterial pathogens to rapidly adapt their

physiology and fitness to the host environment. Work

carried out by our group envisages the identification

and functional characterization of sRNAs and their

mRNA targets, in particular those related to virulence

and resistance to stresses mimicking those faced by

the bacterium during the infection process [35-37].

Work on the nitrogen-fixing bacterium

Sinorhizobium meliloti has also been carried out

envisaging the elucidation of mechanisms involved

in symbiotic host-bacteria interactions [40].

Because live bacteria can be used in cancer

therapy and prognosis in parallel with purified

bacterial products to treat and prevent cancer growth

and metastasis, our research has also been focused

on understanding the role and use of the

Pseudomonas aeruginosa-secreted protein azurin,

as a therapeutic tool to treat poor-prognosis breast

carcinomas [3-5].

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Objectives

Genome-wide and molecular systems biology ap-

proaches were explored during 2012/13 to achieve

an integrative view on how cells interact with their

environment. The main focus of this thematic area

is the implementation and exploitation of post-

genomic approaches to gain a full understanding of

the mechanisms of adaptation and resistance to

environmental challenges in microbial systems, and

to enable their redesign and reconstruction in order

to display functions more suited to specific applica-

tions or exhibiting traits of relevance in Biotechnolo-

gy, Biomedicine and the Environment.

The research strategies employed explore

the interface between “omics” analyses and molec-

ular and cellular biology research to provide an

integrated view of gene/genomic expression, cellu-

lar signalling, and metabolic networks, allowing a

unified perspective of key scientific questions in

Biotechnology, Biomedicine and the Environment,

using microbial model systems or envisaging the

improvement/control of the physiological activity of

microorganisms, as cell factories, food spoilers or

human pathogens. This wide-spanning thematic

line is dedicated to research focusing on an inte-

grative (micro)biology standpoint and integrates a

number of our research lines and projects, as high-

lighted in the next pages and summarized below.

The indicated bibliographic references are those

listed as the BSRG publications in the period 2012-

2013.

Research topics

1. Yeast toxicogenomics

Mechanistic insights and genome-wide views on

the responses to chemicals and environmental

alterations relevant in Environmental Health, Phar-

macology and Biotechnology have been gathered

using the eukaryotic model Saccharomyces cere-

visiae. Such toxicogenomics approaches are im-

portant to assess the response to cytotoxic insults

and have the potential for predictive toxicology and

for guiding bioremediation strategies and biotech-

nological and biomedical research and applications

[16].

This knowledge of the S. cerevisiae cell

response to environmental challenges at the ge-

nome level (exploring transcriptomics, quantitative

proteomics, phosphoproteomics, metabolomics) is

essential to elucidate mechanisms of resistance

and find new targets of pharmaceutical drugs [15],

to select fermentation conditions and to engineer

more robust yeast strains able to improve the

productivity of biotechnological processes, such as

those related to assimilable nitrogen availability,

toxic concentrations of ethanol- and acetic acid-

induced stresses [23,24,33]. It is also essential to

decipher weak acid tolerance mechanisms in the

acidic food spoilage yeast species Zygosaccharo-

myces bailii [23, 31, Palma&Roque et al., submit-

ted], and to understand antifungal drug resistance

in pathogenic Candida species. The genome se-

quence of the highly acetic acid-tolerant Z. bailii

derived interspecies hybrid strain ISA1307, isolated

from a sparkling wine plant, was determined and

annotated and the adaptive response to acetic acid

in this strain was revealed by quantitative prote-

omics [23,31]. The screening of a genomic library

derived from the same strain to identify genes re-

sponsible for acetic acid resistance was also per-

formed [Palma&Roque et al., submitted].

The evolutionary history of the DHA2 (14-

spanner Drug:H+ Antiporter family 2 transporters of

the Major Facilitator Superfamily involved in multi-

drug resistance), ARN (siderophore transporters)

and GEX (glutathione:H+ antiporters) encoding

genes from 31 sequenced yeast strains of 25 differ-

ent hemiascomycetous species, was reconstructed.

A new protein family (DAG) was proposed to span

these three phylogenetic subfamilies of 14-spanner

MFS transporters [12]. The reconstruction of the

Integrative (micro)biology: post-genomic

approaches to boost biosciences research BSR

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evolutionary history of the hemiascomycete DHA1

(12-spanner Drug:H+ Antiporter family 1 transport-

ers) genes was extended to the species comprised

in the CTG phylogenetic complex to get new insights

on how these family genes have evolved in medical-

ly relevant Candida species [Dias and Sá-Correia,

submitted].

2. Genome-wide expression regulation

Our main goal is to understand how different tran-

scription regulatory networks overlap and cross talk

to influence microbial growth and the response to

environmental insults, elucidate the conditions under

which transcription factors are able to bind to pro-

moter binding sites, and explore and develop com-

putational tools to support this research. The

YEASTRACT database, a post-genomic resource at

the forefront of bioinformatics tools that support gene

and genomic regulation analysis and systems biolo-

gy studies in yeast, was updated and upgraded [44].

To get clues into the regulation of hexose transport-

ers encoding genes in response to the recovery of

glucose uptake rates following ammonium supple-

mentation of a nitrogen-limited fermentation, a bi-

clustering analysis was applied to the microarray

data from the transcriptional response of S. cere-

visiae cultivated under different nitrogen availability

conditions [33].

Genome-wide screenings led to the identifica-

tion of small non-coding regulatory RNAs in the op-

portunistic human pathogen Burkholderia cenocepa-

cia [35]. New post-transcriptional regulation mecha-

nisms involving these small non-coding RNAs and

RNA chaperones required for bacterial survival un-

der stress and full virulence in B. cenocepacia were

also described.

3. Bacterial pathogenomics

The proteome expression profiling of B. cenocepa-

cia clonal isolates with different virulence potential,

retrieved from a cystic fibrosis patient during chronic

lung infection, highlighted the involvement of pro-

teins associated with metabolic functions in in-

creased persistence and virulence potential [25]. A

book chapter describing the application of expres-

sion proteomic approaches to studies involving

Burkholderia bacteria and providing a tutorial on the

methodologies available was published [54]. Com-

parative transcriptomic analysis of the B. cepacia

tyrosine kinase bceF mutant revealed a role of this

kinase in tolerance to stress, biofilm formation and

virulence [19].

4. Host-bacteria interactions

The symbiotically relevant gene emrR of the nitrogen

-fixing bacterium Sinorhizobium meliloti was charac-

terized through an expression profiling analysis of

the deletion mutant [40]. This analysis showed a

down-regulation of genes involved in stress respons-

es and biosynthesis of Nod-factor and rhizobactin,

while genes directing the biosynthesis of polysac-

charides were up-regulated, suggesting a role for

EmrR in a regulatory network involved in the S. meli-

loti preparation for symbiosis.

The role and targets of the bacterial protein

azurin from Pseudomonas aeruginosa in a breast

cancer model was elucidated through a tran-

scriptomic profiling of the action of azurin in cells

expressing different levels of P-cadherin, a bad prog-

nosis marker in breast cancer [3].

5. Stem cell research

Quantitative proteomics has also been applied to

other systems of interest to our research unit strate-

gy. Human mesenchymal stem cells (MSC) have

been on the focus of intense clinical-oriented re-

search based on their differentiation potential and

immune-modulatory properties. However, because

ex-vivo expansion in required to reach meaningful

cell number for cellular therapy, the loss of prolifera-

tive, clonogenic and differentiation potential is a

problem associated with cell passaging. In this con-

text, we have used expression proteomics to obtain

mechanistic insights into the effect that culture condi-

tions have on human MSC proteomes, paving the

way to set up a proteome profiling strategy for quality

control to ensure safe and clinically effective expand-

ed stem cells [26].

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Objectives

The success of industrial alcoholic fermentations is

dependent on the ability of S. cerevisiae cells to cope

with the many environmental insults imposed during

the process. In this context, the accumulation of

inhibitory metabolites (e.g. ethanol, acetic acid, long

chain fatty acids) and nutrient limitation (e.g. nitrogen)

resulting directly from the use of unbalanced growth

media or, indirectly, from stress conditions affecting

transmembrane transport, are considered the main

causes of stuck (prematurely arrested) and sluggish

(slow) fermentations. The understanding of the

molecular and physiological basis of yeast response,

adaptation and resistance to stresses imposed during

alcoholic fermentation is essential to guide the design

of rational strategies to increase yeast performance in

an industrial environment. The use of yeast as a

eukaryotic model in pharmacogenomic studies to

elucidate mechanisms of drug resistance and identify

new potential drug targets is also on the focus of our

research.

Research topics

1. Impact of assimilable nitrogen availability in

glucose uptake kinetics

The expression and activity of the different S.

cerevisiae plasma membrane hexose uptake systems

(Hxt) and the kinetics of glucose uptake are

considered essential to industrial alcoholic

fermentation performance. Sugar transporters

inactivation, associated to protein synthesis arrest

and their degradation, even when glucose

concentration is still high, has been proposed as the

major limiting factor of enological fermentation arrest

upon nitrogen depletion. However, the variation of

sugar uptake kinetics throughout nitrogen-limited

sluggish fermentation and the same replenished

fermentation is poorly characterized and it was one of

the topics for research.

2. Global mechanisms of response and resistance to

bioethanol fermentation associated stresses

One of the objectives of our research is the

identification, at a genomic scale, of the genes and

mechanisms of resistance to some of the stresses

occurring during alcoholic fermentation, including high

-glucose (Teixeira et al., 2010, OMICS, 14, 201),

ethanol (Teixeira et al., 2009, Appl Environ Microbiol,

75, 5761) or acetic acid stress (Mira et al., 2010,

Microb Cell Fact, 9, 79). To further understand the

toxic effects caused by ethanol and the molecular

mechanisms triggered by yeast to cope with this

stress, a metabolomic analysis using high

resolution 1H-NMR spectroscopy coupled with

multivariate statistical analysis was explored during

2012-2013 to characterize the alterations occurring in

S. cerevisiae endo- and exo-metabolome under

ethanol induced stress. The effect of the

aquaglyceroporin encoded by FPS1, previously

implicated in ethanol stress resistance, in the yeast

metabolome in the absence or presence of ethanol

stress was also on the focus of this research.

Response and resistance to chemical stress and other

environmental challenges imposed to yeast cells during

alcoholic fermentation and in pharmacogenomic studies BSR

G

Isabel Sá-Correia, Miguel C. Teixeira, Nuno P. Mira, Margarida Palma, Tânia R. Cabrito, Sandra C.

dos Santos

PhD and Msc students: Artur B. Lourenço, Joana F. Guerreiro, Sílvia F. Henriques, Filipa C. Roque, Cláudia P. Godinho,

Filipe B. Silva

S cerevisiae ATCC 201388 (BY4741)expressing Haa1 fused (C-terminal) to GFP(S65T mutant)

Cells were cultivated in MMB (pH 4.0) and harvested after 30 minutes

in the presence or absence of 60 mM acetic acid

Cells were stained with DAPI to a final conc. of 5ng/μl.

Immediately fixed in formaldehyde (1%v/v) for 10min and then visualized.

GFP

DAPI

Merged

Without acetic acid Acetic acid 60mMSubcellular localization in presence/absence of acetic acid

Sub-cellular localization of Haa1 in

response to acetic acid stress

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3. Construction of genetically engineered strains with

improved robustness against high ethanol stress to

be used in high gravity fermentations

Based on the genome-wide identification of

determinants of ethanol resistance the manipulation

of the expression of specific genes in the overall

fermentative capacity is being explored. The

overexpression of the Fps1 aquaglyceroporin, led to

a high ethanol producing strain (Teixeira et al., 2009,

Appl Environ Microbiol, 75, 5761). More recently, the

overexpression of the yeast multidrug resistance

ABC transporter encoded by the PDR18 gene,

proposed to play a role in the incorporation of

ergosterol in the yeast plasma membrane, was

examined. Yeast robustness against growth

inhibitory concentrations of ethanol and bioethanol

productivity in high gravity fermentations was

augmented by increasing the expression level of

PDR18 [3].

4. Screening of natural S. cerevisiae isolates for

acetic acid tolerance

Among the fermentative yeast microbiota it is

expected to find S. cerevisiae isolates better adapted

to the different stresses imposed during alcoholic

fermentation. In this context, to search for strains

with high levels of resistance to acetic acid a

screening among natural wine yeast strains isolated

from spontaneous fermentations of grape must of

Douro wine-producing areas (248 isolates) and

various commercial wine yeasts strains (34 strains),

was performed.

5. Yeast pharmacogenomic studies

The eukaryotic model S. cerevisiae provides an

excellent tool for drug discovery and medicinal

research, contributing to the identification of new

targets and mechanisms of drug action [4]. In recent

years our research in this field has focused on the

antimalarial quinine (dos Santos et al. 2009,

Antimicrob Agents Chemother, 53: 5213-23; dos

Santos et al., 2011, Mol Genet Genomics, 286: 333-

46) and on the anticancer agent imatinib (dos Santos

et al., 2009, Omics, 13: 185-98) [5], based on the

application of genome-wide approaches at a

systems biology level, including transcriptomics,

proteomics and chemogenomics.

Main Achievements

The impact of assimilable nitrogen availability in

glucose uptake kinetics in S. cerevisiae during

alcoholic fermentation was detailed. Results

suggested that glucose transport capacity is maximal

during the early stages of fermentation and

presumably sustained by the low-affinity and high-

capacity glucose transporter Hxt1. During nitrogen-

limited sluggish fermentation, glucose uptake

capacity was reduced to approximately 20% of its

initial values, being presumably sustained by the low

-affinity glucose transporter Hxt3. Results also

suggested that sluggish fermentation recovery after

ammonium supplementation is associated to the

increase of glucose uptake capacity, related to the

de novo synthesis of glucose transporters with

different glucose affinity for glucose and capacity,

presumably of Hxt2, Hxt3, Hxt4, Hxt6 and Hxt7 [1].

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Pdr18

ATP ADP

Pdr11

ATP ADP

Aus1

ATP ADP

Exogenous ergosterolExogenous ergosterol

PM

Free ergosterolFree ergosterol

ER

Vesicular

transport

Non-vesicular

transport

Free ergosterol

Incorporated

ergosterol

Incorporated

ergosterol

Ergosterol

biosynthesis

12

Metabolomic analysis based on quantitative 1H-

NMR revealed extensive metabolomic

reprogramming and the effect of the

aquaglyceroporin Fps1 in ethanol-stressed yeast

[2].

Consistent with the previously described role of

Pdr18 in plasma membrane incorporation of

ergosterol (Cabrito et al., 2011, Biochem J, 440,

195), Pdr18 expression was further seen to reduce

the ethanol induced membrane permeabilization. A

Pdr18-overexpressing yeast strain, in which the

weak PDR18 promoter was replaced by the strong

and fermentation-inducible PDR5 promoter, was

constructed and shown to exhibit increasing

ethanol tolerance and to be able to produce, under

Very High Gravity fermentation, otherwise highly

inhibitory ethanol concentrations [3].

About 16% of the natural isolates of S.

cerevisiae obtained from spontaneous

fermentations of grape must of Douro wine-

producing areas and of the various commercial

wine yeasts strains were able to grow when

cultivated in acetic acid concentrations above 130

mM (pH 4), but only approximately 3% of the

strains tested could grow above 150 mM acetic

acid, with the most resistant isolates (about 1.4%)

growing at concentrations ranging 170-200 mM

acetic acid. The molecular physiology of these

remarkably acetic acid tolerant strains is currently

under study to understand the mechanisms

underlying such remarkable trait.

A quantitative- and phosphoproteomics study

based on 2-dimensional electrophoresis was

designed to monitor the response of yeast cells to

an inhibitory concentration of the tyrosine kinase

inhibitor imatinib [4]. The results highlighted the

importance of gluconeogenesis and glycolytic

pathways, which have recently been associated

with the mechanism of imatinib action in human cell

line studies. The remarkable conservation of the

results obtained in this study, similar to what had

been observed in our previous work with this drug,

further highlights the potential of S. cerevisiae as

model system for pharmacological studies.

Funded projects

ZygoSacAR – Mechanistic insights into acetic

acid resistance in food spoilage yeasts: from the

experimental Saccharomyces cerevisiae to

Zygosaccharomyces spp. PTDC/AGR-

ALI/102608/2008, PI: Isabel Sá-Correia.

INTACT – Integral Engineering of Acetic Acid

Tolerance in Yeast, in the frame of ERA-NET

Industrial Biotechnology, ERA-IB/0002/2010, PI:

Isabel Sá-Correia.

CONTRACT OF TECHNOLOGICAL

DEVELOPMENT, Project FNA (processes for non-

alcoholic fermentation of fruit juices)

SUMOL+COMPAL MARCAS, S.A..

Selected publications

[1] Palma M, Madeira SC, Mendes-Ferreira A, Sá-

Correia I. Impact of assimilable nitrogen availability in

glucose uptake kinetics in Saccharomyces cerevisiae

during alcoholic fermentation, Microbial Cell Factories,

11: 99, 2012.

[2] Lourenço AB, Roque FC, Teixeira MC, Ascenso

JR, Sá-Correia I. Quantitative 1H-NMR-metabolomics

reveals extensive metabolic reprogramming and the effect

of the aquaglyceroporin FPS1 in ethanol-stressed yeast

cells, PLoS One, 8(2), e55439, 2013.

[3] Teixeira MC, Godinho CP, Cabrito TR, Mira NP, Sá-

Correia I. Increased expression of the yeast multidrug

resistance ABC transporter Pdr18 leads to increased

ethanol tolerance and ethanol production in high gravity

alcoholic fermentation, Microbial Cell Factories, 11: 98,

2012.

[4] dos Santos SC, Teixeira MC, Cabrito TR, Sá-

Correia I. Yeast Toxicogenomics: genome-wide

responses to chemical stresses with impact in

Environmental Health, Pharmacology and Biotechnology,

Frontiers in Genetics, 3: 63, 2012.

[5] dos Santos SC, Mira NP, Moreira AS, Sá-Correia I.

Quantitative- and phospho-proteomic analysis of the

yeast response to the tyrosine kinase inhibitor imatinib,

OMICS: A Journal of Integrative Biology, 16(10): 537-

551, 2012.

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Adapted from ref. [5]

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Objectives

Transcriptional regulation depends on the action of

transcription factors (TFs) (activators and/or re-

pressors) that bind to specific DNA sequences in the

promoter region of target genes, thereby modulating

transcription. At a global scale, the transcript level of

a given gene results from the concerted action of

these specific TFs, operating in complex and inter-

twined regulatory networks. In the field of gene and

genomic expression, our main goals are:

1. Understand how different transcription regulatory

networks overlap and cross-talk to influence tran-

scription in yeast under optimal growth conditions and

under stress;

2. Elucidate the molecular mechanisms that control

protein-DNA binding and the action of TFs;

3. Assess the interactions established between TFs

and their target DNA sequences;

4. Development and exploitation of computational

tools to guide gene and genomic regulation studies

(YEASTRACT database) and to build suitable predic-

tive models of transcriptional regulatory networks

active in yeast, and to extrapolate them to other

yeasts and less genetically accessible eukaryotes.

Research Topics

1. Update and upgrade of Yeastract

The YEASTRACT database is an infrastructure for

research on gene and genomic regulation in Saccha-

romyces cerevisiae that was developed in collabora-

tion between our group and the KDBIO Group of IN-

ESC-ID to support the analysis of transcription regu-

latory associations (Teixeira et al., 2006, Nucleic Ac-

ids Res. 34: D446-51). Exclusively developed and

maintained in Portugal since 2006, YEASTRACT has

become an essential tool for Yeast Molecular Biolo-

gists and Systems Biology researchers worldwide,

being the major acknowledged source of regulatory

data of the Yeast Genome Database (SGD), the ma-

jor international database that provides comprehen-

sive integrated biological information for S. cere-

visiae. YEASTRACT progression has been described

in 4 articles that appeared in 4 DataBase Issues of

Nucleic Acids Research (2006, 2008, 2011, 2014).

These papers have been cited over 300 times in ISI

journals since 2006. The YEASTRACT website has

been cited many more times and has become quite

popular, being extensively used by hundreds of re-

searchers worldwide, and receiving around 2,000

monthly visits. The database was very recently updat-

ed and upgraded with new bioinformatic tools and

more detailed regulatory information to facilitate the

exploitation of the gathered material when answering

specific biological questions [1].

We are currently planning the development of

YEASTRACT+ by expanding the scope of application

of the YEASTRACT database to the S. cerevisiae

pan-genome and to other yeasts of biomedical and

biotechnological interest (e.g. Candida species, Zygo-

saccharomyces baillii), and to highlight crucial pro-

cesses underlying adaptation and evolution. This

planned integration and analysis will give unprece-

dented insights into yet unknown regulatory mecha-

nisms and boost the understanding of pathogen–host

interactions and the exploration of biotechnological

interesting species/strains. This knowledge may

deeply impact the treatment of fungal pathogens, the

methods for food spoilage control and the design of

optimized microbes as cell factories.

2. Assessing transcription factor-DNA interactions

using a nanobiotechnology-based approach

The interaction of the S. cerevisiae transcription fac-

tor Haa1 with its target DNA sequence, the Haa1-

responsive element (HRE) 5’-(G/C)(A/C)GG(G/C)G-

3’ [Mira et al., 2011, Nucleic Acids Res. 39:6896-

907], was studied making use of a nanostructured

acoustic wave biosensor developed by IBB/CBME,

from University of Algarve [4]. The biosensor devel-

oped is based on a quartz crystal microbalance

(QCM) analytical method supported by a transmis-

sion line model (TLM) algorithm. Using this new

nanobiosensor it was possible to demonstrate that

Haa1 binding to an oligonucleotide containing the

HRE motif leads to bending of the DNA molecule (by

approximately 37º) and increases the compactness of

Gene and genomic regulation: exploring

bioinformatics and nanobiotechnology tools BSR

G

Isabel Sá-Correia, Miguel C. Teixeira, Nuno P. Mira, Sandra C. dos Santos, Margarida Palma

PhD students: Catarina Costa, Joana Guerreiro, Sílvia F. Henriques, Cláudia P. Godinho

14

the protein-DNA complex. The results obtained in this

work demonstrate the suitability of the QCM to moni-

tor the specific binding of transcription factors to im-

mobilized DNA sequences and provides an analytical

methodology to study protein–DNA biophysics and

kinetics. The interaction of Haa1 with the HRE motif

will be further examined using Force Feedback Mi-

croscopy in collaboration with the CFMC group from

FCUL.

3. Transcription factor engineering for improved yeast

tolerance to acetic acid

The transcription factor (TF) Haa1 was considered

one of the important targets for regulation of specific

TF engineering in order to achieve a reprogramming

of genes controlled by Haa1. In the context of the

INTACT project, the Bremen partner identified mutat-

ed versions of Haa1 by error-prone PCR whose ex-

pression led to increased tolerance to acetic acid,

compared with the wild type Haa1. Our research

group is being examining the reasons for such behav-

iour at the level of: i) Haa1 version protein efficiency

of binding to DNA motif in the promoter regions of

target genes; ii) RNA stability of the different Haa1

versions in presence/absence of acetic acid ; iii) dif-

ferences at the level of sub-cellular localization of

Haa1 versions in yeast cells either or not exposed to

acetic acid; iv) activation of the protein content and

physiological activity of proteins encoded by selected

target genes in response to acetic acid.

4. Biclustering analysis of the transcription profiles of

yeast cells following ammonium supplementation of a

sluggish fermentation

A CCC-biclustering analysis (Contiguous Column

Coherent Biclusters) was applied to the microarray

data previously obtained from the transcriptional re-

sponse of S. cerevisiae PYCC 4072 cultivated under

different nitrogen availability conditions in synthetic

grape juice (Mendes-Ferreira et al., 2007, Appl Envi-

ron Microbiol, 73:3049-60) in order to identify which

hexose (Hxt) transporters may be responsible for glu-

cose uptake in the recovery of the glucose uptake

rates following ammonium supplementation of a slug-

gish fermentation and the involved transcription fac-

tors [2]. CCC-Biclustering is a state of the art biclus-

tering algorithm specifically developed for time series

gene expression data analysis (Madeira et al., 2010,

Trans Comput Biol Bioinform, 7:153-165).

Main Achievements

All regulatory associations stored in the

YEASTRACT database were revisited and new infor-

mation was added regarding the experimental condi-

tions tested and whether the TF is acting as an activa-

tor or repressor. Based on this information, new que-

ries were developed allowing the selection of specific

environmental conditions, experimental evidence or

positive/negative regulatory effect (see Figure). This

release also includes new computational tools that

facilitate the exploitation of the gathered data [1].

YEASTRACT is now linked to the major information

system focused on S. cerevisiae, the SGD

(www.yeastgenome.org). SGD currently displays

data curated by YEASTRACT on its Regulatory data,

providing another avenue for researchers to access

and analyze these data. Further collaborations with

other international databases (Candida Genome Da-

tabase, MIPS-CYGD, etc.) are also planned in the

context of YEASTRACT+ implementation.

A CCC-biclustering analysis provided indications

that the activation of the expression of genes encod-

ing the glucose transporters Hxt2 (during the transi-

tion period to active fermentation) and Hxt3, Hxt4,

Hxt6 and Hxt7 (during the period of active fermenta-

tion) may have a major role in the recovery of the

glucose uptake rate following ammonium supplemen-

tation and suggested a general de-repression of the

glucose-repressible HXT genes, consistent with the

co-down-regulation of the Mig1 and Rgt1 transcrip-

tional repressors [2].

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Figure 4

HXT2

2.52

1.51

0.50

-0.5-1

-1.5-2

HXT5, MIG1, RGT1, GAT1,

GLN3, GZF3, GIS1, MSN4 2.5

21.5

10.5

0-0.5

-1-1.5

-2 LF RF-8h RF-24h

Bicluster Time-points

Period 1 Period 2

Period 1 Period 2

(U) (D)

(D) (N)

No

rmali

zed

ex

pre

ssio

n v

alu

e

(Adapted from ref. [2])

15


12-13

The transcription factor CgPdr1 of the pathogenic

yeast Candida glabrata was found to activate the

transcription of the multidrug transporter CgQDR2 in

C. glabrata cells challenged with clotrimazole or

quinidine [3].

An acoustic nanostructured biosensor developed

IBB/CBME from University of Algarve for the study

of the interaction between S. cerevisiae Haa1 tran-

scription factor and its target DNA sequence was

explored to identify Conformational and Mechanical

changes of DNA upon transcription factor binding

detected by a QCM and transmission line model [4].

Funded projects

FUNDRING - Identification of new biomarkers for

antifungal drug resistance diagnosis in Candida

glabrata: the particular role of multidrug resistance

transporters, PTDC/EBB-BIO/119356/2010, PI: Mi-

guel Teixeira

INTACT - Integral engineering of acetic acid tol-

erance in yeast, ERA-IB/0002/2010, PI at IST: Isa-

bel Sá-Correia

EXPL/FIS-NAN/1395/2013 - Biological physics

studies based on Force Feedback Microscopy, PI

at IST: Isabel Sá-Correia

PTDC/EBB-EBI/108517/2008 - QCMTF-

Biosensor platform for transcription factor DNA bind-

ing activity detection (concluded in September

2013), PI at IST: Isabel Sá-Correia

Selected Publications

[1] Teixeira MC, Monteiro PT, Guerreiro JF, Gonçalves

JP, Mira NP, dos Santos SC, Cabrito T, Palma M, Costa

C, Francisco AP, Madeira SC, Oliveira AL, Freitas AT,

Sá-Correia I, The YEASTRACT database: an upgraded

information system for the analysis of gene and genomic

transcription regulation in Saccharomyces cerevisiae,

Nucleic Acids Res, 42: D161-D166, 2014.

[2] Palma M, Madeira SC, Mendes-Ferreira A, Sá-

Correia I. Impact of assimilable nitrogen availability in

glucose uptake kinetics in Saccharomyces cerevisiae dur-

ing alcoholic fermentation, Microb Cell Fact, 11(1): 99,

2012.

[3] Costa C, Pires C, Cabrito TR, Renaudin A, Ohno M,

Chibana H, Sá-Correia I, Teixeira MC. Candida glabrata

drug:H+ antiporter CgQdr2 (ORF CAGL0G08624g) confers

imidazole drug resistance, being activated by the CgPdr1

transcription factor, Antimicrob Agents Chemother, 57

(7): 3159-67, 2013.

[4] de Carvalho J, Rodrigues RMM, Tomé B, Henriques

SF, Mira NP, Sá-Correia I, Ferreira GNM. Conformational

and Mechanical changes of DNA upon transcription factor

binding detected by a QCM and transmission line mod-

el, Analyst, 139(8): 1847-55, 2014.

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16

Zygosaccharomyces bailii, the most threatening spoilage yeast in food and

beverage industries: genome-wide approaches to understand its exceptional

resistance to the food preservative acetic acid and other remarkable features

Isabel Sá-Correia, Nuno P. Mira, Margarida Palma

PhD students: Joana F. Guerreiro, Filipa C. Roque; Research Assistant: Filipa D. Valada

BSR

G

Objectives

Spoilage resulting from growth and metabolic activity

of ascomycetous yeasts of the Zygosaccharomyces

species is widespread, which leads to significant

economic losses in the food industry and to a

reduction of food supplies worldwide. Z. bailii is one of

the most problematic spoilage yeasts in food and

beverage industries, particularly in acidic foods, soft

drinks, fruit juices, dairy products, salad dressings,

sauces, and some wines [1]. This yeast species ability

to cause spoilage derives from its outstanding capacity

to resist to weak acids widely used as food

preservatives, such as acetic, benzoic, propionic and

sorbic acids, even above the permitted values by

some food legislations.

Understanding the mechanisms of weak acid

resistance is central to the development and

implementation of more effective food and beverage

preservation strategies. Although Z. bailii is the

spoilage yeast that exhibits the highest level of

resistance to acetic acid, most of the scientific

contributions of our research group and others

concerning the mechanisms underlying adaptation and

resistance to acetic acid are essentially focused on the

more susceptible experimental model and spoilage

yeast Saccharomyces cerevisiae. Recent contributions

from our laboratory to the field are highlighted under

topic “Response and resistance to chemical stress and

other environmental challenges imposed to yeast cells

during alcoholic fermentation and in pharmacogenomic

studies”. Given that the mechanistic effects of acetic

acid in Z. bailii are not well understood, in particular

those underlying its remarkable resistance to this food

preservative, our goal is to contribute to accelerate

systems-level understanding of acetic acid resistance

mechanisms through the combination of different

genome-wide approaches.

The yeast species Z. bailii has also attracted

our attention as a potential new host for

biotechnological processes. In particular, it is an

attractive candidate to allow fermentation processes to

be performed under otherwise-restrictive conditions or

to be used in heterologous protein and metabolite

production (in particular organic acids) because of its

high resilience to acidic stress, high specific growth

rate, and high biomass yield. The knowledge of the

genome sequence of the ISA1307 strain during 2013

[2] is expected to inspire and guide novel

biotechnological applications of this yeast species/

strain in fermentation processes, given its robustness

traits.

Research topics

1. Sequencing and annotation of ISA1307 genome

Our research group led a project for the sequencing

and annotation of the genome of the highly acetic acid-

tolerant strain ISA1307 [2]. This strain was isolated

from a sparkling wine continuous production plant and

was formerly considered of the Z. bailii species and

used in the study of Z. bailii remarkable physiological

traits, in particular, its extreme tolerance to acetic acid

stress at low pH and its capacity to co-consume

glucose and acetic acid. This characteristic

distinguishes this species from S. cerevisiae, a

Crabtree-positive yeast in which acetic acid

metabolisation is repressed in the presence of

glucose. The analysis of the genome sequence

revealed that strain ISA1307 is, indeed, an

interspecies hybrid strain between Z. bailii and a

closely related undetermined yeast species. The

0,01

0,1

1

10

100

0 20 40 60

OD

600

nm

Time (h)

Z. bailii -derived interspecies hybrid strain ISA1307

0,01

0,1

1

10

100

0 20 40 60O

D 6

00n

m

Time (h)

S. cerevisiae BY4741

Control

60mM Acetic acid

120mM Acetic acid

0 mM Acetic acid

60mM Acetic acid

120mM Acetic acid

MMB, pH4.0

17


12-13

sequence and annotation of the genome of ISA1307

strain are accessible at http://pedant.helmholtz-

muenchen.de/genomes.jsp?Category=fungal,

including browsing by a GBrowse instance. To allow

a comparative navigation through the genome of the

hybrid strain with the genomes of Z. rouxii CBS732,

S. cerevisiae S288c and Z. bailii CLIB213T, a

GBrowse_syn instance is accessible under http://

mips.helmholtz-muenchen.de/gbrowse2/cgi-bin/

gbrowse_syn/zbailii.

2. Adaptive response of strain ISA1307 to a sub-

lethal growth inhibitory acetic acid concentration

revealed by quantitative proteomics

The first genome-wide expression analysis reported

for an interspecies hybrid strain derived from Z. bailii,

the strain ISA1307, was the quantitative proteomic

analysis published by our group in the journal

Proteomics [3] before the knowledge of the genome

sequence. At that time, ISA1307 was considered of

the Z. bailii species. The objective of this proteomic

analysis was to elucidate the mechanisms

underlying the adaptive response and intrinsic

tolerance to sub-lethal growth inhibitory

concentrations of acetic acid in the highly acetic acid

resistant strain ISA1307, and was based on

quantitative two-dimensional gel electrophoresis (2-

DE) coupled with mass spectrometry for protein

identification. The main goal was to identify

alterations occurring in the protein content in

response to sudden exposure or balanced growth in

the presence of an inhibitory but nonlethal

concentration of acetic acid. Although a total of 111

protein spots were identified by mass spectrometry

with an altered content under the different conditions

examined, only 40% of the differently expressed

proteins could be identified, in part due to the lack of

ISA1307 genome sequence.

3. Screening of a genomic library from strain

ISA1307 to search for genes responsible for acetic

acid resistance

In order to get clues on the genes/proteins involved

in the high level of resistance to acetic acid, we

explored an ISA1307 genomic library previously

constructed that was screened to search for library

plasmids capable to rescue the high susceptibility

phenotype of the deletion S. cerevisiae

BY4741_Δhaa1 with the HAA1 gene encoding the

Haa1 transcription factor deleted. This study, now

submitted for publication [4], identified, at the

genome level, strong candidate genes/proteins

determinants of resistance to acetic acid, also by

exploring the knowledge of the genome sequence.

Main achievements

Quantitative proteomic analysis reinforced the

concept that, when cultivated in a medium containing

glucose and acetate, ISA1307 is able to co-consume

these two carbon sources, with acetate being

channelled through the TCA cycle. This ability to use

acetic acid as a carbon source even in the presence

of glucose, whereas acetate uptake and catabolism

are repressed by glucose in S. cerevisiae, is a

remarkable feature of Z. bailii that is also able to

metabolize sorbate and benzoate. After glucose

exhaustion and in the presence of acetate as the

sole carbon source, an increase in the content of

several proteins involved in gluconeogenesis and

pentose phosphate pathway was registered [3].

The sequencing and annotation of the genome of

the highly acetic acid-tolerant Z. bailii derived

interspecies hybrid strain ISA1307 led to the genome

DNA distributed through 154 scaffolds. The genome

size was found to be around 21.2 Mb,

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Adapted from ref. [2].

18

corresponding to 96% of the genome size estimated

by flow cytometry. Annotation of ISA1307 genome

includes 4385 duplicated genes (about 90% of the

total number of predicted genes) and 1155 predicted

single-copy genes [2]. The availability of the ISA1307

genome sequence also paves the way to a better

understanding of the genetic mechanisms underlying

the generation and selection of more robust hybrid

yeast strains in the stressful environment of wine

fermentations and wines.

The screening of ISA1307 genomic library allowed

the identification of candidate genes for acetic acid

resistance potentially involved in different cellular

processes: cellular transport, transport facilities and

transport routes, protein fate, protein synthesis,

carbohydrate and amino acid metabolism and

transcription [4]. This is consistent with the concept

that the global mechanism behind yeast cells

adaptation and resistance to stress conditions is

multifactorial and do not rely on a single key cellular

process.

Funded Projects

PTDC/AGR-ALI/102608/2008: ZygoSacAR-

Mechanistic insights into acetic acid resistance in

food spoilage yeasts: from the experimental model

Saccharomyces cerevisiae to Zygosaccharomyces

spp. PI: Isabel Sá-Correia


[1] Sá-Correia I, Guerreiro JF, Loureiro-Dias MC, Leão

C, Côrte-Real M. “Zygosaccharomyces”. In: Batt, C.A.,

Tortorello, M.L. (Eds.), Encyclopedia of Food

Microbiology, vol 3. Elsevier Ltd, Academic Press, pp. 849

–855, 2014 (ISBN: 9780123847300)

[2] Mira NP, Münsterkötter M, Valada FD, Santos J,

Palma M, Roque FC, Guerreiro JF, Rodrigues F, Sousa

MJ, Leão C, Güldener U and Sá-Correia I. The genome

sequence of the highly acetic acid tolerant

Zygosaccharomyces bailii-derived interspecies hybrid strain

ISA1307 isolated from a sparkling wine plant, DNA

Research, 1-15, 2014.

[3] Guerreiro JF, Mira NP, Sá-Correia I. Adaptive

response to acetic acid in the highly resistant yeast species

Zygosaccharomyces bailii revealed by quantitative

proteomics, Proteomics, 12, 2303-18, 2012.

[4] Palma M, Roque FC, Guerreiro JF, Queirós L, Mira

NP and Sá-Correia I. Screening of a genomic library from

the Zygosaccharomyces bailii-derived interspecies hybrid

strain ISA1307 to search for genes responsible for acetic

acid resistance, submitted.

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19


12-13

Objectives

Multidrug resistance (MDR) is implicated in the fail-

ure of many therapeutic, food-preservation and crop

protection actions, but can also be explored in the

improvement of biotechnological process productivi-

ty. MDR is often attributed to the action of multidrug

efflux pumps found in the plasma membrane of all

living cells. In this context our research aims to:

1.Carry out the functional analysis of MDR transport-

ers from the Major Facilitator Superfamily (MFS) and

ATP-Binding Cassette (ABC) superfamily in the

model eukaryote Saccharomyces cerevisiae, explor-

ing the obtained data to engineer stress resistant

yeast strains to be used as cell factories.

2.Extend the current knowledge obtained in the

yeast model to the plant model Arabidopsis thaliana,

exploring the obtained information to elucidate the

mechanisms of underlying resistance to chemical

stresses of agricultural interest involving these MDR

transporters and to engineer stress tolerant plants.

3.Extend the current knowledge to the understanding

of antifungal drug resistance in fungal pathogens, in

particular Candida species.

Research Topics

1. Functional analysis of S. cerevisiae MDR trans-

porters of the MFS

Since the release of S. cerevisiae’s genome se-

quence, our research group has been involved in the

functional analysis of new MDR transporters from

the MFS (Sá-Correia et al., 2009, Trends Mibrobiol,

17, 22-31) and ABC superfamily. Ongoing research

pursues a deeper understanding of the physiological

role of these drug efflux pumps and their contribution

to MDR. The evolution of these transporters, based

on sequence and syntenic releationships, are also

being examined, extending current knowledge to

identify and predict the function of MFS-MDR trans-

porters in ascomycetous yeasts. Furthermore, the

functional analysis and engineering of the expres-

sion of these transporters to obtain yeast strains with

an improved stress resistance and fermentation ca-

pacity is also being carried out.

2. Extrapolation of the current knowledge on MDR

transporters to the plant model Arabidopsis thaliana

The knowledge gathered in S. cerevisiae and its use

as a heterologous expression system is being used

to guide the analysis of the role of membrane trans-

porters in A. thaliana resistance to abiotic stress

conditions, in collaboration with the Plant Molecular

Biology (PMB) group, Instituto Gulbenkian de Ciên-

cia (IGC). Several plant MFS transporters are being

functionally and biochemically characterized, focus-

ing on their physiological role and involvement in

stress tolerance (Cabrito et al., 2009, Appl Microbiol

and Biotechnol, 84: 927-936) [1,2]. This joint effort

aims at the elucidation of the biological role of this

poorly characterized family of proteins and the devel-

opment of stress-resistant crops. Due to a recent

collaboration with the Genomics of Plant Stress

Drug efflux pumps in cell defense: from the yeast

model to plant cells BSR

G

Isabel Sá-Correia, Miguel C. Teixeira, Tânia R. Cabrito, Nuno P. Mira, Paulo J. Dias, Sandra

C. dos Santos

PhD and MSc students: Cláudia P. Godinho, Catarina Costa, Joana Guerreiro, Filipa C. Roque, Catarina Prata

Fluorescence microscopy of yeast cells harboring either

the cloning vector or a recombinant plasmid that over-

produces the plant model Pht1;9 fused to the green-

fluorescence protein (GFP) (adapted from ref. [2])

20

(GPlantS) group, Instituto de Tecnologia Química e

Biológica (ITQB), our group is also aiming to contribute

to the functional and biochemical characterization of

membrane transporters from rice, focusing on abiotic

stress responses, namely cold, high salinity and

drought. Given that some of these transporters are

determinants of resistance to compounds of agricultur-

al relevance, such as herbicides, agricultural fungi-

cides, toxic soil contaminant metals, among others,

they can be considered interesting candidates to im-

prove crop growth based on genetic engineering. The

successful cloning and expression of yeast TPO1 in

the plant model Arabidopsis thaliana, resulting in multi-

ple herbicide (2,4-D, barban, metolachlor and alachlor)

and metal (cadmium and aluminium) resistant plant

strains (Patent Application PT 105727, 28/10/2011),

paved the way to the exploitation of yeast MDR efflux

pumps in Plant Biotechnology.

3. Extrapolation of the current knowledge on MDR

transporters to the fungal pathogens of the Candida

genus

The emergence of antifungal drug resistance among

fungal pathogens poses a severe clinical problem.

Drug resistance often results from the action of drug

efflux pumps from the ATP-Binding Cassette and Major

Facilitator Superfamilies (MFS). However, the role of

the putative drug:H+ antiporters (DHA) from the MFS in

fungal pathogens has largely escaped characterization.

The systematic characterization of the DHA

transporters from Candida glabrata is being undertaken

and most of its 10 uncharacterized DHA1 transporters

were indeed found to be implicated in multiple drug

resistance. Altogether, the results obtained during the

ongoing systematic characterization of the forgotten

DHA transporter family in C. glabrata are expected to

improve current understanding of multidrug resistance

in fungal pathogens and to guide de design of new

tools for the diagnosis and treatment of Candida

infections.

Main achievements

The evolutionary history of S. cerevisiae DHA2 (14-

spanner Drug:H+ Antiporter family 2 transporters of the

MFS involved in multidrug resistance), ARN

(siderophore transporters) and GEX (glutathione:H+

antiporters) encoding genes from 31 sequenced yeast

strains of 25 different hemiascomycetous species, was

reconstructed. A common evolutionary root shared by

the encoded proteins was hypothesized and a new

protein family, denominated DAG, was proposed to

span these three phylogenetic subfamilies [3].

The reconstruction of the evolutionary history of the

hemiascomycete DHA1 family genes (Dias et al., 2010,

Omics, 14(6): 701-10) was extended to the species

comprised in the CTG phylogenetic complex to get new

insights on how these family genes have evolved in

medically relevant Candida species [4].

The expression of the S. cerevisiae ABC drug efflux

pump Pdr18, previously found to play a role in the

incorporation of ergosterol in the plasma membrane

(Cabrito et al., Biochem J, 440, 195-202, 2011), was

found to increase yeast ethanol tolerance and

fermentation performance. PDR18 overexpression,

leading to the production of highly inhibitory

concentrations of ethanol, in industrial yeast strains

appears to be a promising approach to improve

alcoholic fermentation performance for sustainable bio-

ethanol production [5].

Through biochemical and functional studies using

the yeast experimental model system, A. thaliana

plasma membrane transporter Pht1;9 was found to

mediate inorganic phosphate acquisition through the

plasma membrane of cells during phosphorus

starvation, being identified as a high-affinity phosphate

Representative images of a wild type and two zifl1 loss-of-function mutant plants grown under

normal water supply or exposed to drought stress (adapted from ref. [1]).

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21


12-13

transporter [2].

The results obtained in the yeast model (Cabrito et

al., Appl Microbiol Biotechnol, 84, 927-936, 2009)

guided the study of ZIFL1 A. thaliana gene in the plant

model. The ZIFL1 gene was found to generate two

alternatively-spliced transcripts, ZIFL1.1 and ZIFL1.3.

ZIFL1.1 was found to be a root tonoplast-localized

transporter that confers resistance to the auxin-like

herbicide 2,4-D, being involved in auxin transport

across the plasma membrane. ZIFL1.3 was found to

be targeted to the plasma membrane of stomatal

guard cells and to regulate drought tolerance [1].

Through heterologous expression and functional

analysis in yeast, another Arabidopsis Tpo1 homolog

gene, ZIF2, was found to be involved in increasing zinc

tolerance and decreasing its intracellular accumulation

in yeast and in A. thaliana [6].

The yeast drug efflux pump Tpo1 was

heterologously expressed in A. thaliana and such

expression was found to reduce the level of growth

inhibition of the transgenic plants exposed to

agricultural pesticides and metal ions (Patent

Application – PT 105727, 28/10/2011). Transgenic

plants that express this yeast gene were found to

accumulate less 2,4-D in the root tips, compared to

plants from the parental strain, when exposed to this

herbicide. These important findings open new avenues

for the use of biotechnology to improve crop tolerance

to chemical and environmental stress.

The Candida glabrata CgQdr2, CgAqr1 and

CgTpo3 were functionally characterized within the

scope of their role in antifungal drug resistance. The

clnical significance of these findings is currently being

inspected (more details in pages 23 and 24) [7-9].

Funded Projects

MFS-stress-Major Facilitator Superfamily transport-

ers in the context of modern agriculture constraints:

exploratory studies (EXPL/AGR-PRO/1013/2013). PI

at IST: Isabel Sá-Correia

FUNDRING – Identification of new biomarkers for

antifungal drug resistance diagnosis in Candida gla-

brata: the particular role of multidrug resistance trans-

porters (PTDC/EBB-BIO/119356/2010). PI: Miguel C

Teixeira

Resistance to pesticides and other chemical stress-

es of agricultural interest: Role of plant Major Facilita-

tor Superfamily Transporters, PTDC/AGR-

AAM/102967/2008, PI at IST: Isabel Sá-Correia


[1] Remy E, Cabrito TR, Baster P, Batista RA, Teixeira

MC, Friml J, Sá-Correia I, Duque P. A Major Facilitator Su-

perfamily transporter plays a dual role in polar auxin transport

and drought stress tolerance in Arabidopsis, The Plant Cell,

25, 901-26, 2013.

[2] Remy E, Cabrito TR, Baptista R, Teixeira MC, Sá-

Correia I, Duque P. The Pht1;9 transporter mediates inor-

ganic Pi acquisition by the Arabidopsis thaliana root during

phosphorus starvation, The New Phytologist, 195, 356–371,

2012.

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Phylogenetic tree of the DHA2, ARN and GEX transportes from Saccharomyces sensu stricto

group

22

[3] Dias PJ, Sá-Correia I, The drug:H+ antiporters of family 2

(DHA2), siderophore ptransporters (ARN) and gluthatione:H+

transporters (GEX) have a common evolutionary origin in

hemiascomycete yeasts, BMC Genomics 14: 901, 2013.

[4] Dias PJ, Sá-Correia I. Phylogenetic and syntetic analysis

of the 12-spanner drug:H+ antiporters family 1 (DHA1) in

pathogenic Candida species: evolution of MDR1 and FLU1

genes. Resubmitted to Genomics, 2014.


Correia I. Increased expression of the yeast multidrug re-

sistance ABC transporter Pdr18 leads to increased ethanol

tolerance and ethanol production in high gravity alcoholic

fermentation, Microbial Cell Factories, 11, 98, 2012.

[6] Remy E, Cabrito TR, Batista RA, Hussein MAM,

Teixeira MC, Athanasiadis A, Sá-Correia I, Duque P.

Intron Retention in the 5'UTR of the Novel ZIF2 Transporter

Enhances Translation to Promote Zinc Tolerance in

Arabidopsis, PLOS Genetics, accepted.

[7] Costa C, Nunes J, Henriques A, Mira NP, Nakayama

H, Chibana H, Teixeira MC. The Candida glabrata drug:H+

antiporter CgTpo3 (ORF CAGL0I10384g): role in azole drug

resistance and polyamine homeostasis, J Antimicrob

Chemother, doi: 10.1093/jac/dku044, 2014





transcription factor, Antimicrob Agents Chemother, 57(7),

3159-67, 2013.

[9] Costa C, Henriques AS, Pires C, Nunes J, Ohno M,

Chibana H, Sá-Correia I, Teixeira, MC. The dual role of

Candida glabrata Drug:H+ Antiporter CgAqr1 (ORF CA-

GL0J09944g) in antifungal drug and acetic acid resistance,

Frontiers in Microbiology, 4, 170, 2013.

[10] dos Santos SC, Teixeira MC, Dias PJ, Sá-Correia I.

MFS transporters required for multidrug/multixenobiotic (MD/

MX) resistance in the model yeast: understanding their

physiological function through post-genomic approaches,

Frontiers in Physiology, 5: 180, 2014.

[11] Costa C, Dias PJ, Sá-Correia I, Teixeira MC. MFS

multidrug transportes in pathogenic fungi: do they have real

clinical impact?, submitted to Frontiers in Physiology,

“Physiological role and regulation of Multidrug/Multixenobiotic

resistance membrane trans-porters”

Issued patent

Sá-Correia I, Cabrito TR, Teixeira MC, Remy E, Duque P.

Utilização de um gene que confere resistência a

xenobióticos em plantas (Use of a gene that confers

resistance to xenobiotics in plants), Provisional national

patent number PT105727. Priority date: 28th October 2011

(Patente de invenção nacional n.º 105727; Date: 2013.06.21)

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Phylogenetic tree of the DHA1 transportes from Saccharomyces sensu stricto group

23


12-13

Antifungal drug resistance and interaction with the

host in the human pathogens Candida glabrata and

Candida albicans BSR

G

Miguel C. Teixeira, Nuno P. Mira

PhD and MsC students: Catarina Costa, Rúben Bernardo, Carla Pires, André Henriques, Joana Nunes, Andreia Ponte,

Diana Cunha

Objectives

Infections caused by Candida species, a problem of

increasing clinical significance, are recognized as the

4th or 5th most common cause of nosocomial infec-

tions. Candida glabrata infections rank second in fre-

quency, after those caused by Candida albicans. The

frequency and relative high mortality levels (up to 45%

for C. glabrata) of these infections are generally at-

tributed to the capacity of these pathogenic yeasts to

efficiently develop multiple drug resistance (MDR). In

the particular case of C. glabrata concern is further

raised by the observation that clinical isolates are of-

ten resistant to azoles, the frontline drugs used in the

treatment and prophylaxis against fungal pathogens.

The key goals of the ongoing research in this field

include the identification of antifungal drug resistance

mechanisms, with focus on the action of multidrug

efflux pumps, aiming the identification of resistance

diagnosis biomarkers and the development of alterna-

tive therapeutic actions. The mechanisms that allow

pathogenic yeast to thrive in the human host, particu-

larly when facing inhibitory weak acid concentrations

are also being inspected.

Research Topics

1. Deciphering antifungal drug resistance mecha-

nisms: the particular role of multidrug transporters of

the DHA family

The emergence of antifungal drug resistance among

fungal pathogens poses a severe clinical problem.

Membrane proteomics is being used to gain further

understanding of the molecular mechanisms underly-

ing the ability of C. glabrata cells to cope with some of

the currently used antifungals. Drug resistance often

results from the action of drug efflux pumps from the

ATP-Binding Cassette and Major Facilitator Super-

families (MFS). However, the role of the putative

drug:H+ antiporters (DHA) from the MFS in fungal

pathogens has largely escaped characterization (more

details in the previous topic).

The systematic characterization of the DHA transport-

ers from Candida glabrata is being undertaken and

most of its 10 uncharacterized DHA1 transporters

were indeed found to be implicated in multiple drug

resistance. In collaboration with the Microbiology Lab

of the Faculty of Medicine of the University of Porto,

the relevance of these transporters in the clinical ac-

quisition of drug resistance is further being analysed.

2. Unveiling the mechanisms of weak acid resistance

in Candida species, and their role in host colonization

To successfully colonize the human host C. albicans

and C. glabrata have to cope with numerous stresses

found in the infection sites which include the presence

of a commensal microbiota. Besides competing for

nutrients, commensal bacteria produce acetic and

lactic acids at concentrations that inhibit growth, spe-

cially in acidic niches such as the vaginal tract. Using

a combination of gene-by-gene and transcriptomic

analyses it was demonstrated that C. glabrata toler-

ance to acetic and lactic acids is largely dependent on

the activity of a novel regulatory system controlled by

the transcription factor CgHaa1. This work was per-

formed in collaboration with the Conway Institute, Uni-

versity College in Dublin (CI-UCD). In the presence of

acetic acid CgHaa1 expression was also found to

increase biofilm formation and adherence of C. gla-

brata to epithelial cells. The elucidation of the mecha-

nism by which CgHaa1 influences biofilm formation

and the adhesive properties of C. glabrata, two well

known virulence traits of this pathogenic yeast, is be-

ing further scrutinized in collaboration with IBB/CEB,

University of Minho.

24

3. Development of molecular-based diagnosis tools for

the detection of systemic candidiasis

The diagnosis of systemic infections caused by C.

albicans and C. glabrata is compromised by the fact

that often the fungal burden present in blood samples

is too low to be detected. The development of more

sensitive diagnosis tools for systemic candidiasis is of

utmost importance since a delay in diagnosis is

associated with an increased risk of death of the

patients. A project aiming the development of a novel

DNA chip that could be used for diagnosis of systemic

candidiasis has been started during 2013 within the

scope of the “PanCandida” project, one of the

awardees of the Gilead Génese 2013 Program. In this

project the transcriptomes of clinical C. glabrata and C.

albicans isolates recovered from the bloodstream of

patients with diagnosed systemic candidiasis are being

compared with the transcriptome of commensal

isolates to identify a set of genes significantly over-

expressed in the systemic isolates that could serve as

a signature of systemic candidiasis and used for

diagnosis. This project is being developed in

collaboration with Centro Hospitalar de Lisboa Central

and CI-UCD.

Main achievements

The expression of CgQDR2 in C. glabrata was found

to confer resistance to imidazole antifungal drugs.

CgQdr2 was found to play a role in the extrusion of

these antifungals from pre-loaded cells. CgQDR2

transcript levels were further seen to be up-regulated

in C. glabrata cells challenged with clotrimazole in the

direct dependency of the CgPdr1 transcription factor

[1].

The Drug:H+ Antiporter CgAqr1 was identified as a

determinant of resistance to the antifungal agent

flucytosine and to reduce the intracellular accumulation

of this drug. CgAqr1 was also found to confer

resistance to acetic acid, which easily accumulates to

inhibitory levels in the vaginal tracts, suggesting that

this transporter may play a role in C. glabrata

persistent colonization in this niche [2].

The DHA CgTpo3 was found to confer resistance to

azole antifungal drugs, catalyzing their extrusion from

within preloaded C. glabrata cells. CgTpo3 was further

found to confer resistance to polyamines, contributing

to decrease its intracellular accumulation in spermine-

challenged cells. Since this compound may reach

inhibitory concentrations in the male genital tract, it is

hypothesized that CgTpo3 may play a role in C.

glabrata persistent colonization in this niche [3].

The role played by the CgHaa1-dependent signaling

system in C. glabrata response and tolerance to acetic

acid stress was characterized. Around 25% of the C.

glabrata genes up-regulated under acetic acid stress

were found to be regulated, directly or indirectly, by

CgHaa1. Several of the CgHaa1-regulated were

required for maximal C. glabrata tolerance to acetic

acid including genes involved in regulation of internal

pH and in multidrug-resistance.

Funded Projects

FUNDRING – Identification of new biomarkers for

antifungal drug resistance diagnosis in Candida glabra-

ta: the particular role of multidrug resistance transport-

ers (PTDC/EBB-BIO/119356/2010). PI: Miguel C.

Teixeira

PanCandida- Towards the development of a pange-

nomic DNA chip for the early detection of systemic

candidiasis caused by C. albicans and C. glabrata

(PGG/035/2013). PI: Nuno P. Mira






transcription factor, Antimicrobial Agents and Chemothera-

py, 57(7), 3159-67, 2013.


Chibana H, Sá-Correia I, Teixeira MC. The dual role of Can-

dida glabrata Drug:H+ Antiporter CgAqr1 (ORF CA-

GL0J09944g) in antifungal drug and acetic acid resistance,

Frontiers in Microbiology, 4, 170, 2013.

[3] Costa C, Nunes J, Henriques A, Mira NP, Nakayama H,

Chibana H, Teixeira MC. The Candida glabrata drug:H+ anti-

porter CgTpo3 (ORF CAGL0I10384g): role in azole drug re-

sistance and polyamine homeostasis, Journal of Antimicro-

bial Chemotherapy, 2014, doi: 10.1093/jac/dku044.

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12-13

Objectives

The activities of the BSRG in the area of Environmen-

tal Biotechnology and Chemistry are developed in the

fields of Environmental Microbiology and Environmen-

tal Toxicology, aiming:

1. To characterize the microbial populations and

their dynamics from processes of biotechnological

interest, such as anaerobic digesters and microbial

fuel cells.

2. To improve knowledge on bacterial biodegrada-

tion of s-triazine herbicides, viewing optimization of

bioremediation strategies that will help to decontami-

nate soils polluted with s-triazine-based herbicidal

formulations (due mainly to overuse, careless dispos-

al or accidental spills). These processes may help to

prevent contamination of adjacent freshwater com-

partments with the ecotoxic herbicides and respective

chlorinated metabolites (please see highlight in page

27).

3. To develop bioassays for the assessment of the

toxicity of xenobiotics (e.g. pesticides, synthetic dyes

and others) based on the two simple models of the

eukaryotic cell, the yeast Saccharomyces cerevisiae

and the nematode Caenorhabditis elegans.

4. To identify molecular biomarkers of toxicity and

to gain insights into potential mechanisms of response

to xenobiotic compounds (e.g. pesticides) based on

global gene expression profiling in the eukaryotic

model S. cerevisiae.

Research Topics

1. Molecular characterization of microbial communi-

ties

Microbial populations from UASB reactors operated

continuous or intermittently (collaborative work with

CESAM and DEP - Univ. Aveiro), and from Microbial

Fuel Cells producing electricity from wastewaters

(collaboration with BERG—IST) are being character-

ized by molecular methods based on 16S rDNA se-

quences. FISH methodologies are being used to

quantify key bacterial members of methane-producing

microbial consortia in UASB reactors treating dairy

industry wastewaters [3, 4, and Nadais et al., 2011,

Energy, 36: 2146-2168].

2. Yeast- and C. elegans-based toxicity testing

The single-cell eukaryotic model S. cerevisiae

(endpoints: growth inhibition, and modification of ex-

pression of selected gene biomarkers) and the simple

animal model C. elegans (endpoint: inhibition of repro-

duction) have been exploited in the assessment of the

potential toxicity of xenobiotics, namely of pesticides

and of synthetic dyes. For example, the levels of tox-

icity of textile-dyes and respective reaction products,

formed during microbial and/or enzymatic biotransfor-

mation processes were compared, in collaboration

with ITQB [Mendes et al., 2011, Bioresour Technol,

102: 9852-9859], and more recently in collaboration

with ENVERG–CEBQ (in course).

Environmental Microbiology and Toxicology

BSR

G

Cristina A. Viegas, Jorge H. Leitão, Christian G. Ramos, Sílvia A. Sousa

PhD and MSc students: Fátima N. Gil, André M. Grilo, Joana R. Feliciano, Mafalda Cardoso;

Research assistant: Vera P. Silva

26

3. Xenobiotic response in S. cerevisiae based on

gene expression profiling

Genome-wide expression profiling of the yeast re-

sponse to exposure to equitoxic concentrations

(20%-growth inhibitory concentrations) of six pesti-

cides from different chemical families

(chloroacetanilide, phenylurea, chlorophenoxyacetic

acid, carbamate and anylinopyrimidine families) has

been analysed in order to identify molecular bi-

omarkers of exposure and toxicity which may be

useful for development of yeast-based bioassays for

preliminary toxicity testing or for assessment of pes-

ticide toxicity in environmental samples, and to pre-

dict novel mechanisms of response to pesticide

moderate toxicity that may be relevant for non-target

eukaryotes, is being explored (collaboration with

IGC) [Gil et al., 2011, Environ Toxicol Chem, 30:

2506-2518].

Main Achievements

A FISH methodology was established to quantify

Synthrophomonadaceae in microbial populations

from UASB reactors [3,4].

Comparison of the datasets of differentially ex-

pressed genes in yeast in response to the six pesti-

cides under study highlighted the potential of the

transcriptional profiling approach to distinguish be-

tween the toxicological effects of structurally different

pesticides in the yeast and allowed the identification

of potential biomarkers of toxicity [1,2]

A toxicity bioassay based on measurements by

reverse transcriptase RT-PCR of transcripts levels

alterations in yeast pyrimethanil-responsive genes

was established [1,2], and its feasibility to assess the

toxicity of simulated runoff samples has been exam-

ined. These samples were generated using a semi-

field simulator of soil contamination with a pyrime-

thanil commercial formulation mimicking worse-case

scenarios (prototype developed by the project part-

ners from IMAR/Univ. Coimbra). Comparison of

data from the gene expression bioassay with con-

ventional ecotoxicity endpoints obtained with the

animal model C. elegans, and using other ecologi-

cally relevant standard aquatic test-organisms

(collaboration with IMAR/Univ. Coimbra), has ena-

bled us to discuss usefulness and limitations of the

yeast and the C. elegans bioassays in environmental

samples biomonitoring (in course).

Funded projects

Studies on the efficacy and scale-up of bioremedi-

ation strategies for soils contaminated with the herbi-

cide terbuthylazine, PTDC/AAC-AMB/111317/2009,

PI: Cristina A. Viegas

Development of bioassays for herbicide toxicity

based on gene expression profiling using yeast cells,

PTDC/AMB/64230/2006, PI: Cristina A. Viegas.

Enzymatic degradation and synthesis of azo and

anthraquinonic dyes, PTDC/BIO/72108/2006, PI at

IST: Cristina A. Viegas.

Optimization and control of intermittent UASB re-

actors and microbial population dynamics, PTDC/

AMB/65025/2006, PI at IST: Jorge H. Leitão.


[1] Gil FN, Gonçalves AC, Becker JD, Viegas CA. Ge-

nome-wide transcriptional response to pesticide exposure

in the model yeast Saccharomyces cerevisiae, the FEBS

journal, 279(S1):230, 2012.

[2] Gil FN, Gonçalves AC, Becker JD, Viegas CA. Pesti-

cide toxicity studies using gene expression profiling in the

model yeast Saccharomyces cerevisiae, 7th European

Conference on Pesticides and Related Organic Micropollu-

tants in the Environment & 13th Symposium on Chemistry

and Fate of Modern Pesticides – Pesticides2012, Porto 7-

10 October, Portugal (oral communication).

[3] Nadais MH, Capela I, Arroja L, Leitão JH, Grilo AM,

Couras C. Effects of operational shocks on UASB microbi-

al populations, 8th Conference on sustainable development

of energy, water and environment systems. September 22-

27, Dubrovnik, Croatia, 2013

[4] Ramos CG, Grilo AM, da Costa PJP, Nadais H,

Leitão JH. Extraction and Purification of DNA from UASB

Reactors Sludge Samples. In: DNA Binding and DNA Ex-

traction: Methods, Applications and Limitations (Chunxu

Zhou and Xia Ling Eds.). Nova Publishers. 2012. ISBN 978

-1-61470-958-9.

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12-13

Objectives

s-Triazine herbicides (e.g. atrazine-ATZ, and ter-

buthylazine-TBA) have been used extensively world-

wide for weed control in a variety of crops, and in

non-agriculture applications. In the European Union

(EU), about 3600 tons of s-triazine herbicides have

been used each year, representing 4.3% of the total

of herbicides. ATZ is moderately mobile through soil,

and accidental spills or careless uses can lead to

contamination of sediments and aquatic systems

due to leaching and runoff events. Indeed, ATZ and

its toxic N-dealkylated metabolites are among the

most frequently detected herbicides on water com-

partments in quantities exceeding the values set up

by regulatory agencies for drinking water [1]. As a

consequence, ATZ has recently been banned in the

EU countries due to the precautionary principle,

though it is still used in the USA, Africa, Latin Ameri-

ca, Asia and Australia. In the last years, mainly in the

EU, TBA has replaced ATZ in herbicidal formula-

tions, hence gaining agricultural relevance. It has

been considered as having more environment-

friendly properties than ATZ (e.g. lower mobility and

bioavailability in soil). However, its higher sorption to

soil organic matter may increase the potential to

reach waters and sediments due to runoff/drainage

events, with special relevance for sites where care-

less disposal or accidental spills may occur, thus

raising ecological risk concerns [1].

In this project, we aim to improve knowledge

and exploit the catabolic ability of two soil s-triazine–

degrading bacterial strains, Pseudomonas sp. ADP

and Arthrobacter aurescens TC1 [1], viewing optimi-

zation of biodegradation of s-triazine herbicides in

soil, mainly focused on ATZ and TBA. Ultimate goal

is to optimize bioremediation strategies that will con-

tribute to decontaminate soils polluted with s-triazine

-based herbicidal formulations (due mainly to over-

use, careless disposal or accidental spills), thus al-

lowing to prevent contamination of adjacent freshwa-

ter compartments with the herbicides and their toxic

chlorinated metabolites.

Main Achievements

As part of a framework for rational bioremedia-

tion of ATZ-contaminated land, we have presented

evidences that a cleanup tool combining soil bio-

augmentation with the ATZ-mineralizing bacterium

Pseudomonas sp. strain ADP and biostimulation

with citrate is effective under worst-case scenarios

of soil contamination with an ATZ-commmercial

formulation (Atrazerba FL) in bench-scale soil mi-

crocosms [1, and Lima et al., 2009, Chemosphere

74:187-192]. To assess the impact of bioremedia-

tion treatments on soil habitat function and soil re-

tention function, an integrated approach has been

used with ecotoxicity tests with standard soil and

aquatic organisms to assess the potential toxicity of

soil and water samples (e.g. eluates, leachates, and

runoffs), respectively [1, and Chelinho et al., 2010,

Soils Sediments, 10:568-578] (collaboration with

IMAR/Univ. Coimbra).

During 2012, it was demonstrated that the appli-

cation of this bioremediation tool at a larger scale

and under more realistic semifield conditions, was

also clearly effective at removing the herbicide from

soil contaminated with high Atrazerba FL (mimicking

misuse applications or accidental spills) [2]. More

importantly, it contributed to reduce the potential

ecological risks of ATZ for both soil and aquatic

compartments in just 7 days [2].

Bioremediation strategies for soils contaminated

with s-triazine herbicides

Cristina A. Viegas, Arsénio M. Fialho

PhD and MSc students: Fátima N. Gil, Carla A. Mateus, Janete Gonçalves, Viviane Varela, Mafalda Cardoso;

Research assistant: Vera P. Silva

BSR

G

1

10

100

0 2 4 6 8

Pseudom

onas

sp

AD

P

x 1

07

CF

U/g

of

soil

Time (d)

28

We also presented evidences that presence in

soil of concentrations of the chloroacetanilide herbi-

cide S-metolachlor (S-MET) representative of high

doses (up to 50xRD) of a commercial formulation

containing both ATZ and S-MET as active substanc-

es (Primextra-S-Gold), did not affect neither Pseudo-

monas sp. ADP survival nor its ability to mineralize

ATZ, in soil [3]. Consistently, biodegradation experi-

ments in soil microcosms spiked with 20x or 50xRD

of this double commercial formulation revealed rapid

ATZ removal from soil. However, it was not accom-

panied by soil detoxification, as indicated by toxicity

of eluates prepared from samples of treated soil

against a microalgae species. This high toxicity may

be due to residues of S-MET and/or its metabolites

remaining in the soil, which appear to be not degrad-

ed by Pseudomonas sp. ADP [3]. These results indi-

cate that caution should be taken when considering

bioremediation strategies for land polluted with mix-

tures of pesticides, particularly when the contami-

nants that may be mixed with ATZ cannot be biode-

graded by the bioaugmentation bacteria applied.

Concerning TBA biodegradation by Pseudomonas

sp ADP, it was found to be slower and less extensive

than that of ATZ, and this appears not to be related

with significant TBA toxicity towards the bacterial

strain [Carla Mateus, MSc in Biotechnology, IST,

2012].

The efficacy of another s-triazine-biodegrading

bacterial strain Arthrobacter aurescens TC1 over

TBA biodegradation in soil have been under focus in

our more recent research. Arthrobacter aurescens

TC1 appears to be better suited than Pseudomonas

sp ADP for TBA biodegradation in soil. Work in

course aims to optimize inocula preparation in order

to obtain and preserve, in advance, high quantities of

physiologically active cells to be used for the bioaug-

mentation of TBA-contaminated soils in semifield and

field scale trials. Studies have mainly focused the

optimization of growth media, and also of cells for-

mulation and conservation methods (e.g. freeze-

drying, vermiculite-adsorption, or refrigeration at 4ºC)

[4, and Viviane Varela, MSc in Biological Engineer-

ing, IST, 2013]. In addition, the bioremediation effica-

cy of a bioremediation tool based on soil bioaugmen-

tation with Arthrobacter aurescens TC1 (refrigerated

pellet cake, 1 month-preservation) has been exam-

ined at semifield scale in soil microcosms contami-

nated with a TBA commercial formulation (10xRD),

combining chemical and ecotoxicity data.

Funded project

Studies on the efficacy and scale-up of bioremedi-

ation strategies for soils contaminated with the herbi-

cide terbuthylazine, PTDC/AAC-AMB/111317/2009,

PI: Cristina A. Viegas.


[1] Viegas CA, Chelinho S, Moreira-Santos M, Costa C,

Gil FN, Silva C, Lima D, Ribeiro R, Sousa JP, Fialho AM.

Bioremediation of soils contaminated with atrazine and

other s-triazine herbicides: current state and prospects, In:

Justin A. Daniels (ed), Advances in Environmental Re-

search - Volume 6, Nova Science Publishers, Inc., NY,

USA, pp. 1-49 (ISBN 978-1-61728-163-1), 2012.

[2] Chelinho S, Moreira-Santos M, Silva C, Costa C,

Viana P, Viegas CA, Fialho AM, Ribeiro R, Sousa JP.

Semi-field testing of a bioremediation tool for atrazine con-

taminated soils: evaluating the efficacy on soil and aquatic

compartments, Environmental Toxicology and Chemis-

try, 31: 1564-1572, 2012.

[3] Viegas CA, Costa C, André S, Viana P, Ribeiro R,

Moreira-Santos M. Does S-metolachlor affects the

performance of Pseudomonas sp. Strain ADP as bioaug-

mentation bacterium for atrazine-contaminated soils?,

PLoS ONE 7(5): e37140, doi:10.1371/

journal.pone.0037140, 2012.

[4] Silva V, Mateus C, Gonçalves J, Varela V, Viegas CA.

A bactéria Arthrobacter aurescens TC1 como ferramenta

de biorremediação para ambientes contaminados com os

herbicidas terbutilazina e atrazina, In: Borrego C, Miranda

AI, Arroja L, Fidélis T, Castro EA, Gomes AP (eds), Actas

da 10ª Conferência Nacional do Ambiente (ISBN: 978-989-

98673-0-7), Departamento de Ambiente e Ordenamento da

Universidade de Aveiro, Aveiro, Portugal, 2013.

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1.5

2

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Mic

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th r

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(d

-1)

Primextra S-Gold dose (xRD)

0 20 50

Atrazerba FL dose (xRD)

29


12-13

Objectives

Cystic Fibrosis (CF) patients have a predisposition to

recurrent and chronic respiratory infections that con-

tribute significantly to disease progression. Although

the large majority of respiratory infections among CF

patients are caused by Pseudomonas aeruginosa,

infections caused by Burkholderia cepacia complex

(Bcc) bacteria are especially threatening and feared

since they are largely associated with a worse prog-

nosis and increased risk of death, in particular with

the so called “cepacia syndrome”. These opportunistic

pathogens are inherently resistant to most antibiotics

and can adapt to adverse environmental conditions,

rendering their eradication from the CF lung almost

impossible. Our research in the field aims to contrib-

ute to the understanding of Bcc pathogenesis in CF

patients, in particular regarding the mechanisms of

persistence and adaptive strategies employed during

long-term residence in the CF lung, occurring along-

side with disease progression. Our research has fo-

cused not only on the most frequent B. cenocepacia

and B. multivorans Bcc species, but also on B. cepa-

cia, B. dolosa, B. stabilis and B. contaminans. These

species are rarely isolated worldwide and are there-

fore poorly studied. We expect to contribute to the

identification of potential determinants of virulence as

targets for new therapeutics in CF and potential tar-

gets for inactivation to enhance antimicrobial activity

and limit persistent infections by these bacteria.

Research Topics

1. Surveillance and epidemiology of Bcc bacteria

amongst a Portuguese CF population followed at

Hospital de Sta Maria

Although transient infections may occur for some pa-

tients, acquisition of Bcc typically results in chronic

infection. Our research group has maintained a 2 dec-

ade-long collaboration with the major Portuguese CF

Treatment Centre at Hospital de Santa Maria (HSM),

in Lisbon, for the microbiological surveillance of Bcc

respiratory infections (Coutinho et al., 2011, Front Cell

Inf Microbio, 1:1-11; Coutinho et al., 2011, Infect Im-

mun, 79:2950-2960). We have published the first

studies describing the epidemiology of Bcc in CF res-

piratory infections in Portugal and gathered a large

collection of over 700 clinical isolates, which includes

numerous clonal variants retrieved during long-term

infection of several CF patients.

During 2012-13, the collaboration with the

HSM CF Treatment Centre and Clinical Pathology

Service was continued and the epidemiological sur-

vey of Bcc respiratory infection amongst the CF popu-

lation followed in this hospital. Also, we have provided

support to Portuguese hospitals regarding the identifi-

cation of the Bcc species to which clinical isolates

belong, using molecular biology approaches, whenev-

er this was considered critical.

Species identification as well as strain geno-

typing and monitoring of clonal variants during long-

term colonization are critical tasks for the systematic

epidemiological surveillance of Bcc bacteria obtained

from CF patients at Hospital de Sta Maria, during the

hospital routine. We have been using state of the art

techniques for species identification and differentia-

tion of specific lineages based on multilocus se-

quence typing (MLST). However, this technical ap-

proach is still limited to the analysis of a small number

of isolates due to the high cost and time-consuming of

this procedure. During 2012-2013, we contributed to

the optimization of a protocol (the SNaPBcen assay)

for extensive bacterial population studies. The strate-

gy used for the SNaPBcen assay is based on target-

ing single nucleotide polymorphisms (SNPs) located

in MLST genes instead of sequencing full MLST se-

quences.

2. Adaptive evolution and persistence in the CF lung

during chronic infection

Bcc bacteria have remarkable genome plasticity,

providing an invaluable advantage for adaptation to

the highly stressful CF lung environment. Widespread

positive selection across the genome of Bcc bacteria

leads to the emergence of multiple phenotypic vari-

ants of the clonal population exhibiting phenotypic

alterations relevant to bacterial pathogenesis. Under-

standing the mechanisms of microbial genetic adapta-

tion during chronic infection, the corresponding impli-

cations for different Bcc species/strains, and the im-

pact of Bcc infections on clinical outcome and disease

Burkholderia cepacia complex bacteria in cystic fibro-

sis: epidemiology, virulence, and adaptive evolution

during chronic lung infection

Isabel Sá-Correia, Carla C. Coutinho, Sandra C. dos Santos

PhD students: Ana S. Moreira, Rita Maldonado

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progression, is vital for the improvement of infection

control policies and therapeutic approaches by CF

Centres.

We have been involved in the characterization

of relevant features in the pathogenesis of sequential

Bcc clonal variants for which patient history is availa-

ble, together with the exploitation of genome-wide

expression approaches and comparative genomic

analyses. Significantly, this includes Bcc species that

are rarely isolated and therefore are poorly studied, in

spite of their damaging effects on CF patients. In fact,

the chronically colonized Portuguese CF population

followed at Hospital de Sta Maria is remarkably atypi-

cal since it includes a high representation of rare Bcc

species in CF, namely, B. cepacia, B. dolosa and B.

contaminans. In particular, the abnormal prevalence

of B. cepacia, a unique case worldwide, was associ-

ated with intrinsically contaminated non-sterile saline

solutions for nasal application, detected, with our col-

laboration, during routine market surveillance by the

Portuguese competent authority (Cunha et al., 2007,

J Clin Microbiol, 45:1628-33).

We have also contributed to the study of mi-

crobial adaptation and evolution within the host envi-

ronment based on extensive phenotypic, genotypic

and genome-wide expression analysis of selected B.

cenocepacia clonal variants (Coutinho et al., 2011,

Infect Immun, 79:2950-2960); Madeira et al., 2011,

Proteomics, 11:1313-1328; Mira et al., 2011, PLoS

ONE, 6:e28831) [1], recently extended to the pheno-

typic variation of B. dolosa [2] and B. cepacia species

(unpublished results). Previous studies have focused

on 11 sequential B. cenocepacia clonal isolates re-

trieved from a chronically colonized CF patient, from

the onset of infection until his death 3.5 years later

with cepacia syndrome. Three of these 11 isolates

have been particularly scrutinized by genome-wide

expression analyses. The virulence of these 3 iso-

lates was also compared, and it was found that the 2

isolates retrieved during late-stage disease are signif-

icantly more virulent [1]. We also used quantitative

proteomics, at both the intracellular and extracellular

levels, to identify mechanisms that could be associat-

ed with this increased virulence and ability to persist

in the lung. Our findings reinforce the crucial role of B.

cenocepacia adaptation to the host environment in

the development of chronic infections and highlight

the importance of metabolic reprogramming in the

adaptation and increased pathogenic potential of B.

cenocepacia during the course of a chronic infection.

Comparative analysis of the endometabolome of dif-

ferent Bcc clinical isolates can be helpful to gain nov-

el insights into the contribution of metabolites to Bcc

virulence in a CF context. As such, we are currently

comparing the endo-metabolome of four sequential B.

cenocepacia clonal variants using an analytical plat-

form based on nuclear magnetic resonance spectros-

copy.

3. Systems biology approach: integration of the re-

sults and more in-depth analyses

We are currently employing an integrated systems

biology strategy, where metabolomic, proteomic, tran-

scriptomic and genomic data are integrated to

achieve a global perspective of Bcc pathogenesis and

evolution within the CF airways (see Figure). The

most promising research lines are being further ex-

plored by in-depth studies. In particular, genomic al-

terations that are identified by the ongoing compara-

tive analysis can be linked with critical phenotypes in

the respective Bcc clonal variants. Similarly, proteins,

metabolites, transcriptional networks and metabolic

pathways of interest emerging from these studies are

being explored in selected Bcc isolates from our col-

lection, to ascertain their relevance in a clinical con-

text and across different Bcc species.

In this context, during 2012 we were partic-

ularly interested in the impact of adaption to micro-

aerophilic conditions in the persistence and viru-

lence of Bcc bacteria in the CF lung, the variation

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12-13

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of lipopolysaccharide (LPS) structure among differ-

ent isolates, and the related immune and pro-

inflammatory responses (in the context of interna-

tional collaborations in the framework of a COST

Action). We have already compared the chemical

structure of the LPS of studied B. cenocepacia

clonal variants, and described the chemical struc-

ture of the lipooligosaccharide (LOS) endotoxin

from a B. dolosa isolate [3]. Given the importance

of LPS biosynthesis and structure in virulence and

persistent infections, this is a topic that we are

highly interested in pursuing.

Main Achievements

The virulence potential of 3 thoroughly charac-

terized sequential B. cenocepacia isolates

(IST439, IST4113 and IST4134) was compared in

human epithelial cell lines, and it was found that

isolates IST4113 and IST4134, retrieved during

late-stage disease, are significantly more virulent.

The proteomic profiling of these 3 variants was

performed and 52 proteins were found to be differ-

entially expressed in the two last isolates com-

pared with IST439. These proteins are involved in

metabolic functions, nucleotide synthesis, transla-

tion and protein folding cell envelope biogenesis

and iron homeostasis. The quantitative compari-

son of the different proteomes suggests an im-

portant role played by metabolic reprogramming in

the virulence potential and persistence of B. ceno-

cepacia, in particular regarding bacterial adapta-

tion to microaerophilic conditions [1].

The exoproteome of isolates IST439, IST4113

and IST4134, was quantitatively compared using

an experimental design that mimics growth in the

CF lung under oxygen limitation in biofilm-like lay-

ers. NMR-based metabolomics was also explored

to compare the metabolome of these same clonal

variants (unpublished results).

The chemical structure of the LPSs of the above

referred variants was also compared in the context

of COST BM1003 action and results indicate a

marked variation between early and late isolates

LPSs (unpublished results).

Whole-genome sequences of sequential B.

cenocepacia clonal variants with marked pheno-

typic differences and virulence level were obtained

and are currently being analyzed by comparative

genomics. This analysis is expected to provide

clues into virulence mechanisms and into the ge-

nomic evolution underwent by B. cenocepacia

during chronic colonization of the patient’s lung,

and how it translates to a clinical setting.

A novel chemical structure and the strong pro-

inflammatory activity of the lipooligosaccharide of

an isolate rom the rarely found species B. dolosa

species, obtained in Portugal from a cystic fibrosis

patient, were described [3].

The phenotypic assessment of 14 clonal iso-

lates of B. dolosa obtained over a period of 5.5

years of surveillance from the only CF patient in-

fected with B. dolosa in the major Portuguese CF

center, during the last 2 decades, was carried out.

Results reinforce the concept that clonal expansion

occurs during long-term colonization. The first iso-

late retrieved was found to be more susceptible to

antibiotics and to exhibit a higher swarming motility

compared with most of the isolates obtained during

the later stages of disease progression and antimi-

crobial therapy [2].

32

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Contribution to the development of the SNaPBCen

assay based on targeting SNPs located in B. cenoce-

pacia MLST genes, envisaging the genotyping of B.

cenocepacia strains and clonal variants. This method

allows the differentiation of specific lineages and is

capable to reveal isolates with microvariation, and the

presence of multiple clonal variants in patients chroni-

cally colonized with this species [4].

Our longstanding work in the field of expression

proteomic analyses applied to microbiological research

led to the publication of an invited chapter in the book

“Burkholderia: From Genomes to Function”, describing

the methodologies currently available and providing

researchers with a simple tutorial on the application of

two-dimensional electrophoresis-based expression

proteomics, as well as presenting a broad literature

review on the application of proteomic approaches to

different Burkholderia species in fields such as diag-

nostics, vaccine development, infection and patho-

genicity, and biodegradation [5].

In the context of COST BM1003 action, we have

also contributed to the development of an internation-

al Pseudomonas aeruginosa reference panel [6].

Funded projects

ADHRES - Signature Project: Expression profiling of

adhesive (AHD) and resistance (RES) genes in biofilm

lifestyle in P. aeruginosa, P. putida and B. cenocepacia,

in the frame of the ERA-NET Patho- genomics, ERA-

PTG/SAU/0001/2008, PI: Isabel Sá -Correia.

COST BM1003 action: Microbial cell surface determi-

nants of virulence as targets for new therapeutics in Cyst-

ic Fibrosis, PI at IST: Isabel Sá-Correia.

PhysBioFFM - Biological physics studies based on

Force Feedback Microscopy, EXPL/FIS-NAN/1395/2013,

PI at IST: Isabel Sá-Correia.


[1] Madeira A, dos Santos AC, Santos PM, Coutinho CP,

Tyrrell J, McClean S, Callaghan M, Sá-Correia I. Proteomic

profiling of Burkholderia cenocepacia clonal isolates with differ-

ent virulence potential retrieved from a cystic fibrosis patient

during chronic lung infection, PLoS One, 8(12): e83065, 2013.

[2] Moreira AS, Coutinho CP, Avezedo P, Lito L, Melo-

Cristino J, Sá-Correia I. Burkholderia dolosa phenotypic varia-

tion during the decline in lung function of a cystic fibrosis patient

along 5.5 years of chronic colonization, Journal of Medical

Microbiology, 63: 594-601, 2014 (Week’s Best Articles: Cystic

Fibrosis February 5-12 2014, according to MDLinx Pulmonolo-

gy).

[3] Di Lorenzo F, Silipo A, Lanzetta R, Fazio LL, Paciello I,

Coutinho CP, Sá-Correia I, Bernardini M, Molinaro A. Struc-

tural elucidation and Biological activity of the lipooligosaccharide

of a Burkholderia dolosa isolate from a cystic fibrosis patient,

ChemBioChem 14(9): 1105-15, 2013.

[4] Eusébio N, Coutinho C, Sá-Correia I, Araújo R. SNaP-

Bcen: a novel and practical tool for genotyping Burkholderia

cenocepacia, Journal of Clinical Microbiology, 51(8): 2646-

53,2013.

[5] Sá-Correia I, dos Santos SC, Madeira A. “Burkholderia

Proteomics: methodologies, challenges, and applications” in

Burkholderia: From Genomes to Function (ed. Tom Coenye &

Eshwar Mahenthiralingam), Horizon Press, 2014.

[6] De Soyza A, Hall A, Mahenthiralingam E, Drevinek P,

Kaca W, Drulis-Kawa Z, Stoitsova S, Toth V, Coenye T, Zlos-

nik J, Burns J, Sá-Correia I, De Vos D, Pirnay JP, Kidd T,

Reid D, Manos J, Lockgether J, Wiehlmann L, Tummler B,

McClean S, Winstanley C. Developing an internation-

al Pseudomonas aeruginosa reference panel, Microbiology

Open, 2(6): 1010-1023, 2013.

Structure of Burkholderia dolosa lipooligosaccharide [3].

33


12-13

Objectives

Long-term infection of the airways of Cystic Fibrosis (CF)

patients with Burkholderia cepacia complex bacteria has

been associated with emergence of mucoid morphotype

variation. The outcome of disease progression seems to

be dependent on the morphotype of the isolates. Our aim

is to understand the cues and the molecular mechanisms

triggering mucoid morphotype variation by following ex-

perimental approaches covering the areas of genomics,

transcriptomics and phenomics.

Research Topics

1. Mucoid morphotype variation of Burkholderia multi-

vorans during chronic persistence in the airways of cystic

fibrosis patients

We have studied two Burkholderia multivorans clonal iso-

lates displaying different morphotypes from a chronically

infected CF patient to evaluate traits development during

lung infection. Expression profiling of mucoid D2095 and

nonmucoid D2214 isolates, using custom Burkholderia

microarrays, revealed downregulation of genes encoding

products related to virulence-associated traits and metab-

olism in D2214. Furthermore, D2214 showed no exopoly-

saccharide production, lower motility and chemotaxis,

lower virulence in Galleria mellonella, and more biofilm

formation. Overall, adaptation during Bcc chronic lung

infections gives rise to genotypic and phenotypic variation

among isolates, contributing to their fitness while main-

taining their capacity for survival in this opportunistic hu-

man niche.

2. Stress conditions triggering the emergence of mucoid

morphotype variation in Burkholderia and effect on antimi-

crobial compounds resistance and biofilm formation in

vitro

We have shown that in vitro, the mucoid clinical isolate B.

multivorans D2095 gives rise to stable nonmucoid vari-

ants in response to nutrient limitation, presence of antibi-

otics, and osmotic and oxidative stresses. Furthermore,

colony morphotype variation within the Burkholderia ge-

nus occurred in Bcc and non-Bcc strains irrespectively of

their clinical or environmental origin. Infection of Galleria

mellonella larvae with mucoid parental isolates and non-

mucoid variants was isolate specific. Survival under nutri-

ent limitation, exposure to oxidative stress and iron limita-

tion was comparable either for the mucoid isolate or the

respective nonmucoid variant. In addition, most of the

tested nonmucoid variants produced more biofilm bio-

mass. Altogether, these findings suggest that important

virulence traits are increased or not significantly affected

by the nonmucoid morphotype of the Burkholderia isolates

and perhaps their virulence potential is preserved.

Mucoid morphotype variation in Burkholderia

cepacia complex clinical isolates

Leonilde M. Moreira, Inês N. Silva, Ana S. Ferreira

MSc student: Andreia C. Tavares

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3. Comparative genomics of Burkholderia multi-

vorans clinical isolates 3. Comparative genomics

of Burkholderia multivorans clinical isolates

Bcc bacteria are characterized by large size ge-

nomes comprising a high number of protein-

encoding genes that confer bacteria the ability to

rapidly adapt and colonize new environmental

niches. To unveil the global genetic differences

that can explain the mucoid phenotypic variation

and give some insights into the evolution of ge-

nome content during chronic lung infection, a

comparative genomic study of two sequential B.

multivorans clinical isolates – the mucoid D2095

and the nonmucoid D2214 was carried out. Whole

-genome sequence and de novo assembly

showed a genome size of 6.7 and 6.5 Mb for B.

multivorans D2095 and D2214, respectively. Com-

parative genomics identified a large deletion of

175 kb in D2214 genome. The remaining muta-

tions are single base-pair mutations or small inser-

tion/deletions. Some of the mutations are likely to

cause partial or complete loss-of-function in the

protein encoded by the altered gene. Our data

confirms genome reduction during chronic infec-

tion as observed for other CF pathogens and

opens new hypotheses for the mechanisms trig-

gering mucoid-to-nonmucoid variation in Bcc.

Main Achievements

Evidences on the genotypic and phenotypic

traits variation of Burkholderia cepacia complex

bacteria during chronic lung colonization.

Demonstration that nutrient limitation, presence

of antibiotics, and osmotic and oxidative stresses

are triggering Bcc mucoid morphotype variation in

vitro.

Sequence determination, assembly and ge-

nome annotation of the two clinical isolates: mu-

coid B. multivorans D2095 and nonmucoid D2214

in collaboration with Prof. Pedro Santos from Uni-

versity of Minho.

Identification of several mutations putatively

involved in mucoid morphotype variation by com-

parative genomic approaches.

Funded projects

KINASE- Phosphoproteomics in Burkholderia

to assess the role of tyrosine phosphorylation in

virulence determinants expression, PTDC/QUI-

BIQ/118260/2010, PI: Leonilde M. Moreira.


Silva IN, Tavares AF, Ferreira AS, and Moreira LM.

Stress conditions triggering mucoid morphotype varia-

tion in Burkholderia species and effect on virulence in

Galleria mellonella and biofilm formation in vitro. PLoS

One. 8(12): e825222. doi: 10.1371/

journal.pone.0082522, 2013.

35


12-13

Objectives

Extracellular polysaccharides (EPS) of bacterial

origin have diverse functions in nature, such as in

bacterium-plant interactions, virulence factors or

protective agents. Due to their unique properties as

rheology modification agents, stabilizers, emulsifiers

and gelling agents, they also represent an important

class of polymeric materials with interest in Biotech-

nology. Research carried out by our group is fo-

cused on the proteins directing EPS biosynthesis,

on EPS-mediated interaction with hosts either in

symbiosis or pathogenesis and on EPS potential

industrial applications. The biological systems under

study are the exopolysaccharide cepacian from

Burkholderia and succinoglycan from Sinorhizobium

meliloti.

Research Topics

1. Proteins required for exopolysaccharide biosyn-

thesis in Burkholderia cepacia complex

The Burkholderia cenocepacia J2315 bceN gene

was shown to encode a protein with GDP-D-

mannose 4,6-dehydratase enzyme activity

(E.C.4.2.1.47). The protein is only active when in

the tetrameric form and catalyzes the conversion of

GDP-D-mannose into GDP-4-keto-6-deoxy-D-

mannose. This sugar nucleotide is the intermediary

necessary for the biosynthesis of GDP-D-rhamnose,

one of the sugar residues of cepacian. The enzyme

kinetics characterization by NMR spectroscopy re-

vealed that the enzyme requires Mg2+ for its activity,

is strongly inhibited by the substrate, and follows a

non-Michaelis-Menten kinetics model. The lack of a

functional bceN gene in a mutant derived from B.

cepacia IST408 slightly reduced cepacian produc-

tion. However, in the B. multivorans ATCC 17616

strain that harbors bceN as the single gene in its

genome with predicted GDP-mannose dehydratase

activity, a bceN mutant did not produce cepacian,

indicating that the bceN gene product is required for

cepacian biosynthesis.

2. Analysis of the Burkholderia cepacia tyrosine

kinase bceF mutant reveals a role in tolerance to

stress, biofilm formation and interaction with host

cells

The bacterial tyrosine (BY) kinase family comprises

the major group of bacterial enzymes endowed with

tyrosine kinase activity. We have previously shown

that BceF protein from Burkholderia cepacia IST408

belongs to this BY-kinase family and is involved in

the biosynthesis of the exopolysaccharide cepacian.

However, little was known about the extent of regu-

lation by this protein kinase activity. In order to ex-

amine this we performed a comparative transcrip-

tome profile between the bceF mutant and the wild-

type B. cepacia IST408. Genes with decreased ex-

pression in the bceF mutant were related to stress

response, motility, cell adhesion, and carbon and

Bacterial exopolysaccharides: biosynthesis

and biological role

Leonilde M. Moreira, Jorge H. Leitão, Ana S. Ferreira, Sílvia A. Sousa, Inês N. Silva

PhD and MSc students: Mário R. Santos, Joana R. Feliciano, Vítor H. Oliveira, Sílvia Matos, Micaela Torrado; Research

Assistant: Andreia Silva

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energy metabolism. Genes with increased expres-

sion were related to intracellular signaling, type VI

secretion and lipid metabolism. Mutation of bceF

gene led to reduced survival under heat-shock

stress and UV-light exposure, reduced swimming

motility and alteration of biofilm architecture when

grown in vitro. The bceF mutant showed decreased

ability to adhere and invade human lung epithelial

cells and to disrupt tight junctions, which enables

bacteria to reach paracellularly the basolateral sur-

face. As homologues of BceF occur in a number of

pathogenic and plant-associated Burkholderia

strains, the modulation of bacterial behavior through

tyrosine kinase activity is most likely a widely occur-

ring phenomenon.

3. Sinorhizobium meliloti proteins with a role in ex-

opolysaccharide biosynthesis and in nitrogen fixation

symbiosis

Members of the TetR family of repressors are known

to control genes whose products are involved in mul-

tidrug resistance, catabolic pathways, biosynthesis

of antibiotics, osmotic stress and pathogenicity. We

have characterized a TetR member of S. meliloti

encoded by gene SMc03169. A deletion mutant ex-

hibited lower tolerance to acidic conditions and in-

creased susceptibility to detergents and oxidative

stress agents. This mutant also showed defects in

symbiosis with Medicago sativa, since the number of

pink nodules decreased by one-half and it was una-

ble to directly compete with the wild-type strain for

nodulation. Expression profiling showed downregula-

tion of genes involved in Nod factor and siderophore

biosynthesis, phosphate uptake systems, and genes

belonging to the heat-shock regulon. Among the

upregulated genes in the mutant we found genes

directing succinoglycan biosynthesis, lipopolysac-

charide modification and genes whose products are

involved in metabolism.

Main Achievements

Functional and kinetics characterization of the

GDP-mannose 4,6-dehydratase from Burkholderia

cepacia complex bacteria [1,2].

Demonstration that the BceF tyrosine kinase from

Burkholderia cepacia is required for exopolysaccha-

ride biosynthesis, stress response, biofilm formation

and virulence [3].

Demonstration that Burkholderia cepacia BceF

protein is required for enhancement of CFBE41o-

cell invasion and paracellular translocation and mod-

ulation of the proinflammatory response.

Demonstration that the Sinorhizobium meliloti

TetR repressor encoded by SMc03169 is involved in

regulatory networks targeting cell envelope and me-

tabolism and is required for the biological nitrogen

fixation symbiosis with Medicago plants [4].

Funded projects

ROOT-INT– Role of a two-component regulatory

system in the early interaction between Sinorhizobi-

um meliloti and plant root hairs, PTDC/BIA-

MIC/113733/2009, PI: Leonilde M. Moreira.

Exploiting the type II phosphomannose isomerise

BceAJ as a new target for the development of new

antimicrobials and for biotechnological applications,

PTDC/EBB-BIO/098352/2008, PI: Jorge H. Leitão.

How do Bacterial Phosphotyrosine Phosphatase

Proteins Modulate Host-Pathogen Interactions? Eu-

ropean Society of Clinical Microbiology and Infec-

tious Diseases (ESCMID) PI: Ana S. Ferreira.


[1] Sousa SA, Feliciano JR, Pinheiro PF, Leitão JH.

Biochemical and functional studies on the Burkholderia

cepacia complex bceN gene, encoding a GDP-D-mannose

4, 6-dehydratase. PLoS One 8 (2), e56902, 2013.

[2] Sousa SA, Feliciano JR, Grilo AM, Leitão JH.

Bioinformatics: a molecular microbiologist’s perspective.

Current Bioinformatics 9:8-17, 2014.

[3] Ferreira AS, Silva IN, Oliveira VH, Becker JD,

Givskov M, Ryan RP, Fernandes F, Moreira LM. Com-

parative transcriptomic analysis of the Burkholderia cepacia

tyrosine kinase bceF mutant reveals a role in tolerance to

stress, biofilm formation and virulence. Applied and Envi-

ronmental Microbiology, 79, 3009-3020, 2013.

[4] Santos MR, Tomás AT, Becker JD, Moreira LM. Sino-

rhizobium meliloti EmrR regulator is required for efficient

colonization of Medicago sativa root nodules. Molecular

Plant-Microbe Interactions, 27, 388-399, 2014.

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12-13

Objectives

Our research focused on studying surface exposed

proteins, namely trimeric autotransporter adhesins

(TAAs), in Burkholderia pathogenicity associated

with infection of the human respiratory tract. The

goal of the project is the molecular and functional

characterization of these TAAs, aiming to gain a

better understanding of how pathogenic Burkholderia

initiate infections. The research specifically focused

on three TAA encoding genes that are clustered

together and belong to a unique genomic region of

the cystic fibrosis epidemic strain Burkholderia

cenocepacia J2315.

Research topics

1. The trimeric autotransporter BCAM0224: a Swiss

army knife of Burkholderia cenocepacia

We show that BCAM0224 occurs on the bacterial

surface and adopts a trimeric conformation. Further,

we demonstrated that BCAM0224 is needed for the

earlier stages of biofilm formation and is required for

swarming motility. In addition, BCAM0224 plays an

important role in evasion of the human innate

immune system, providing resistance against the

bactericidal activity of serum via the complement

classical pathway. Finally, BCAM0224 mediates

bacterial adhesion to and invasion of cultured human

bronchial epithelial cells. Together, our data reveal

the high versatility of the BCAM0224 protein as a

virulence factor in pathogenic bacterium B.

cenocepacia [3].

In collaboration with Prof. Yves Dufrêne,

Institute of Condensed Matter and Nanosciences,

Université Catholique de Louvain, Belgium, Atomic

force microscopy (AFM) has been used to examine

the interactions between B. cenocepacia BCAM0224

-BCAM0224 and between B. cenocepacia

BCAM0224 and living airway epithelial cells [4].

We have shown that the adhesin forms

homophilic trans-interactions engaged in bacterial

aggregation, and that it behaves as a spring capable

to withstand high forces. We also founded that

BCAM0224 binds collagen, a major extracellular

component of host epithelia. Both homophilic and

heterophilic interactions displayed low binding

affinity, which could be important for epithelium

colonization. We also demonstrated that BCAM0224

recognizes receptors on living pneumocytes, and

leads to the formation of membrane tethers that may

play a role in promoting adhesion. Collectively, our

results showed that BCAM0224 is a multifunctional

adhesin endowed with remarkable binding

Unraveling the involvement of trimeric autotransporter

adhesins in Burkholderia cenocepacia host-cells interactions

Arsénio M Fialho, Dalila Mil-Homens

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Gene organization of the TAA gene cluster in B. cenocepacia J2315 [1,2 and Mil-Homens et al., 2010, Microbiology, 156, 1084-96; Mil-Homens et al., 2011, Front Cell Infect Microbiol, 1:13].

(A) - BCAM0224 structure prediction. Computer modelling

of the putative 3D structure for the membrane-anchor

region of BCAM0224. (B) – Membrane localization (M-

monomers; T – trimmers) of BCAM0224 in the wild type B.

cenocepacia K56-2. (C) - Fluorescence confocal

microscopy images of B. cenocepacia K56-2 and

BCAM0224::Tp mutant using the anti-BCAM0224 antibody.

38

properties, which may represent a general

mechanism among TAAs for strengthening bacterial

adhesion [4].

2. Identifying host cellular receptors targeted by B.

cenocepacia TAA adhesins

The research specifically focuses on identifying host

cell receptors for B. cenocepacia TAAs. To gain

insights into the nature of such receptors, we are

developing a combination of genetic, cellular,

molecular and imaging techniques. As a first

approach and since lipid rafts are primary interaction

sites for pathogens, we are interested in analyzing

the involvement of such microdomains in

Burkholderia-host cell interaction events. In this way,

we have started examining if a pretreatment with

Methyl-b-cyclodextrin (MβCD) (a lipid raft disrupter)

can impair the adhesion of B. cenocepacia to host

cells. As a second approach, we are focusing our

experiments in various putative TAA host cell

receptors, such as glycolipids (aGM1, aGM2,

globoside, Gb2 and Gb3), mucins (mucus

glycoproteins) and proteins (TNF receptor 1, TLR2,

cytokeratin 13). Then, we have examined whether

pretreatment of host cells with enzymes and

antibodies that degrade/block such receptors,

prevents/impairs B. cenocepacia (wild type and TAA

mutants) adhesion. Our ultimate goal in this research

is to gain an improved understanding of the molecular

basis of host-specificity and virulence of B.

cenocepacia and identify potential protein targets for

more effective disease management.

Funded project

Fundação para a Ciência e a Tecnologia (FCT),

Portugal, Grant PTDC/BIA-MIC/118386/2010.


[1] Mil-Homens D, Bernardes N, Fialho AM. The

antibacterial properties of docosahexaenoic omega-3 fatty

acid against the Cystic Fibrosis multi-resistant pathogen

Burkholderia cenocepacia, FEMS Microbiology Letters

328:61-69, 2012

[2] Mil-Homens D, Fialho AM. A BCAM0223 Mutant of

Burkholderia cenocepacia is Deficient in Hemagglutination,

Serum Resistance, Adhesion to Epithelial Cells and

Virulence, PLoS One, 7(7):e41747, 2012.

[3] Mil-Homens D, Leça MI, Fernandes F, Pinto SN,

Fialho AM. Characterization of BCAM0224, a

multifunctional trimeric autotransporter from the human

pathogen Burkholderia cenocepacia, Journal of

Bacteriology, 2014 (doi:10.1128/JB.00061-14)

[4] El-Kirat-Chatel S, Mil-Homens D, Beaussart A, Fialho

AM, Dufrêne YF. Single-molecule atomic force microscopy

unravels the binding mechanism of a Burkholderia

cenocepacia trimeric autotransporter adhesin, Molecular

Microbiology, 89(4):649-659, 2013.

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BCAM0224 from B. cenocepacia K56-2, a multifaceted protein as

revealed by its different biological properties.

39


12-13

Objectives

Small non-coding RNAs are increasingly recognized

as powerful tools used by bacterial pathogens to

rapidly adapt their physiology and fitness to the host

environment. Work carried out by our group envisag-

es the identification and functional characterization of

sRNAs and their mRNA targets, particularly of those

related to virulence and resistance to stresses mim-

icking those faced by the bacterium during the infec-

tion process. Our goal is to identify sRNAs and path-

ways which might be exploited as targets for thera-

peutic intervention against infections caused by bac-

teria of the Burkholderia cepacia complex.

Research Topics

1. Identification and validation of sRNAs from bacte-

ria of the Burkholderia cepacia complex.

A total of 24 sRNAs from B. cenocepacia J2315 were

identified experimentally, based on the co-purification

of sRNAs with the bacterium Hfq protein, followed by

conversion into cDNA, cloning, computational analy-

sis of sequences and validation by Northern blot

analysis. The sRNAs identified escaped previous

identification in studies based on transcriptomic or

bioinformatics analyses. Three sRNAs were found

exclusively within genome sequences of bacteria of

the Burkholderia cepacia complex and have no hom-

ologues in other bacteria, while the other 21 share

homology, at a different extent, to sRNAs of other

bacterial species.

2. Functional characterization of h2cR

The novel cis-encoded sRNA h2cR from the human

opportunistic pathogen Burkholderia cenocepacia

J2315 was identified and functionally characterized.

The sRNA was found to negatively regulate the hfq2

mRNA, through binding to part of the 5’-UTR region

of the hfq2 mRNA, resulting in accelerated hfq2

mRNA decay and reduced protein levels in exponen-

tially growing cells. Both the h2cR transcript and the

hfq2 mRNA are stabilized by the other B. cenocepa-

cia RNA chaperone, Hfq. Infection experiments using

the nematode Caenorhabditis elegans revealed that

down-regulation of Hfq2 by h2cR decreases the bac-

terium ability to colonize and persist within the nema-

Small non-coding RNAs in Burkholderia cepacia complex

Jorge H. Leitão, Christian G. Ramos, Sílvia A. Sousa

PhD students: André M. Grilo, Joana R. Feliciano

BSR

G

40

tode, suggesting a role for h2cR on bacterial per-

sistence in the host.

3. Functional characterization of MtvR

The 136 nt-long small non-coding RNA MtvR from

the Bcc member Burkholderia cenocepacia J2315

was identified and characterized. MtvR homo-

logues are restricted to the Burkholderia genus.

The mRNA levels corresponding to 17 out of a total

of 19 selected genes were affected when MtvR was

either overexpressed or silenced. MtvR was found

to interact with its target hfq mRNA by binding ex-

clusively to the 5’-UTR of the hfq mRNA, requiring

nucleotides 8-16 and 125-129 for pairing. This in-

teraction resulted in decreased protein synthesis,

suggesting a negative regulatory effect of MtvR on

the RNA chaperone Hfq. Bacterial strains with

MtvR silenced or overexpressed exhibited plei-

otropic phenotypes related to growth and survival

to several stresses, swimming and swarming motili-

ties, biofilm formation, resistance to antibiotics

(especially to aminoglycosides), and ability to colo-

nize and kill the nematode Caenorhabditis elegans.

MtvR is proposed to be a major global regulatory

sRNA in Bcc bacteria.

Main Achievements

A methodology for the experimental identifica-

tion of sRNAs based on their co-precipitation with

Hfq-like proteins was established. This methodolo-

gy led to the identification of 24 sRNAs from the

epidemic strain B. cenocepacia J2315 [1].

The cis-encoded sRNA h2cR was identified.

h2cR was found to be a negative regulator of the

mRNA levels of hfq2, depending on the RNA chap-

erone Hfq for its stability [2].

The sRNA MtvR was functionally characterized.

Work performed showed that this sRNA affects the

transcripts levels of multiple genes pointing out

MtvR as a global regulatory sRNA in bacteria of the

Burkholderia cepacia complex [3].

Funded project

RNomics-Bcc: RNomics studies to identify

small non-coding RNAs of Burkholderia cepacia

complex involved in host-pathogen interactions,

PTDC/BIA-MIC/119091/2010, PI: Jorge H. Leitão.


[1] Ramos CG, Grilo AM, da Costa PJP, Leitão JH.

Experimental identification of small non-coding regulatory

RNAs in the opportunistic human pathogen Burkholderia

cenocepacia J2315. Genomics 101(2):139-148, 2013.

[2] Ramos CG, da Costa PJP, Döring G, Leitão JH.

The novel cis-encoded small RNA h2cR is a negative

regulator of hfq2 in Burkholderia cenocepacia. PLOS One

7(10): e47896, 2012.

[3] Ramos CG, Grilo AM, da Costa PJP, Feliciano JR,

Leitão JH. MtvR is a global regulatory sRNA in

Burkholderia cenocepacia. Journal of Bacteriology, 195

(16), 3514–3523, 2013.

[4] Ramos CG, Leitão JH. “Caenorhabditis elegans as a

research tool to unveil bacterial virulence determinants:

lessons from the Burkholderia cepacia complex”, In: Nem-

atodes: Morphology, Functions and Management

Strategies (Fausto Boeri and Jordan A. Chung, Eds.),

Nova Science Publishers, Inc., NY, USA, 135-156, 2012

(ISBN: 978-1-61470-784-4).

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h2cR

hfq2

70

70

h2cR

hfq2

70

70

X

XcepR

Hfq

Exponential Stationary

RNase RNase

Hfq

Hfq

? ?

h2cR

hfq2

70

70

h2cR

hfq2

70

70

X

XcepR

Hfq

Exponential Stationary

RNaseRNaseRNase RNaseRNaseRNase

Hfq

Hfq

? ?

41


12-13

Our research, in collaboration with IPATIMUP,

Portugal, focused on studying the bacterial protein

azurin as an anti-cancer agent. Azurin is a low

molecular weight (14kDa), water-solube protein

produced by the bacterium Pseudomonas

aeruginosa. It has cytotoxicity towards cancer cell

lines in vitro and in vivo. Azurin can enter

preferentially into cancer cells, acting as a

multivalent agente [1-3]. On entry into cancer cells,

azurin forms a complex with p53, stabilizing it and

inducing apoptosis. Moreover, azurin inhibits

angiogenesis through inhibition of the

phosphorylation of VEGFR-2, FAK and AKT. p28, a

peptide of azurin, has recently completed a Phase I

clinical trial with promising results [4,5].

Research topics

We aimed to study the molecular and cellular events

occurring when breast cancer cells overexpressing

P-cadherin, were treated with azurin. P-cadherin

overexpression occurs in about 30% of all breast

carcinomas representing one of the most

aggressive sub-type of breast cancer.

1. Azurin impairs invasion and causes a specific

decrease of P-cadherin levels in human breast

cancer cell lines

A sub-lethal single dose of azurin (with cell viability

of at least 80%) produced a decrease in the

invasion of two P-cadherin expressing breast cancer

cell models, the luminal MCF-7/AZ.Pcad and the

triple negative basal-like SUM 149 PT through a

Matrigel artificial matrix. In both cell lines, the

decrease in invasion was associated with a

decrease in the total P-cadherin protein levels and a

concomitant decrease of its membrane staining,

whereas E-cadherin remains not altered with high

expression levels and with normal membrane

localization [6].

2. Azurin decreases soluble forms of P- and E-

cadherin

Metalloproteinases (MMPs) are involved in the

degradation of the extracellular matrix components.

The active form of MMP2 has been detected in half

of all human breast carcinomas. In azurin treated

cells the activity of MMP2 in the extracellular media

of cells was decreased. The proteolytic activity of

MMP2 acts, in part, by shedding P-cadherin

extracellular domain itself, releasing a soluble form

of P-cadherin, sPcad12, which was also reduced in

the extracellular media of azurin-treated cells [6].

3. Signaling pathways and matrix remodeling

triggered by azurin

We investigated signaling pathways triggered by

azurin in cancer cell treatments. FAK and Src are

non-receptor tyrosine kinases that mediate a

significant number of signaling pathways with

relevance to cell-cell and cell-matrix adhesions. We

observed that azurin leads to a decrease in the

phosphorylation levels of both these proteins [6].

Bacterial Proteins in Cancer Therapy: azurin, a therapeutic

protein that interferes with oncogenic signaling and blocks

tumor progression

BSR

G

Arsénio M. Fialho, Nuno Bernardes

MSc student: Sofia Abreu

Azurin multivalent action against two P-cadherin expressing breast cancer cell models.

Azurin, a bacterial protein with anti-cancer properties

42

4. Array-based gene expression profile of azurin

treated breast cancer cells

Microarrays analysis reveals genetic pathways

modulated by azurin that are important contributors

to cell proliferation in breast cancer, in particular

pathways that regulate cell-cell adhesion and cell-

matrix interactions. We have performed a microar-

ray analysis to infer about the signaling pathways

that are altered in a p53 wild-type and P-cadherin

overexpressing breast cancer cell model upon az-

urin treatment. Our results strengthen the hypothe-

sis that azurin has a multivalent action towards

cancer cells, promoting the endocytosis of cell sur-

face receptors and the interruption of signaling

pathways hyper activated in cancer cells. The

blockage of these signaling pathways in P-cadherin

overexpressing breast cancer cell models leads to

the abrogation of tumor cell invasion, providing a

possible new therapeutic approach to this specific

type of breast cancer [7].

Altogether, our data show that azurin exhibits a

specific preference to decrease P-cadherin expres-

sion levels, abrogating its invasive effects and,

therefore, may have a potential use in the treat-

ment of breast carcinomas overexpressing this

protein.

Financed project

Fundação para a Ciência e a Tecnologia (FCT),

Portugal, Grant PTDC/EBB-BIO/100326/2008.


[1] Fialho AM, Salunkhe P, Manna SK, Mahali S,

Chakrabarty AM. Glioblastoma Multiforme: Novel Thera-

peutic Approaches, ISRN Neurology, Article ID 642345,

2012.

[2] Fialho AM, Bernardes N, Chakrabarty AM. Recent

patents on live bacteria and their products as potential

anticancer agents, Recent Patents on Anti-Cancer Drug

Discovery 7: 31-55, 2012.

[3] Bernardes N, Chakrabarty AM, Fialho AM. Engi-

neering of bacterial strains and their products for cancer

therapy, Applied Microbiology and Biotechnology, 97

(12):5189-5199, 2013.

[4] Fialho AM, Chakrabarty AM. Patent controversies

and court cases: Cancer diagnosis, therapy and preven-

tion, Cancer Biology and Therapy, 13: 1229 – 1234,

2012.

[5] Avner BS, Fialho AM, Chakrabarty AM. Overcoming

drug resistance in multi-drug resistant cancers and micro-

organisms: A conceptual framework, Bioengineered,

3:262-270, 2012.

[6] Bernardes N, Ribeiro AS, Abreu S, Mota B, Matos

RG, Arraiano CM, Seruca R, Paredes J, Fialho AM.

The bacterial protein azurin impairs invasion and FAK/Src

signaling in P-cadherin-overexpressing breast cancer cell

models, PLoS One, 8 (7), e69023, 2013.

[7] Bernardes N, Ribeiro AS, Abreu S, Vieira AF, Car-

reto L, Santos M, Seruca R, Paredes J, Fialho AM.

High-throughput molecular profiling of a P-cadherin over-

expressing breast cancer model reveals new targets for

the anti-cancer bacterial protein azurin, The International

Journal of Biochemistry & Cell Biology, 50: 1-9, 2014.

Issued patent

Fialho AM, Bernardes N, Gonçalves J, Santos AC.

Compositions to treat HIV infections and methods there-

for, 1126/MUM/2011, March 2012.

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ese

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Array-based gene expression profile of azurin treated

breast cancer cells.

http://www.ncbi.nlm.nih.gov/pubmed/22954683



http://www.landesbioscience.com/journals/bioe/article/21130/



43


12-13

Articles in International Peer-Reviewed

Journals

[1] Avner BS, Fialho AM, Chakrabarty AM. Overcoming

drug resistance in multi-drug resistant cancers and microor-

ganisms: A conceptual framework, Bioengineered, 3, 262-

270, 2012.

[2] Barbas A, Popescu A, Frazão C, Arraiano CM, Fialho

AM. Rossmann-fold motifs can confer multiple functions to

metabolic enzymes: RNA binding and ribonuclease activity

of a UDP-glucose dehydrogenase, Biochemical & Bio-

physical Research Communications, 430(1): 218-24,

2013.

[3] Bernardes N, Ribeiro AS, Abreu S, Vieira AF, Carreto

L, Santos M, Seruca R, Paredes J, Fialho AM. High-

throughput molecular profiling of a P-cadherin overexpress-

ing breast cancer model reveals new targets for the anti-

cancer bacterial protein azurin, The International Journal

of Biochemistry and Cell Biology, 2014 (doi: 10.1016/

j.biocel.2014.01.023).

[4] Bernardes N, Ribeiro AS, Abreu S, Mota B, Matos

RG, Arraiano CM, Seruca R, Paredes J, Fialho AM. The

bacterial protein azurin impairs invasion and FAK/Src sig-

naling in P-cadherin-overexpressing breast cancer cell

models, PLoS One, 8(7): e69023, 2013.

[5] Bernardes N, Chakrabarty AM, Fialho AM. Engineer-

ing of bacterial strains and their products for cancer thera-

py, Applied Microbiology and Biotechnology, 97(12):

5189-99, 2013.

[6] Chelinho S, Moreira-Santos M, Silva C, Costa C,

Viana P, Viegas AC, Fialho AM, Ribeiro R, Sousa JP.

Semi-field testing of a bioremediation tool for atrazine-

contaminated soils: evaluating the efficacy on soil and

aquatic compartments, Environmental Toxicology and

Chemistry, 31, 1564-1572, 2012.

[7] Costa C, Nunes J, Henriques A, Mira NP, Nakayama

H, Chibana H, Teixeira MC. The Candida glabrata drug:H+

antiporter CgTpo3 (ORF CAGL0I10384g): role in azole drug

resistance and polyamine homeostasis, Journal of Antimi-

crobial Chemotherapy, 2014 (doi: 10.1093/jac/dku044).


Chibana H, Sá-Correia I, Teixeira MC. Candida glabra-

ta drug:H+ antiporter CgQdr2 (ORF CAGL0G08624g) con-

fers imidazole drug resistance, being activated by the CgP-

dr1 transcription factor, Antimicrobial Agents and Chem-

otherapy, 57(7), 3159-67, 2013.


Chibana H, Sá-Correia I, Teixeira MC. The dual role

of Candida glabrata Drug:H+ Antiporter CgAqr1

(ORFCAGL0J09944g) in antifungal drug and acetic acid

resistance, Frontiers in Microbiology, 4, 170, 2013.

[10] de Carvalho J, Rodrigues RMM, Tomé B, Henriques

SF, Mira NP, Sá-Correia I, Ferreira GNM. Conformational

and Mechanical changes of DNA upon transcription factor

binding detected by a QCM and transmission line mod-

el, Analyst, 139(8): 1847-55, 2014.

[11] De Soyza A, Hall A, Mahenthiralingam E, Drevinek

P, Kaca W, Drulis-Kawa Z, Stoitsova S, Toth V, Coenye

T, Zlosnik J, Burns J, Sá-Correia I, De Vos D, Pirnay JP,

Kidd T, Reid D, Manos J, Klockgether J, Wiehlmann L,

Tummler B, McClean S, Winstanley C. Developing an

international Pseudomonas aeruginosa reference panel,

Microbiology Open, 2, 1010-23, 2013.

[12] Dias PJ, Sá-Correia I. The drug:H+ antiporters of fami-

ly 2 (DHA2), siderophore transporters (ARN) and glutathi-

one:H+ antiporters (GEX) have a common evolutionary

origin in hemiascomycete yeasts, BMC Genomics, 14, 901,

2013.

[13] Di Lorenzo F, Silipo A, Lanzetta R, Fazio LL, Paciel-

lo I, Coutinho CP, Sá-Correia I, Bernardini M, Molinaro

A. Structural elucidation and biological activity of the lipooli-

gosaccharide of a Burkholderia dolosa isolate from a cystic

fibrosis patient, ChemBioChem, 14(9), 1105-15, 2013.

[14] dos Santos SC, Teixeira MC, Dias PJ, Sá-Correia I.

MFS transporters required for multidrug/multixenobiotic

(MD/MX) resistance in the model yeast: understanding their

physiological function through post-genomic approaches,

Frontiers in Physiology, 5:180, 2014.

[15] dos Santos SC, Mira NP, Moreira AS, Sá-Correia I.

Quantitative- and phospho-proteomic analysis of the yeast

response to the tyrosine kinase inhibitor imatinib, OMICS: A

Journal of Integrative Biology, 16, 537-51, 2012.

[16] dos Santos SC, Teixeira MC, Cabrito TR, Sá-Correia

I. Yeast Toxicogenomics: genome-wide responses to chem-

ical stresses with impact in Environmental Health, Pharma-

cology and Biotechnology, Frontiers in Genetics, 3, 63,

2012.

[17] El-Kirat-Chatel S, Mil-Homens D, Beaussart A, Fi-

alho AM, Dufrêne YF. Single-molecule atomic force mi-

croscopy unravels the binding mechanism of

a Burkholderia cenocepacia trimeric autotransporter adhe-

sin, Molecular Microbiology, 89(4): 649-59, 2013.

[18] Eusébio N, Coutinho C, Sá-Correia I, Araújo R.

SNaPBcen: a novel and practical tool for genotyp-

ing Burkholderia cenocepacia, Journal of Clinical Microbi-

ology, 51(8), 2646-53, 2013.

[19] Ferreira AS, Silva IN, Oliveira VH, Becker JD,

Givskov M, Ryan RP, Fernandes F, Moreira LM. Compar-

ative transcriptomic analysis of the Burkholderia cepacia

tyrosine kinase bceF mutant reveals a role in tolerance to

stress, biofilm formation and virulence. Applied and Envi-

ronmental Microbiology, 79, 3009-3020, 2013.

Publications

44

[20] Fialho AM, Bernardes N, Chakrabarty AM. Recent

patents on live bacteria and their products as potential anti-

cancer agents, Recent Patents on Anti-Cancer Drug Dis-

covery, 7, 31-55, 2012.

[21] Fialho AM, Chakrabarty AM. Patent controversies and

court cases: Cancer diagnosis, therapy and prevention, Can-

cer Biology and Therapy, 13, 1229 - 1234, 2012.

[22] Fialho AM, Salunkhe P, Manna SK, Mahali S,

Chakrabarty AM. Glioblastoma Multiforme: Novel Therapeu-

tic Approaches, ISRN Neurology, Article ID 642345, 10

pages, 2012.

[23] Guerreiro JF, Mira NP, Sá-Correia I. Adaptive re-

sponse to acetic acid in the highly resistant yeast species

Zygosaccharomyces bailii revealed by quantitative prote-

omics, Proteomics, 12, 2303-18, 2012.

[24] Lourenço AB, Roque FC, Teixeira MC, Ascenso JR,

Sá-Correia I. Quantitative 1H-NMR-metabolomics reveals

extensive metabolic reprogramming and the effect of the

aquaglyceroporin FPS1 in ethanol-stressed yeast

cells, PLoS One, 8(2), e55439, 2013.

[25] Madeira A, dos Santos SC, Santos PM, Coutinho CP,

Tyrrell J, McClean S, Callaghan M, Sá-Correia

I. Proteomic profiling of Burkholderia cenocepacia clonal

isolates with different virulence potential retrieved from a

cystic fibrosis patient during chronic lung infection, PLoS

One 8(12), e83065, 2013.

[26] Madeira A, da Silva CL, Dos Santos F, Camafeita E,

Cabral JMS, Sá-Correia I. Human Mesenchymal Stem Cell

Expression Program upon Extended Ex-Vivo Cultivation, as

Revealed by 2-DE-Based Quantitative Proteomics, PLoS

One, 7(8), e43523, 2012 (highlighted in Mesenchymal Cell

News 4.34).

[27] Matos RG, Fialho AM, Giloh M, Schuster G, Arraiano

CM. The rnb Gene of Synechocystis PCC6803 Encodes a

RNA Hydrolase Displaying RNase II and Not RNase R Enzy-

matic Properties, PLoS One, 7(3), e32690, 2012.

[28] Mil-Homens D, Leça MI, Fernandes F, Pinto SN, Fi-

alho AM. Characterization of BCAM0224, a multifunctional

trimeric autotransporter from the human pathogen Burkhold-

eria cenocepacia, Journal of Bacteriology, 2014

(doi:10.1128/JB.00061-14).

[29] Mil-Homens D, Bernardes N, Fialho AM. The antibac-

terial properties of docosahexaenoic omega-3 fatty acid

against the Cystic Fibrosis multi-resistant pathogen

Burkholderia cenocepacia, FEMS Microbiology Letters,

328, 61-69, 2012.

[30] Mil-Homens D, Fialho AM. A BCAM0223 Mutant of

Burkholderia cenocepacia is Deficient in Hemagglutination,

Serum Resistance, Adhesion to Epithelial Cells and Viru-

lence, PLoS One, 7(7), e41747, 2012.

[31] Mira NP, Münsterkötter M, Valada FD, Santos J, Pal-

ma M, Roque FC, Guerreiro JF, Rodrigues F, Sousa MJ,

Leão C, Güldener U and Sá-Correia I. The genome se-

quence of the highly acetic acid tolerant Zygosaccharomyces

bailii-derived interspecies hybrid strain ISA1307 isolated from

a sparkling wine plant, DNA Research, 1-15, 2014.

[32] Moreira AS, Coutinho CP, Azevedo P, Lito L, Melo-

Cristino J, Sá-Correia I. Burkholderia dolosa phenotypic

variation during the decline in the lung function of a cystic

fibrosis patient along 5.5 years of chronic colonization. Jour-

nal of Medical Microbiology, 63: 594-601, 2014 (Week’s

Best Articles: Cystic Fibrosis February 5-12 2014, according

to MDLinx Pulmonology).

[33] Palma M, Madeira SC, Mendes-Ferreira A, Sá-Correia

I. Impact of assimilable nitrogen availability in glucose uptake

kinetics in Saccharomyces cerevisiae during alcoholic fer-

mentation, Microbial Cell Factories, 11, 99, 2012.

[34] Pereira SG, Rosa AC, Ferreira AS, Moreira LM, Pro-

ença DN, Morais PV, Cardoso O. Virulence factors and

infection ability of Pseudomonas aeruginosa isolates from a

hydropathic facility and respiratory infections, Journal of

Applied Microbiology, 2014 (doi: 10.1111/jam.12463).

[35] Ramos CG, Grilo AM, da Costa PJ, Leitão JH. Experi-

mental identification of small non-coding regulatory RNAs in

the opportunistic human pathogen Burkholderia cenocepacia

J2315, Genomics, 101(2):139-148, 2013.

[36] Ramos CG, Grilo AM, da Costa PJP, Feliciano JR,

Leitão JH. MtvR is a global regulatory sRNA in Burkholderia

cenocepacia, Journal of Bacteriology, 195 (16), 3514–

3523, 2013.

[37] Ramos CG, da Costa PJ, Döring G, Leitão JH. The

novel cis-encoded small RNA h2cR is a negative regulator of

hfq2 in Burkholderia cenocepacia, PLoS One, 7(10):

e47896, 2012.

[38] Remy E, Cabrito TR, Baster P, Batista RA, Teixeira

MC, Friml J, Sá-Correia I, Duque P. A Major Facilitator

Superfamily Transporter Plays a Dual Role in Polar Auxin

Transport and Drought Stress Tolerance in Arabidopsis, The

Plant Cell, 25, 901-26, 2013.

[39] Remy E, Cabrito TR, Baptista R, Teixeira MC, Sá-

Correia I, Duque P. The Pht1;9 transporter mediates inor-

ganic Pi acquisition by the Arabidopsis thaliana root during

phosphorus starvation, The New Phytologist, 195, 356–

371, 2012.

[40] Santos MR, Tomás AT, Becker JD, Moreira LM. Sino-

rhizobium meliloti EmrR regulator is required for efficient

colonization of Medicago sativa root nodules, Molecular

Plant-Microbe Interactions, 27, 388-399, 2014.

[41] Silva IN, Tavares AF, Ferreira AS, Moreira LM. Stress

conditions triggering mucoid morphotype variation in

Burkholderia species and effect on virulence in Galleria

mellonella and biofilm formation in vitro, PLoS One 8(12),

e825222, 2013 (doi: 10.1371/journal.pone.0082522).

[42] Sousa SA, Feliciano JR, Grilo AM, Leitão JH. Bioin-

formatics: a molecular microbiologist’s perspective, Current

Bioinformatics, 9 (1):8-17, 2014.

[43] Sousa SA, Feliciano JR, Pinheiro PF, Leitão JH.

Biochemical and functional studies on the Burkholderia cepa-

cia complex bceN gene, encoding a GDP-D-mannose 4,6-

dehydratase, PLoS One, 8: e56902, 2013.

45


12-13

[44] Teixeira MC, Monteiro PT, Guerreiro JF, Gonçalves

JP, Mira NP, dos Santos SC, Cabrito T, Palma M, Costa

C, Francisco AP, Madeira SC, Oliveira AL, Freitas AT, Sá

-Correia I. The YEASTRACT database: an upgraded infor-

mation system for the analysis of gene and genomic tran-

scription regulation in Saccharomyces cerevisiae, Nucleic

Acids Research, 42, D161-D166, 2014.


Correia I. Increased expression of the yeast multidrug re-

sistance ABC transporter Pdr18 leads to increased ethanol

tolerance and ethanol production in high gravity alcoholic

fermentation, Microbial Cell Factories, 11, 98, 2012.

[46] Viegas SC, Mil-Homens D, Fialho AM, Arraiano CM.

The virulence of Salmonella enterica serovar typhimurium in

the insect model Galleria mellonella is impaired by mutations

in RNase E and RNase III, Applied and Environmental

Microbiology, 79(19): 6124-33, 2013.

[47] Viegas CA, Costa C, André S, Viana P, Ribeiro R,

Moreira-Santos M. Does S-metolachlor affect the perfor-

mance of Pseudomonas sp. strain ADP as bioaugmentation

bacterium for atrazine-contaminated soils?, PLoS One, 7(5),

e37140, 2012.

Book Chapters

[48] Leitão JH, Sousa SA, Ramos CG, Feliciano JR, Grilo

AM, da Costa PJP. “Experimental and bioinformatics

approaches to identify potential targets and develop novel

strategies to fight infections caused by multiresistant

Burkholderia cepacia complex bacteria”. In: Microbial

pathogens and strategies for combating them: science,

technology and education, vol 1, Formatex Research

Center, Badajoz, Spain, 2013. Pages 372-383 (ISBN 978-84

-939843-9-7).

[49] Leitão JH, Simões N. "Identification of novel

antimicrobials using a live-animal infection model". In: M.C.

Barreto and N. Simões (ed), Determination of Biological

Activities: A Laboratory Manual, Universidade dos Açores,

Ponta Delgada, 2012. Pages 29-39 (ISBN 978-972-8612-82-

5).

[50] Mira NP, Teixeira MC, Sá-Correia I. “Characterization

of complex regulatory networks and identification of promoter

regulatory elements in yeast: in silico and wet-lab”, In:

Methods in Molecular Biology - Transcriptional

Regulation: Methods and Protocols (Vancura A, Ed),

Springer, vol. 809, 27-48, 2012. (ISBN 978-1-61779-375-2).

[51] Ramos CG, Leitão JH. “Caenorhabditis elegans as a

research tool to unveil bacterial virulence determinants:

lessons from the Burkholderia cepacia complex”, In:

Nematodes: Morphology, Functions and Management

Strategies (Fausto Boeri and Jordan A. Chung, Eds.), Nova

Science Publishers, Inc., NY, USA, 135-156, 2012 (ISBN:

978-1-61470-784-4).

[52] Ramos CG, Grilo AM, da Costa PJP, Nadais H,

Leitão JH. “Extraction and Purification of DNA from UASB

Reactors Sludge Samples”, In: DNA Binding and DNA

Extraction: Methods, Applications and Limitations

(Chunxu Zhou and Xia Ling, Eds.) Nova Science Publishers,

Inc., NY, USA, 123-139, 2012 (ISBN: 978-1-61470-958-9).

[53] Sá-Correia I, Guerreiro JF, Loureiro-Dias MC, Leão

C, Côrte-Real M. “Zygosaccharomyces”. In: Encyclopedia

of Food Microbiology, Batt, C.A. & Tortorello, M.L. (Eds.),

vol 3. Elsevier Ltd, Academic Press, pp. 849–855, 2014

(ISBN: 9780123847300)

[54] Sá-Correia I, dos Santos SC, Madeira A.

“Burkholderia Proteomics: methodologies, challenges, and

applications” in Burkholderia: From Genomes to Function

Tom Coenye & Eshwar Mahenthiralingam (Eds.), Horizon

Press, 2014 (ISBN: 978-1-908230-35-5)

[55] Teixeira MC. "Complex regulatory interplay between

multidrug resistance and oxidative stress response in yeast:

the FLR1 regulatory network as a systems biology case-

study", In: Oxidative Stress - Molecular Mechanisms and

Biological Effects, Lushchak V.I. & Semchyshyn H. (Eds),

INTECH, Vienna, Austria, 323-340, 2012 (ISBN: 978-953-51-

0554-1).

[56] Viegas CA, Chelinho S, Moreira-Santos M, Costa C,

Gil FN, Silva C, Lima D, Ribeiro R, Sousa JP, Fialho AM.

“Bioremediation of soils contaminated with atrazine and other

s-triazine herbicides: current state and prospects”, In:

Advances in Environmental Research, Justin A. Daniels

(ed), vol 6, Nova Science Publishers, Inc., NY, USA, 2012,

pp. 1-49 (ISBN 978-1-61728-163-1).

Edition of Special Issues

Fontes CMGA, Sá-Correia I, Romão MJ, Sá-Nogueira I,

Prates JAM, Ferreira LMA. Special issue of Biocatalysis

and Biotransformation, vol. 30, issue 3, 2012, with the

proceedings of the 9th Carbohydrate Bioengineering Meeting

(CBM9)

Mira NP, Teixeira MC. Microbial Mechanisms of Tolerance

to Weak Acids, Special issue published in Frontiers in Mi-

crobiology – Section of Microbial Physiology and Metab-

olism (11 articles included), 2013.

Sá-Correia I, Teixeira MC. “Physiological role and regula-

tion of Multidrug/Multixenobiotic resistance membrane trans-

porters”, Frontiers in Physiology, 2014.

Issued Patents

Fialho AM, Bernardes N, Gonçalves J, Santos AC.

Compositions to treat HIV infections and methods therefor,

Indian patent 1126/MUM/2011. Priority date: March 2012.

Sá-Correia I, Cabrito TR, Teixeira MC, Remy E, Duque P.

Utilização de um gene que confere resistência a xenobióticos

em plantas (Use of a gene that confers resistance to

xenobiotics in plants), Provisional national patent number

PT105727. Priority date: 28th October 2011 (Patente de

invenção nacional n.º 105727; Date: 2013.06.21)

46

Patent applications

Martins AMSD, Leitão JH, Alves LGAR, Pinheiro PBF,

Feliciano JRR. “Síntese e uso de ciclamas com atividade

antibacteriana”. Pedido de Patente Nacional nº 107018.

Priority date: 20th June 2013.

Dissertations

Ph.D. Theses

Andreia Madeira, “Genome wide expression approaches

applied to biomedical research: long-term adaptation

Burkholderia cenocepacia to cystic fibroses patient's air-

ways and extended ex-vivo cultivation of human mesenchy-

mal stem cells”, PhD in Biotechnology, IST/UTL

(supervisors: Isabel Sá-Correia; co-supervisor: Joaquim

Sampaio Cabral), 2012.

Catarina Roma Rodrigues, "Global responses to phenol

and myrcene in Pseudomonas at the level of cell membrane

proteome unveiled by quantitative prote-

omics" (supervisor: Isabel Sá-Correia; co-supervisor: Pedro

Santos), 2012.

Dalila Mil-Homens, “Burkholderia cenocepacia pathogene-

sis: unravelling the virulence functions of trimeric autotrans-

porter adhesins and development of alternative therapies to

target this pathogen”, PhD in Biotechnology, IST/UTL

(supervisor: Arsénio M. Fialho), 2012.

Inês Nunes Silva, “Comparative genomics and tran-

scriptomics to study mucoid morphotype variation in

Burkholderia cepacia complex clinical isolates”, PhD in

Biotechnology, IST/UTL (supervisor: Leonilde M. Moreira;

co-supervisor: Jӧrg D. Becker ), 2012.

Nuno Bernardes “Bacterial protein azurin as a new thera-

peutic tool to treat poor prognosis breast cancer with over-

expression of P-cadherin” PhD in Biotechnology, IST/UTL

(supervisor: Arsénio M. Fialho; co-supervisor: Joana F.

Paredes), 2013.

M.Sc. Theses

Ana Cristina Taborda Gomes de Almeida, “Functional

analysis of the yeast multidrug resistance transporters Tpo1

and Pdr18 under stress induced by the agricultural fungicide

mancozeb”, MSc in Biotechnology (supervisor: Isabel Sá-

Correia; co-supervisor: Tânia R. Cabrito), 2012.

André Marques Sales Henriques, “Role of the multidrug

efflux pumps CgAqr1 and CgTpo2/3 in Candida

glabrata resistance to flucytosine and azole drugs”, MSc in

Biomedical Engineering (supervisor: Miguel C. Teixeira),

2012.

Andreia Filipa Campos Tavares, “Biological relevance of

mucoid vs. nonmucoid morphotype variation by

Burkholderia cepacia complex”, MSc in Cellular Biology and

Biotechnology, Faculdade de Ciências, Universidade de

Lisboa (Supervisor at IST: Leonilde M. Moreira; supervisor

at FC-UL: Carlos Farinha), 2012.

Andreia Ponte, "Genome-wide identification of

mechanisms of resistance to the antifungal drug flucytosine:

from S. cerevisiae to C. glabrata", MSc in Biotechnology,

IST (supervisor: Miguel C. Teixeira), 2013.

Camille Payre, “Les méchanismes moléculaires à l'origine

des différences de tolérance à l'acide acétique et

d'utilization du glucose et de l'acide acétique chez

Saccharomyces cerevisiae et Zygosaccharomyces baillii”,

Genie Biologique, Analyses Biologiques et Biochimiques,

IUT Dijon-Auxerre (supervisor: I. Sá-Correia; co-supervisor:

Margarida Palma), 2012.

Carla Alexandra Nunes Mateus, “Study of bioremediation

for soils contaminated with s-triazine herbicides: the

bioaugmentation bacteira Pseudomonas sp. ADP

and Arthrobacter aurescens TC1 compared”, MSc in

Biotechnology (supervisor: Cristina A. Viegas), 2012.

Cláudia Sofia Pires Godinho, “Studies on the role and

regulation of the multidrug resistance efflux pumps Tpo1

and Pdr18 in yeast tolerance to ethanol and weak acid

stresses, to increase yeast robustness”, MSc in Applied

Microbiology, Faculdade de Ciências, Universidade de

Lisboa (supervisor at IST: Isabel Sá-Correia; supervisor

at FC-UL: Ana Tenreiro), 2012.

Cláudia Rita Ferreira Maldonado, "Adaptive strategies

of Burkholderia cenocepacia to long term residence in

the lungs of cystic fibrosis patients: genome-wide

analyses", Applied Microbiology, FC-UL (supervisor at

IST: Isabel Sá-Correia; supervisor at FC-UL: Ana

Tenreiro).

Eunice Penas “Avaliação da interdependência entre

histidinas cinases híbridas (BCAM0218, BCAM0227) e

adesinas triméricas autotransportadas presentes no

agrupamento genético TAA do microrganismo

patogéneo Burkholderia cenocepacia J2315”, Mestrado

em Doenças Infecciosas Emergentes da Faculdade de

Medicina da Universidade de Lisboa (supervisor at IST:

Arsénio M. Fialho; co-supervisor at FM-UL: José

Augusto Cristino), 2013.

Filipa Gomes de Almeida Dias, “Functional analysis of

the Burkholderia cepacia complex bceOSU genes

encoding putative polysaccharide O-acyltransferases”,

MSc in Biotechnology (supervisor: Leonilde M. Moreira;

co-supervisor: Ana Sofia Ferreira), 2012.

Filipe Bravo da Silva, “Improvement of alcoholic

fermentation performance based on highly acetic acid

tolerant wine isolates of Saccharomyces cerevisiae and

genetically engineered strains”, MSc in Biological

Engineering (supervisor: Isabel Sá-Correia; co-

supervisor: Margarida Palma), 2012.

Gonçalo Emanuel Fialho Mourata da Silva

“Discovering and exploiting bacterial proteins as

anticancer agentes”, Mestrado em Biologia Humana e

Ambiente, Faculdade de Ciências da Universidade de

Lisboa (Supervisor at IST: Arsénio M. Fialho, Supervisor

at FC-UL: Ana M. Viegas), 2013.

47


12-13

Grégoire Chardon, “La résistance à l’acide acétique

chez Zygosaccharomyces bailii”, Diplôme Universitaire

de Technologie - Genie Biologique, Analyses Biologiques

et Biochimiques, IUT Dijon-Auxerre (supervisor: Isabel Sá

-Correia; co-supervisor: Joana F. Guerreiro), 2013.

Guida Filipa Bartolomeu de Campos Camacho,

“Contribution to the functional analysis of the paralogous

gene-pairs CgTpo1_1 and CgTpo1_2 and CgFlr1_1 and

CgFlr1_2 from the human pathogen Candida glabrata”,

MSc in Biological Engineering (supervisor: Miguel C.

Teixeira), 2012.

Janete Simão Gonçalves, "Estratégias de

biorremediação para solos contaminados com o

herbicida terbutilazina com base na bioadição de

Pseudomonas sp. ADP", MSc in Applied Microbiology,

Faculty of Sciences of the University of Lisbon

(supervisor at IST: Cristina A. Viegas; supervisor at FC-

UL: Manuela Carolino; ), 2013.

João Miguel Rodrigues da Silva Brazão, “Functional

analysis of Arabidopsis thaliana major facilitator

superfamily (MFS) transporters through heterologous

expression in yeast”, MSc in Biotechnology, IST

(supervisor: Isabel Sá-Correia), 2013.

Jonathan Ribeiro, "Profiling antifungal drug resistance in

a collection of Candida glabrata clinical isolates:

correlation with the expression of multidrug efflux pumps",

MSc in Biomedical Engineering, IST (supervisor: Miguel

C. Teixeira), 2013.

Kaur Alasoo, “Elucidating the transcriptional regulatory

network controlling the TPO1 response to benzoic acid in

yeast”, euSYSBIO Erasmus Mundus Master´s in Systems

Biology, IST (supervisor: Isabel Sá-Correia; co-

supervisor: Tânia R. Cabrito) , 2012.

Maria Raquel Moita, “Towards the production of levulinic

and itaconic acids in Saccharomyces cerevisae: a

contribution for the understanding of the molecular

mechanisms of toxicity of the acids in producing cells”,

MSc in Biotechnology (supervisor: Nuno P. Mira), 2013.

Rúben Bernardo, “Functional analysis of the Candida

glabrata CgHaa1 (ORF CAGL0L09339g) transcription

factor: role in acetic acid resistance and in biofilm

formation”, MsC thesis in Biotechnology. (supervisor:

Nuno P. Mira) , 2013.

Sofia Patrão Neves Alves, “Contribution to the

functional analysis of genes involved in cyclic GMP

signaling in bacteria of the Burkholderia cepacia

complex”, MSc in Human Biology and Environment,

Faculdade de Ciências da Universidade de Lisboa

(supervisor at IST: Jorge H. Leitão; supervisor at FC-UL:

Deodália Dias), 2013.

Sofia de Almeida Santos de Castro e Abreu, “Bacterial

protein azurin as a new candidate anticancer drug by

decreasing cell adhesion through integrins”, Mestrado em

Biologia Celular e Molecular, Faculdade de Ciência e

Tecnologia, Universidade de Coimbra (supervisor at IST:

Arsénio M. Fialho; supervisor at FCT-UC: Carmen Maria

Martins de Carvalho Alpoim), 2013.

Vítor Hugo Jorge de Oliveira, “Studies on the

involvement of quorum sensing in the regulation of

exopolysaccharide biosynthesis by Burkholderia cepacia

complex isolates”, MSc in Applied Microbiology,

Faculdade de Ciências, Universidade de Lisboa

(supervisor at IST: Leonilde M. Moreira, supervisor at FC-

UL: Mário Santos), 2012.

Viviane Motta Varela, "Studies on s-triazines degrading

bacteria: Influence of three methods of culture formulation

on Arthrobacter aurescens TC1 ability to biodegrade the

herbicides terbuthylazine and atrazine", MSc in Biological

Engineering (supervisor: Cristina A. Viegas), 2013.

Other Publications in International

Journals

Editorials


Prates JAM, Ferreira LMA. Editorial: Biocatalysis and Bio-

transformation, 30(3): 273, 2012 (DOI:

10.3109/10242422.2012.681858)

Mira NP, Teixeira MC. "Microbial mechanisms of tolerance

to weak acid stress", Frontiers in Microbiology (Editorial),

4, 416, 2013.

Papers in Conference Proceedings

Grilo AM, Ramos CG, Sousa SA, Arroja L, Capela I,

Leitão JH, Nadais H. Optimizing Energy Production from

Wastewater by Intermittent Operation of Anaerobic Reac-

tors, European Biomass Conference and Exhibition Pro-

ceedings, pp. 1721 – 1724, 2011. DOI:

10.5071/19thEUBCE2011-VP2.6.4. ISBN-13:978-88-89407-

55-4

Silva V, Mateus C, Gonçalves J, Varela V, Viegas CA. A

bactéria Arthrobacter aurescens TC1 como ferramenta de

biorremediação para ambientes contaminados com os herbi-

cidas terbutilazina e atrazina, In: Borrego, C., Miranda, A.I.,

Arroja, L., Fidélis, T., Castro, E.A., Gomes, A.P. (eds), Actas

da 10ª Conferência Nacional do Ambiente (ISBN: 978-989

-98673-0-7), Departamento de Ambiente e Ordenamento da

Universidade de Aveiro, Aveiro, Portugal, 2013.

Viegas CA, Silva V, Mateus C, Chelinho S, Moreira-

Santos, Gonçalves J, Varela V, Ribeiro R, Fialho AM, and

Sousa JP. Bioremediation strategies based on Pseudomo-

nas sp. strain ADP for worst-case scenarios of soil contami-

nation with herbicidal formulations containing chlorinated s-

triazines. In proceedings of “BioMicroWorld2013 - V Inter-

national Conference on Environmental, Industrial and

Applied Microbiology”, Madrid, Spain, 2013.

Abstracts in international journals

Costa C, Pires C, Ribeiro J, Teixeira MC. "Antifungal drug

resistance mediated by Candida glabrata Drug:H+ Anti-

porters", Yeast, 30(S1), S175, 2013.

48

Bernardes N, Ribeiro AS, Mota B, Matos RG, Arraiano

CM, Seruca R, Paredes J and Fialho AM. “Azurin impairs P

-cadherin-dependent invasion of breast cancer cells via de-

creased FAk/Src signaling”, EACR22- from Basic Research

to Personalized Cancer Treatment, Barcelona, July 7-10,

European Journal of Cancer, 48: 3133:3318, 2012.

Ferreira AS, Silva IN, Becker JD, McClean S, Callaghan

M, Moreira LM. “Role of BceF BY-kinase in host-pathogen

interaction, biofilm formation and survival to stress in the

opportunist pathogen Burkholderia cepacia IST408: Tran-

scriptomic and phenotypic approaches”, FEBS Journal 279,

502-502, 2012.

Ferreira AS, Oliveira VH, Silva, IN, Becker JD, McClean S,

Callaghan M, Moreira LM. “Role of BceF BY-kinase in host–

pathogen interaction, biofilm formation and survival to stress

by the cystic fibrosis pathogen Burkholderia cepacia IST408:

Transcriptomic and phenotypic approaches”, Journal of

Cystic Fibrosis 12, S77, 2013.

Gil FN, Gonçalves AC, Becker JD, Viegas CA. “Genome-

wide transcriptional response to pesticide exposure in the

model yeast Saccharomyces cerevisiae, FEBS Journal, 279

(S1), 230, 2012.

Grilo AM, Ramos CG, Leitão JH. “Identification of regulato-

ry small non-coding RNAs in Burkholderia cepacia J2315”,

FEBS Journal, 279(S1), 510, 2012.

Pinto-de-Oliveira A, Coutinho CP, Ramos CG, Sousa SA,

de Carvalho CCCR, Leitão JH, Sá-Correia I. “The

Burkholderia cepacia small colony variants (SCV) are a more

pathogenic bacterial form that may facilitate persistent respir-

atory infections in CF patients”, Journal of Cystic Fibrosis,

12(S1), S76, 2013.

Ramos CG, Grilo AM, da Costa PJP, Feliciano JR, Leitão

JH. “Unveiling the roles of small non-coding RNAs and RNA

chaperones on the biology of opportunistic pathogens of the

Burkholderia cepacia complex”, Journal of Cystic Fibro-

sis,12 (S1), S77, 2013.

Silva IN, Tavares AC, Ferreira AS, Moreira, LM. “Mucoid

morphotype variation of Burkholderia multivorans: adaptation

to cystic fibrosis lung environment”, FEBS Journal 279, 237-

237, 2012.

Sousa, SA, P Soares-Castro, JR Feliciano, CG Ramos,

JLC Martinez, PM Santos, JH Leitão. “Unveiling the

Burkholderia cenocepacia J2315 RNA chaperone Hfq2 in-

teractome”, Journal of Cystic Fibrosis, 12(S1), S77, 2013.

Sousa SA, Feliciano JR, Ramos CG, Leitão JH. “The

Burkholderia cenocepacia J2315 small non-coding RNA

MavA is required for fitness and virulence”, Journal of Cyst-

ic Fibrosis, 12(S1), S76, 2013.

Papers in National Journals

Bernardes N, Fialho AM. Biotecnologia e inovação

terapêutica: bactérias e produtos seus derivados como

agentes anticancerígenos” Boletim de Biotecnologia, 4: 42-

45, 2013.

Ramos CG, Grilo AM, Sousa SA, Feliciano JR, Leitão JH.

"Post-transcription regulation of gene expression in the hu-

man opportunistic pathogens of the Burkholderia cepacia

complex". SPM Magazine, 3(1), 2014.

Sousa SA, Ramos CG, Grilo AM, Feliciano JR, da Costa

PJP, Leitão JH. “Estratégias de identificação de novos alvos

para combater as infecções por bactérias do complexo

Burkholderia cepacia”, Boletim de Biotecnologia, 4 (série

2), 41-43, 2013.

Teixeira MC, Cabrito TR, Brazão J, Sá-Correia I. “Yeast as

model eukaryote and expression host system: is it still

useful?”, Microbiologia - SPM Magazine, 2 (2), 2013.

Online publications

The YEASTRACT database (http://www.yeastract.com/) was

very recently updated and upgraded with new bioinformatic

tools and more detailed regulatory information to facilitate the

exploitation of the gathered material when answering specific

biological questions. New options were provided for the

ranking of transcription factors according to their relevance in

the regulation of specific gene sets. Furthermore, information

on the environmental condition and experimental setup

underlying each documented regulatory association was

gathered, thus allowing a fine tuning of the queries offered for

the analysis of gene and genomic regulation.

http://www.yeastract.com/

49


12-13

Communications in International Confer-

ences

Invited Conferences

Leitão JH, “RNA chaperones and sRNAs from bacteria of the

Burkholderia cepacia complex”. FEBS-ASM Workshop on:

Biology of RNA in host-pathogen interactions. 26-29 January

2014, Tenerife, Spain.

Sá-Correia I, "Toward a systems-based understanding of

yeast response and tolerance to acetic acid" in: Yeast and

Food Microbiology Symposium, FEMS Congress 2013, 21-25

July, Leipzig, Germany.

Sá-Correia I, "Genomics of adaptation to the lung” in:

Burkholderia cepacia complex: still a problem? Symposium,

36th European Cystic Fibrosis Conference, 12-15 June 2013,

Lisbon, Portugal.

Oral Communications

Gil FN, Gonçalves AC, Becker JD, Viegas CA. “Pesticide

toxicity studies using gene expression profiling in the model

yeast Saccharomyces cerevisiae”, 7th European Conference

on Pesticides and Related Organic Micropollutants in the

Environment & 13th Symposium on Chemistry and Fate of

Modern Pesticides, 7-10 October 2012, Porto, Portugal.

Matos RG, Fialho AM, López-Viñas E, Gomez-Puertas P,

Schuster G, Arraiano CM. “The evolution of the RNase II-

family of exoribonucleases: singular features in Archaea and

Cyanobacteria” FASEB Meeting: Post-transcriptional control

of gene expression: mechanisms of mRNA decay, June 24-

29 2012, Steamboat Springs, Colorado.

Mil-Homens D and Fialho AM. “Trimeric autotransporter

adhesins in the human pathogen Burkholderia cenocepacia: a

multifunctional family of proteins implicated in virulence” 3rd

Scientific Meeting of Institute for Biotechnology and Bioengi-

neering, 16-17 March 2012, Instituto Superior Técnico, Lis-

boa.

Nadais MH, Capela I, Arroja L, Leitão JH, Grilo AM, Cour-

as C. “Effects of operational shocks on UASB microbial popu-

lations”, 8th Conference on sustainable development of ener-

gy, water and environment systems. 22-27 September 2013,

Dubrovnik, Croatia.

Ramos CG, Grilo AM, da Costa PJP, Feliciano JR,

Leitão JH. “Towards the unveiling of the biological roles

played by small non-coding RNAs in the opportunistic patho-

gens of the Burkholderia cepacia complex”. FEBS Internation-

al Workshop “New Developments in RNA Biology, 1-4 Sep-

tember 2012, Tavira, Portugal.

Silva IN, Santos PM, Becker JD, Moreira LM. “Within-cystic

fibrosis patient genoptypic variation in Burkholderia multi-

vorans sequential isolates”, International Burkholderia cepa-

cia working group (IBCWG), 9-12 April 2014, Nimes, France.

Silva V, Mateus C, Gonçalves J, Varela V, Viegas CA.

“Arthrobacter aurescens TC1 as an efficient bioaugmentation

bacterium for soils contaminated with s-triazine herbicides”, V

International Conference on Environmental, Industrial and

Applied Microbiology, 2-4 October 2013, Madrid, Spain.

Teixeira MC. “Systematic functional analysis of the drug:H+

antiporter family from the pathogenic yeast C. glabrata”,

Yeast Genetics and Molecular Biology Meeting, July 31 -

August 5 2012, Princeton, NJ, USA.

Viegas CA, Chelinho S, Moreira-Santos M, Silva V, Ma-

teus C, Ribeiro R, Fialho AM, Sousa JP. “Pseudomonas sp.

strain ADP as a bioaugmentation bacterium in soil micro-

cosms contaminated with herbicidal formulations containing s

-triazines”, Symposium on Pollutant degradation under envi-

ronmental stress: towards rational bioaugmentation -

BACSIN2012, 29-30 March 2012, Amsterdam, The Nether-

lands.

Viegas CA, Chelinho S, Silva V, Mateus C, André S,

Moreira-Santos M, Viana P, Ribeiro R, Fialho AM, Sousa

JP. “Performance of a bioremediation tool based on Pseudo-

monas sp. strain ADP in soils contaminated with s-triazine

herbicides”, 7th European Conference on Pesticides and Re-

lated Organic Micropollutants in the Environment & 13th Sym-

posium on Chemistry and Fate of Modern Pesticides, 7-10

October 2012, Porto, Portugal.

Viegas CA, Silva V, Mateus C, Chelinho S, Moreira-

Santos M, Gonçalves J, Varela V, Ribeiro R, Fialho

AM, Sousa JP. “Bioremediation strategies based on

Pseudomonas sp. Strain ADP for worst-case scenarios of

soil contamination with herbicidal formulations containing

atrazine or terbuthylazine”, V International Conference on

Environmental, Industrial and Applied Microbiology, 2-4

October 2013, Madrid, Spain.

Poster Communications

Costa C, Pires C, Henriques A, Camacho G, Nunes J,

Teixeira MC. “Systematic functional analysis of the

drug:H+ antiporter family from the pathogenic yeast C.

glabrata”, Yeast Genetics and Molecular Biology Meeting,

July 31 - August 5 2012, Princeton, NJ, USA.

Costa C, Cabrito TR, Pires, C., Sá-Correia I, Teixeira

MC. “Role of the uncharacterized Candida glabrata

drug:H+ antiporter CgQdr2 (ORF CAGL0G08624g) in

antifungal drug resistance: a functional analysis”, 11th

ASM Conference on Candida and Candidiasis, 29 March -

2 April 2012, San Francisco, California, USA.

Costa C, Henriques A, Pires C, Nunes J, Sá-Correia I,

Teixeira MC. “The Candida glabrata drug:H+ antiporter

CgAqr1 plays a role in acetic acid and antifungal drug re-

sistance”, Fifth FEBS Advanced Lecture Course in Human

Communications

50

Fungal Pathogens, 25-31 May 2013, La Colle sur Loup,

France.

Costa C, Pires C, Ribeiro J, Teixeira MC. “Antifungal drug

resistance mediated by Candida glabrata Drug:H+ Anti-

porters”, 26th International Conference on Yeast Genetics and

Molecular Biology, 29 August - 3 September 3 2013, Frank-

furt, Germany.

da Costa PJP, CG Ramos, AM Grilo, JH Leitão.

“Experimental identification of regulatory small non-coding

RNAs from bacteria of the Burkholderia cepacia complex”,

FEBS International Workshop: New Developments in RNA

Biology, 1-4 September 2012, Tavira, Portugal.

da Costa PJP, Ramos CG, Grilo AM, Feliciano JR, Leitão

JH. “The small noncoding RNA MtvR is a global regulator of

gene expression in Burkholderia cenocepacia”, The Non

Coding Genome EMBO-EMBL Symposium, 9-12 October

2013, Heidelberd, Germany.

Dias PJ, Sá-Correia I. “New Insights on the Evolution of the

Hemiascomycete Yeasts Major Facilitator Superfamily Multi-

drug Resistance Transporters, Experimental Approaches to

Evolution and Ecology using Yeast”, EMBL Advance Training

Centre, 17-21 October, 2012, Heildelberg, Germany.

dos Santos DJVA, Leitão JH, Guedes RC. “Development

and validation of a homology model of Type II phosphoman-

nose isomerase”, 6th International Theoretical Biophysics

Symposium (TheoBio 2013), 24-27 June 2013, Gothenburg,

Sweden.

Ferreira AS, McClean S, Callaghan M, Moreira LM.

"Involvement of the bacterial tyrosine kinase BceF and the

phosphotyrosine phosphatase BceD in Burkholderia cepacia

interaction with the cystic fibrosis epithelial cell line

CFBE41o", EMBO/FEBS Host-Microbes Interactions course,

30 August – 7 September 2013, Spetses, Greece.

Gil FN, Gonçalves AC, Becker JD, Viegas CA. “Genome-

wide transcriptional response to pesticide exposure in the

model yeast Saccharomyces cerevisiae”, 37th FEBS and 22nd

IUBMB Congress, 4-9 September 2012, Seville, Spain.

Grilo AM, CG Ramos, JH Leitão. “Identification of regulatory

small non-coding RNAs in Burkholderia cepacia J2315”. 37th

FEBS and 22nd IUBMB Congress, 4-9 September 2012, Se-

ville, Spain.

Grilo AM, Ramos CG, da Costa PJP, Feliciano JR, Leitão

JH. “Experimental identification of small non-coding RNAs

from the human pathogen Burkholderia cenocepacia”, The

Non Coding Genome EMBO-EMBL Symposium, 9-12 Octo-

ber 2013, Heidelberd, Germany.

Guerreiro JF, Mira NP, Sá-Correia I. “Adaptive response to

acetic acid in the highly tolerant yeast species Zygosaccharo-

myces bailii, revealed by quantitative proteomics”, EMBO

Meeting 2012, 22-25 September, Nice, France.

Madeira A, dos Santos SC, Santos PM, Coutinho CP,

Tyrrell J, McClean S, Callaghan M, Sá-Correia I. “Adaptive

mechanisms associated with increased virulence and persis-

tence of Burkholderia cenocepacia during chronic lung infec-

tion: A quantitative proteomic analysis”, 36th European Cystic

Fibrosis Conference, 12-15 June 2013, Lisbon, Portugal.

Maldonado RF, dos Santos SC, and Sá-Correia I.

“Quantitative exoproteomic analysis to better understand the

mechanisms underlying Burkholderia cenocepa-

cia persistence and virulence in CF lung infections”, 36th Eu-

ropean Cystic Fibrosis Conference, 12–15 June 2013, Lisbon,

Portugal.

Mil-Homens D, Fialho AM. “Unraveling the involvement of

trimeric autotransporter adhesins in Burkholderia cenocepa-

cia host-cells interactions”, EMBO|EMBL Symposium: New

Approaches and Concepts in Microbiology, 14 - 16 October

2013, EMBL Heidelberg, Germany.

Mira NP, Henriques SF, Teixeira MC, Palma M, dos San-

tos SC, Sá-Correia I. “Haa1p-dependent regulatory network

of the yeast response to acetic acid stress”, Yeast Genetics

and Molecular Biology Meeting, July 31 - August 5 2012,

Princeton, NJ, USA.

Mira NP, Madeira A, Santos P, Coutinho CP, Moreira AS,

Pinto-de-Oliveira A, Sá-Correia I. “Transcriptomic and pro-

teomic analyses reveal Burkholderia cenocepacia adaptive

strategies to long-term colonization of the lungs of a cystic

fibrosis patient under antimicrobial therapy”, 3rd Seminar on

EraNet Pathogenomics, 23-25 January 2012, Tenerife, Spain.

Moreira AS, Coutinho CP, de Carvalho CCCR, Azevedo P,

Lito L, Melo-Cristino J, Sá-Correia I. “Phenotypic variation

of Burkholderia dolosa along 5.5 years of chronic colonization

with decline of lung function of a cystic fibrosis patient”.


Lisbon, Portugal.

Pereira M, Coutinho CP, Lopes A, Sá-Correia I, Videira

PA, Cabral MG. “Modulation of dendritic cell functions by

clonal variants of Burkholderia cenocepacia isolated from a

cystic fibrosis patient”, 36th European Cystic Fibrosis Confer-

ence, 12-15 June 2013, Lisbon, Portugal.

Pinto-de-Oliveira A, Coutinho A, Ramos CG, Sousa SA,

de Carvalho CCCR, Leitão JH, Sá-Correia I.

“The Burkholderia cepacia small colony variants (SCV) are a

more pathogenic bacterial form that may facilitate persistent

respiratory infections in CF patients”, 36th European Cystic

Fibrosis Conference, 12-15 June 2013, Lisbon, Portugal.


JH. “The MtvR sRNA is a major regulator of Burkholderia

cenocepacia cell physiology”. FEBS-ASM Workshop on: Biol-

ogy of RNA in host-pathogen interactions, 26-29 January

2014, Tenerife, Spain.

Ramos CG, Grilo AM, Feliciano JR, Leitão JH. “Post-

transcriptional regulation in bacterial pathogens: Small non-

coding RNAs and RNA chaperones”, Molecular Biology in

Portugal and EMBL, 18 July 2013, Lisbon, Portugal.

Ramos CG, da Costa PJP, Döring G, Leitão JH. “Hfq and

sRNAs: A new layer of complexity in virulence regulation”,

The 4th EMBO Meeting, 22-25 September 2012, Nice,

France.

Ramos CG, da Costa PJP, Leitão JH. “The Burkholderia

cenocepacia RNA chaperone Hfq2 is negatively regulated by

the novel cis-encoded small RNA h2cR”, FEBS International

Workshop: New Developments in RNA Biology, 1-4 Septem-

ber 2012, Tavira, Portugal.

51


12-13

Santos MR, Marques AT, Becker JD, and Moreira LM.

“Characterization of the TetR transcriptional regulator

SMc03169 and its contribution to symbiosis between Sinorhi-

zobium meliloti and leguminous plants”, 10th European Nitro-

gen Fixation Conference, 2-5 September 2012, Munich, Ger-

many.

Viegas SC, Mil-Homens D, Fialho A, Arraiano CM.

“Mutations in RNase E and RNase III attenuate S. Typhimuri-

um virulence”, FEBS-ASM Workshop on: Biology of RNA in

host-pathogen interactions, 26-29 January 2014. Tenerife,

Canary Islands, Spain.

Viegas SC, Mil-Homens D, Silva IJ, Saramago M,

Domingues S, Fialho A, Arraiano CM. “Endoribonucleases

mutations impair Salmonella typhimurium virulence in the

model host Galleria mellonella”, FEBS International Work-

shop on “New Developments in RNA Biology" 1 – 4 Septem-

ber 2012, Tavira, Portugal.

Viegas SC, Mil-Homens D, Silva IJ, Saramago M,

Domingues S, Fialho A and Arraiano CM. “The virulence of

Salmonella typhimurium is attenuated by mutations in endori-

bonucleases E and III”, in FASEB Meeting: Post-

transcriptional control of gene expression: mechanisms of

mRNA decay, from 24-29 June 2012, Steamboat Springs,

Colorado, USA.

Communications in National Conferences

Invited Conferences and Seminars

Fialho AM. “Bacterial proteins, a new class of multifunctional

anticancer drugs” 3rd PhD Meeting ITQB, 10-12 October

2012, Oeiras.

Leitão JH. “Post-transcription regulation in bacterial patho-

genesis: roles played by RNA chaperones and sRNAs”, Mi-

crobiotec´13 Portuguese Congress of Microbiology and Bio-

technology, 6-8 December 2013, Aveiro.

Leitão JH, “Small non-coding regulatory RNAs from

Burkholderia cepacia complex bacteria:

potential targets for therapeutic intervention?” DBE Seminars,

Instituto Superior Técnico, 28 May 2012, Lisboa.

Mira NP. “Involvement of the Haa1p-dependent regulatory

network in S. cerevisiae response to acetic acid stress: ge-

nome-wide approaches guided by bioinformatic analysis”, 1st

symposium in Bioinformatics, Universidade de Trás-os-

Montes e Alto Douro, 15 February 2012, Vila Real.

Sá-Correia I. "Systems-based understanding of chemical

stress defense mechanisms in yeast: challenges and opportu-

nities", António V. Xavier Seminars, ITQB, 29 November

2012, Oeiras.

Teixeira MC. "Unveiling antifungal drug resistance mecha-

nisms in the human pathogen Candida glabrata: from gene to

genome-wide approaches", CBAA seminars, Instituto Superi-

or de Agronomia, 30 January 2013, Lisboa.

Teixeira MC. "Antifungal drug resistance in yeasts: elucida-

tion of the biomolecular mechanisms", DBE Seminars, Insti-

tuto Superior Técnico, 16 April 2012, Lisboa.

Oral Communications

Costa C, Ponte A, Teixeira MC. “Unveiling 5-flucytosine

resistance mechanisms in yeast, using chemogenomics and

membrane proteomics approaches”, Microbiotec'13 Portu-

guese Congress of Microbiology and Biotechnology, 6-8 De-

cember 2013, Aveiro.

Costa C, Pires C, Henriques A, Camacho G, Cabrito T, Sá-

Correia I, Teixeira MC. “CgQdr2 drug:H+ antiporter from the

pathogenic yeast Candida glabrata: role and regulation in

azole drug resistance”, XIX Jornadas de Leveduras Prof.

Nicolau van Uden, 15-16 June 2012, Caparica.

Dias PJ, Sá-Correia I. “Evolution of the 12-spanner drug:H+

antiporters of family 1 (DHA1) in the pathogenic Candida

species: phylogenetic analysis of Mdr1 and Flu1 proteins”,

XIX Jornadas de Biologia de Leveduras “Nicolau van Uden”,

15-16 June 2012, Caparica.

Dias PJ, Sá-Correia I. “The major facilitator superfamily mul-

tidrug resistance transporters (mfs-mdr) in hemiascomy-

cetous yeasts: phylogenetic and syntenic analyses”, Microbi-

otec’13 Portuguese Congress of Microbiology and Biotechnol-

ogy , 6-8 December 2013, Aveiro.

dos Santos SC, Palma M, Tenreiro S, Mira NP, Moreira

AS, Becker J and Sá-Correia I. "Yeast as a model in phar-

macogenomics: genome-wide studies applied to quinine and

imatinib", XIX Jornadas de Biologia de Leveduras Professor

Nicolau van Uden, 15-16 June 2012, Caparica.

Ferreira AS, Silva IN, Becker JD, McClean S, Callaghan M,

and Moreira LM. “Involvement of the BceF tyrosine kinase in

stress response, biofilm formation and virulence in Burkhold-

eria cepacia IST408 clinical isolate”, XXXVII Jornadas de

Genética, 28-30 May 2012, Lisboa.

Henriques SF, Mira NP, Matos R, Arraiano C and Sá-

Correia I. “Gene expression regulation in yeast mediated by

the transcription factor Haa1p: identification of residues re-

quired for functional Haa1-DNA associations and role of cop-

per in DNA-binding”, XIX Jornadas de Biologia de Leveduras

Professor NIcolau van Uden, 15-16 June 2012, Caparica.

Matos RG, Fialho AM, Schuster G, Arraiano CM. ”The

evolution of the RNase II-family of exoribonucleases selected

enzymes with peculiar features: examples from Archaea and

Cyanobacteria”, XXXVII Jornadas Portuguesas de Genética,

28-30 May 2012, Lisboa.

Mira NP, Münsterkötter M, Dias-Valada F, Santos J, Palma

M, Roque FC, Guerreiro JF, Rodrigues F, Sousa MJ, Leão

C, Guldener U, Sá-Correia I. “Genome sequence and anno-

tation of the highly acetic acid-tolerant Zygosaccharomyces

bailii derived interspecies hybrid strain

ISA1307”, Microbiotec’13 Portuguese Congress of Microbiolo-

gy and Biotechnology, 6-8 December 2013, Aveiro.

Palma M, Madeira S C, Mendes-Ferreira A and Sá-Correia

I. “Impact of assimilable nitrogen availability in glucose up-

take kinetics in Saccharomyces cerevisiae during alcoholic

fermentation”, XIX Jornadas de Biologia de Leveduras Pro-

fessor NIcolau van Uden, 15-16 June 2012, Caparica.

52

Palma M, Roque F, Guerreiro JF, Mira N and Sá-Correia I.

“Screening of a genomic library of the Zygosaccharomyces

bailii derived interspecies hybrid strain ISA1307 to search for

genes responsible for acetic acid resistance”, Microbiotec’13

Portuguese Congress of Microbiology and Biotechnology, 6-8

December 2013, Aveiro.


JH. “The MtvR sRNA is involved in the regulation of Hfq”,

Microbiotec'13 Portuguese Congress of Microbiology and

Biotechnology, 6-8 December 2013, Aveiro.

Santos MR, Marques AT, Becker JD, and Moreira LM.

“Involvement of the TetR transcriptional regulator SMc03169

in pH sensitivity and contribution to nodulation competitive-

ness in Sinorhizobium meliloti”, 3rd Meeting of the Institute for

Biotechnology and Bioengineering, 16-17 March 2012, Lis-

boa.

Silva IN, Ferreira AS, Becker JD, Tavares AC, and Moreira

LM. “Genotype and phenotype variation of Burkholderia multi-

vorans sequential isolates during long-term colonization of the

airways of cystic fibrosis patients”, XXXVII Jornadas de Gen-

ética, 28-30 May 2012, Lisboa.

Silva V, Mateus C, Gonçalves J, Varela V, Viegas CA.

“Arthrobacter aurescens TC1 as a bioaugmentation tool for

bioremediation of environments contaminated with the herbi-

cides atrazine and terbuthylazine”, 10ª Conferência Nacional

do Ambiente, 6-8 November 2013, Aveiro.

Poster Communications

Bernardes N, Ribeiro AS, Matos R, Arraiano CM, Seruca

R, Paredes J, Fialho AM. “Azurin impairs P-cadherin-

dependent invasion of breast cancer cells via decreased FAK/

Src signalling” I3S Scientific retreat, 10-11 May 2012, Póvoa

do Varzim.

Ferreira AS, Oliveira VH, Moreira LM. “Quorum-Sensing

inhibitors as antimicrobial agents against bacteria belonging

to Burkholderia cepacia complex”, XXXVII Jornadas de Gen-

ética, 28-30 May 2012, Lisboa.

Ferreira AS, Silva IN, Becker JD, McClean S, Callaghan M,

Pilkington R, Givskov M, Ryan RP, Fernandes F, Moreira

LM. “Involvement of the BceF tyrosine kinase in stress re-

sponse, biofilm formation and virulence in Burkholderia cepa-

cia IST408 clinical isolate” 3rd Meeting of the Institute for Bio-

technology and Bioengineering, 23-24 November 2012, Lis-

boa.

Grilo AM, Ramos CG, da Costa PJP, Feliciano JR, Leitão

JH. “Identification of small non-coding RNAs and their roles

on the biology of opportunistic pathogens of the Burkholderia

cepacian complex”, Microbiotec’13 Portuguese Congress of

Microbiology and Biotechnology, 6-8 December 2013, Aveiro.

Isidoro D, Santos MR, Ferreira AS, and Moreira LM.

“Studies on gellan gum modification aiming new biotechno-

logical applications”, XXXVII Jornadas de Genética, 28-30

May 2012, Lisboa.


JH. “The MtvR sRNA is involved in the regulation of Hfq”,

Microbiotec’13 Portuguese Congress of Microbiology and

Biotechnology, 6-8 December 2013, Aveiro.

Ramos CG, Grilo AM, Silva IN, Ferreira AS, Becker JD, da

Costa PJP, Feliciano JR, Sousa SA, Moreira LM, Leitão

JH. “Building an Atlas of Hfq-dependent samll non-coding

RNAs from the opportunistic human pathogen Burkholderia

cenocepacia J2315”, Microbiotec´ 13 Portuguese Congress of

Microbiology and Biotechnology, 6-8 December 2013, Aveiro.

Santos MR, Marques AT, Becker JD, Moreira LM.

“Characterization of the TetR transcriptional regulator

SMc03169 and its contribution to symbiosis between Sino-

rhizobium meliloti and leguminous plants”, XXXVII Jornadas

de Genética, 28-30 May 2012, Lisboa.

Tavares AC, Silva IN, Becker JD, Ferreira AS, Moreira LM.

“Mucoid morphotype variation of Burkholderia isolates under

stress conditions: Effects on gene expression, phenotypic

properties and virulence”, IV Symposium on Bioengineering,

23-24 November 2012, Porto.

Tavares AC, Silva IN, Becker JD, Ferreira AS, Moreira LM.

“Effect of mucoid morphotype variation of Burkholderia iso-

lates on gene expression, resistance to antimicrobial environ-

ments and virulence”, XXXVII Jornadas de Genética, 28-30

May 2012, Lisboa.

Teixeira MC, Monteiro PT, Guerreiro JF, Gonçalves JP,

Mira NP, dos Santos SC, Cabrito TR, Palma M, Costa C,

Francisco AP, Madeira SC, Oliveira AL, Freitas AT, Sá-

Correia I. YEASTRACT: an upgraded information system for

the analysis of gene and genomic transcription regulation in

Saccharomyces cerevisiae, Microbiotec'13 Portuguese Con-

gress of Microbiology and Biotechnology, 6-8 December

2013, Aveiro.

53


12-13

Guest Editors of International Journals


Prates JAM, Ferreira LMA - Guest editors of the special

issue “Biocatalysis and Biotransformation”, Proceedings of

the 9th Carbohydrate Bioengineering Meeting (CBM9), 2012.

Mira NP, Teixeira MC. Guest editors of the special issue

“Microbial Mechanisms of Tolerance to Weak Acids”, Fron-

tiers in Microbiology – Section of Microbial Physiology

and Metabolism (11 articles included), 2013.

Sá-Correia I, Teixeira MC. Guest editors of the special issue

“Physiological role and regulation of Multidrug/Multixenobiotic

resistance membrane transporters”, Frontiers in Physiolo-

gy, 2014.

Editorial boards of International Journals

“OMICS: A Journal of Integrative Biology” (2012-2013),

“FEMS Yeast Research” (2012-2013), “Journal of Biomedi-

cine and Biotechnolology/BioMed Research Internation-

al” (2012-2013), “International Journal of Microbiology” (2012-

2013) , “Microbial Cell” (2013) (I. Sá-Correia)

“International Journal of Microbiology”, “The Open Microbiolo-

gy Journal”, “Bioengineered Bugs” (A.M. Fialho)

“Dataset papers in Science”, Hindawi Publishing Corporation

(J.H. Leitão)

Editorial boards of National Journals

“Magazine de MICROBIOLOGIA” (http://

www.spmicrobiologia.pt/ ), the journal of the Portuguese Soci-

ety of Microbiology (I. Sá-Correia and C.G. Ramos)

“Boletim de Biotecnologia” (J.H. Leitão)

Organization of Scientific Events

5th PYFF- Physiology of Yeast and Filamentous Fungi, 4-7

June 2013, Montpellier, France (I. Sá-Correia, scientific com-

mittee and chairperson of session “Response and tolerance

to stress”)


Lisboa, Portugal (I. Sá-Correia, scientific & organizing com-

mittees and chairperson of the Symposium "Translating what

we have learned from bacterial genomics to CF care")

FEMS Congress 2013, July 21-25, Leipzig, Germany (I. Sá-

Correia, co-chair of “Yeast and Food Microbiology” Symposi-

um)

2012 and 2013 editions of the Winter School in Systems Biol-

ogy held within the framework of the Erasmus Mundus

euSYSBIO Masters´ in Systems Biology, Instituto Superior

Técnico-Alameda (Organizers: I. Sá-Correia and M.C.

Teixeira)

Coordination of post-graduation degrees

Master’s program in Microbiology, University of Lisbon (IST

and the Faculties of Sciences, Medicine and Veterinary Medi-

cine) (I. Sá-Correia, coordinator)

Master’s Program in Biotechnology, IST (I. Sá-Correia, coor-

dinator)

Ph.D. Program in Biotechnology, IST (I. Sá-Correia, coordi-

nator)

Erasmus Mundus euSYSBIO Master’s Programme in Sys-

tems Biology (Instituto Superior Técnico, KTH (Sweden) and

Aalto University (Finland)) (I. Sá-Correia, coordinator at IST)

Other Scientific Activities

2012 2013

54

General and Committee Chairs or Committee

Memberships

Portuguese Society of Microbiology (I. Sá-Correia, Presi-

dent; A.M. Fialho, 2nd secretary)

Council of the Federation of European Microbiological Soci-

eties (FEMS) (I. Sá-Correia, member)

Study group on fungal infections of the European Society of

Clinical Microbiology and Infectious Diseases (ESCMID)

(N.P. Mira)

Biological Sciences Research Group of IBB at Instituto Su-

perior Técnico (I. Sá-Correia, coordinator)

Department of Bioengineering of IST (I. Sá-Correia, vice-

president)

Scientific Board of IST, Universidade de Lisboa (I. Sá-

Correia, member)

General Council of Universidade de Lisboa (I. Sá-Correia,

member)

Evaluation panels

Member of the Evaluation Panel "Genetics, Genomics,

Bioinformatics and Systems Biology" of Advanced

grants, European Research Council (ERC)(2012

applications) (I. Sá-Correia)

Coordinator/Member of the evaluation panel "Biology, Bio-

chemistry and Biotechnology" of University study cycles for

the Portuguese Agency for the Evaluation and Accreditation

of Higher Education (A3ES) (I. Sá-Correia)

Awards

Miguel C. Teixeira - Honorable Mention within the scope of

the UTL/Santander scientific awards 2012, in the area of

Biochemical Engineering, Biochemistry and Biotechnology,

as a recognition of the impact of his scientific work during

the past five years.

Sandra C. dos Santos - Young Researcher Award (ex-

equeo) SPM - Isabel Spencer-Martins, awarded by the Por-

tuguese Society of Microbiology, 2012.

Sandra C. dos Santos - Best Poster Award, granted by the

European Cystic Fibrosis Society at the 36th European Cyst-

ic Fibrosis Conference, with the communication “Adaptive

mechanisms associated with increased virulence and per-

sistence of Burkholderia cenocepacia during chronic lung

infection: A quantitative proteomic analysis”, 2013.

Nuno P. Mira - Best oral communication Award (ex-aqueo),

awarded by Portuguese Society of Microbiology and

Alfagene at the Microbiotec13 meeting with the oral commu-

nication “Genome sequence and annotation of the highly

acetic acid-tolerant Zygosaccharomyces bailii-derived inter-

species hybrid strain ISA1307”, 2013.

55


12-13

Faculty Staff Isabel Sá-Correia Arsénio M. Fialho Cristina A. Viegas Jorge H. Leitão Leonilde M. Moreira Miguel C. Teixeira Nuno P. Mira

Post-doctoral Fellows Sílvia A. Sousa Ana S. Ferreira Sandra C. dos Santos Margarida Palma Paulo J. Dias Carla P. Coutinho Tânia R. Cabrito Christian G. Ramos Dalila Mil-Homens Inês N. Silva

PhD Students Catarina Rodrigues Andreia Madeira Mário R. Santos Nuno Bernardes Fátima Gil Catarina Costa André M. Grilo Ana S. Moreira Joana F. Guerreiro Joana R. Feliciano Sílvia F. Henriques Filipa C. Roque Rita Maldonado

Research Assistants Vera P. Silva Filipa Valada Paulo JP da Costa Andreia Silva

Master Students Ana Almeida André Henriques Andreia Tavares Andreia Ponte Carla Alexandra Mateus Carla Pires Cláudia P. Godinho Eunice Penas Filipa G. Dias Filipe Silva Gonçalo Silva Guida Camacho Janete Gonçalves João Brazão Jonathan Ribeiro Kaur Alasoo Maria Moita Rúben Bernardo Sofia Abreu Sofia Alves Vítor Oliveira Viviane Varela

Technical Assistants Mónica Rato Sílvia Rana

BSRG Members (2012-2013)

56

BSRG


Institute for Biotechnology and Bioengineering

Instituto Superior Técnico

Av. Rovisco Pais

1049-001 Lisbon

Portugal

http://ibb.pt/bsrg/

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BSRG 2012-2013

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