Comparative Genomics The FinaleAngela Pena, Ambily Sivadas, Amit RupaniShimantika Sharma, Juliette Zerick Keerti Surapaneni, Artika Nath, Hema Nagrajan

Outline

Results

• Goal 1 – PCR Assay

• Goal 2 – Comparative genome analysis

• Goal 3 – Haemolysis study

• Goal 4 – Virulent factors

• Discussion

Goal 1Identification and characterization of target genes for PCR Assay

Identification of target genes

A CB

A C

Hhae

NTHi

PCR products of different size

Fw primer Rv primer

One copy of B or multiple copies? Is A-C organized the same way in both organisms?Identify candidate clusters/genes for assay development and conserved regions for primer/probe design

Cluster Analysis – Genome Set

Cluster statistics

Total clusters: 8402 Total common to all genomes : 361 Total unique to Hhae: 82 Total unique to Hinf: 38 Total unique to Pathogenic strains: 0

Protein sequences were clustered using Blastclust

Metabolism protein syntheis /post transtransportDNA repair replication chaperone/protein foldingproteolysis tRNA processing DNA repair/ replication secretioncompetencecell division cell cycle regulationHypothetical ATP/DNA bindingunknown transcriptionstress response secretionantiobiotic resistence

Common clusters – Functional breakdown

Target identification - Protocol 1

1. Take all proteins common to all 25 genomes

• Common = Most conserved proteins

1. Cluster Analysis: Take all proteins common to all 25 genomes

• Common = Most conserved proteins

2. Compute and compare inter-cluster distances for Hhae vs Hinf

• Look for species specific patterns

• Look for including unique genes

Target identification - Protocol 1

Protocol II:BLAST Everything

Our method is this: for every unique Hhae gene, we will locate its corresponding contig

We checked the flanking regions (on the contig) for conserved genes.

We will then locate the conserved genes in the Hinf genome and see if they are adjacent.

Since a wide net can be cast with BLASTn searches, this includes homologs.

If a pistol just isn't working for you . . .

Start with a set of Hhae

genes

Select a unique Hhae gene from

the set

Get the locations of the conserved flanking genes in the Hinf

genome

Search the set of common Hhae/Hinf

(conserved) genes for genes in the flanking

regions

Is there at least one conserved gene in each

flanking region?

Are the conserved genes adjacent or “close enough” in the Hinf genome?

Reject gene and start over

We found a (more) unique gene!

NO

NO

YES

YES

PCR Assay: Results

Target 1

PCR product

No duplication was found for these genes

A B DC

A B D

Hh

NTHi

3-oxoacyl-(acyl carrier protein) synthase III

Nucleic acid binding protein (hypothetical)

fatty acid/phospholipid

synthesis protein

50S ribosomal

protein1020 bp

170 bp

Hh

NTHi

1250 bp

380 bp

PCR Assay: Results

Target 2

PCR product

A B E

A 1 E

Hh

NTHi

predicted membrane

protein

fructose-biphosphate

aldolase

1451 bp

1934 bp

Hh

NTHi

1934 bp

1451 bp

C D

D2 3 4 5

purine nucleoside

phosphorylase

6

Target validation by Insilico PCR

H. haemolyticus5 strains

Non TypableH. influenzae19 strains + 1 Typeable

1 1775870 2749

Step 1: Multiple Sequence Alignment by ClustalW2 - Overview

905 ntsTarget 1

Step 2: Phylogenetic analysis Neighbor Joining Tree Percentage of Identity usingJalview

H. haemolyticus5 strains

Non TypableH. influenzae20 strains

1 1775870 2749

Forward

Reverse

5’-CTCACTTACGCCACCACGTA-3’

3’-TGCAACAATAATCAGTTCAATATCT-5’

Step 3: Finding primers

Product length: 1354

M21621

Product length: 487

AAZD00000000

Non Typable H. influenzae

H. haemolyticus

In silico PCR Analysis

In silico PCR Analysis

STRAIN PRODUCT LENGTH START STOP

M19107 1363 654 673

M19501 1364 654 673

M21127 1363 654 673

M21621 1354 655 673

M21709 487 654 673

CP000671 489 653 672

L42023 487 654 673

Sequence (5'->3') FORWARD

Non TypableH. influenzae20 strains

H. haemolyticus5 strains

1 5372

MSA – Target 2

Goal 2Comparative genomic analysis

Horizontal Gene Transfer• Horizontal gene transfer (HGT), also lateral gene transfer (LGT) refers to the transfer of 

genetic material between organisms

Alien Hunter• Predicts putative horizontally transferred regions.• Standalone software• Available at http://www.sanger.ac.uk/Software/analysis/alien_hunter

Usage:./alien-hunter <input_file> <output_file>

INPUT: raw genomic sequence

PREDICTION: HGT regions based on Interpolated Variable Order Motifs (IVOMs)

.sco file

Last time, we got many hits with varied scores that covered almost 90% of the genes in each genome. Hence, we decided to place a threshold on the scores.

• We studied the distribution of scores for each genome by plotting histograms for each genome based on the scores.

• We decided to place a threshold of >70 after studying all the histograms.

Screenshot of M21621

HGT gene count

1 2 3 4 5 60

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

before filteringafter filtering

before filtering after filtering

1699 360

2709 145

3065 225

5601 253

1140 108

2717 185

Insertion elements

• An Insertion element is a short DNA sequence that acts as a simple transposable element.

• A transposable element (TE) is a DNA sequence that can change its relative position (self-transpose) within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste".

IS Finder

FASTA sequencesWe retrieved FASTA sequences by submitting the accession IDs in NCBI

BLASTWe blasted these insertion sequences against each of the strains and got the location of the insertion sequences in the strain.

A PERL script was written to extract the insertion sequences from their respective contigs in each strain.

Comparative Analysis TableFeature /Strain

 Tools M19107 M19501 M21127 M21621 M21639 M21709 Average

Genome size -  1774129 1809865 2029793 1959123 2397857 1808157 1963154

GC content % IGIPT 39.39 38.23 39.11 39.11 38.30 38.73 38.81

Total Number of genes

-  1973 1785 2086 1923 2669 1840 2046

Operons -  76 (27) 69 (14) 71 (25) 79 (21) 87 (36) 70 (14) 73

Virulence factors    124  115 116  115   124 144    115

HGT gene count Alien Hunter

360 145 225 253 108 185 213

Pathogenic  - No No Yes Yes Yes No - 

Insertion elements IS Finder 6 6 - - 15 - 4.5

Hemolytic activity -  Y  N  Y  Y  N  N  - 

M19107 – Circular alignment using BRIG

M19501 – Circular alignment using BRIG

M21127– Circular alignment using BRIG

M21621– Circular alignment using BRIG

M21639 – Circular alignment using BRIG

M21709 – Circular alignment using BRIG

Goal 3Identification and Characterization of Haemolysin in Hhae

AIM #1

Look for the hemolysin BA operon present in the H.haemolyticus strains and characterize it as present/absent in the hemolytic and non hemolytic strains

HEMOLYSIN • Hemophilus ducreyi, requires two adajecent genes, hhdB

and hhdA for hemolysis .

• hhdB is an outer membrane protein, which is required for secretion and activation of the hemolysin structural protein, hhdA.

• Once secreted, hhdA interacts with target cell membranes, oligomerizes, and forms pores 2.5 to 3.0 nm in diameter, which lyse the target cell

TWO PROTEIN SECRETION SYESTEM

OUR STRATEGY

•Downloaded the Fasta files of all hemolysin protein sequence of the Pasteurellaceae family from NCBI protein database.

•Blasted the predicted protein sequences of the six strains against these.

Cut off threshold: Identity 70% Coverage 80%

RESULTSStrain Hemolysis Gene A /contig Gene B /contif

Haemophilus haemolyticus M19107

Y 51_11|7343|115961417 amino acidsZP_09185204.1| hemolysin [Haemophilus [parainfluenzae]

51_1216|11855|13366503 amino acidsZP_09185203.1| hemolysin activation/secretion protein [Haemophilus [parainfluenzae]

Haemophilus haemolyticus M21127

Y 20_113|106934|111307|1457 amino acids ZP_09185204.1| hemolysin [Haemophilus [parainfluenzae]

20_112|105150|106760536 amino acids ZP_09185203.1| hemolysin activation/secretion protein Haemophilus [parainfluenzae

Haemophilus haemolyticus M21621

Y 1_361|369207|3735801457 amino acidsZP_09185204.1| hemolysin [Haemophilus [parainfluenzae]

1_362|373754|375349|531 amino acids |ZP_09185203.1| hemolysin activation/secretion protein [Haemophilus [parainfluenzae]

Haemophilus haemolyticus M19501

N None None

Haemophilus haemolyticus M21639

N None None

Haemophilus influenzaM21709

N None None

All hits had 70% and more identity and 95-100 coverage

AIM# 2

•Characterize the domains/motifs/residue in hemolysin.

•Depict the secondary structures in hemolysin.

•Predict the 3D structure of hemolysin.

SIGNAL PEPTIDE & HAEMAGGLUTINATION ACTIVITY DOMAIN

• A signal peptide (25 aa) to transport the hemolysin to outer membrane or periplasm. LipoP cleavage site Spase I at 25-26. NOT LIPOPROTEIN

• Haemagglutination activity domain -suggested that the haemagglutination activity domain is a carbohydrate-dependent haemagglutination activity site which is found in a range of haemagglutinins and haemolysins

N’ terminal

Signal Peptide

Haemagglutination activity domain

HAEMAGLUTININ REPEAT• Haemaglutinin repeat is a highly divergent repeat that

occurs in number of proteins implicated in cell aggregation

hemolysins/cytolysins ShlA of Serratia marcescens, HpmA of Proteus mirabilis, EthA of Edwardsiella tarda, HhdA of Haemophilus ducreyi, the large supernatant proteins LspA1 and LspA2 of H. ducreyi, and the HecAadhesin of E. chrysanthemi .Clantin et al., 2004. The crystal structure of filamentous hemagglutinin secretion domain and its implications for the two-partner secretion pathway.PNAS.

TPS DOMAIN All TPS-secreted proteins contain a distinctive N-proximal module essential for secretion, the TPS domain. TpsA proteins display two conserved regions, C1 and C2, and two less-conserved regions, LC region. ANPNL and NPNGIS is found in this region

Does the TPS domain exist in H.haemolyticus strains?

211272162119107Fha30EthA HpmALspA1LspA2 ShlAHhdA.

Fha30H.HH.HH.H

HhdAEthA

ShlAHpmALSpA1LSPA2

CONSERVED RESIDUES IN TPS DOMAIN

NPNL & NPNGI These motifs form type I beta -turns, which might play important stabilizing roles. The conserved residues of the TPS domain serve to drive the folding of the TPS domain into a beta -helix and to stabilize the helix

HAD 39-159-PfamOr

TPS 39-270

TPS

ANPNL

NPNLGI

STRATEGY SECONDARY STRUCTURE

AIM #3

•Identify the domains in the hemolysin activator gene •Determine the secondary and 3D

structure of hemolysin activator gene

HEMOLYSIN ACTIVATOR PROTEIN TRANSMEMBRANE PROTEIN

MEMBRANE PROTEINS

α-helical β-barrel

β-barrel membrane protein class are located in the outer membrane of Gram-negative bacteria.

These proteins have membrane spanning segments formed by antiparallel β-strands, creating a channel in the form of a barrel that spans the outer membrane.

DOMAIN IS HEMOLYSIN ACTIVATOR

SP (LipoP) – SPI cleavage site between pos. 19 and 20. NOT LIPOPROTEIN

POTRA_2- polypeptide-transport-associated domain. In ShlB this domain has a chaperone-like function over ShlA.

Activator domain in ShlB is shown to interacts with ShlA during secretion and imposes a conformational change in ShlA to form the active hemolysin.ShlA/B: Serratia marcescens

POTRA_2

Activator Domain SP

Strategy

• Prediction of TransMembrame Beta Barrels (PRED TMBB) Method is powerful when used for discrimination purposes, as it can discriminate with a high accuracy the outer membrane proteins from water soluble in large datasets

• The 'TransMembrane protein Re-Presentation in 2 Dimensions' tool, automates the creation of uniform, two-dimensional, high analysis graphical images/models of alpha-helical or beta-barrel transmembrane proteins.

Work Flow

Sequence

Discrimination scoreSequence

Step 1: Find Discrimination Score

Haemophilus haemolyticus M19107 –hemolystic Contig : 51_1216 Start/End: |11855|13366 Length: 503 amino acids

STEP2Predicted structure 2D of hemolysin activator Haemophilus haemolyticus M19107 –hemolystic

CYTOPLASMIC

Coloring by Hydrophobic Potential

+ -More hydrophobic Hydrophilic

3D STRUCTRES

STRUCTURE PREDICTION METHODS

Homology

Modelling

De novo protein

structure prediction

Protein Threadin

g

Requires a template with a high

percentage identity

Models the structure based on general

principles that govern protein

folding energetics

Works when homology modelling

fails

PROTEIN THREADING APPROACH • It follows Protein Threading approach to predict the

structure of the target protein sequence.

• In practice, when the sequence identity in a sequence alignment is low (i.e. <25%), homology modeling may not produce a significant prediction. In this case, if there is distant homology found for the target, protein threading can generate a good prediction.

• Protein threading, also known as fold recognition, is a method of protein modeling (i.e. computational protein structure prediction) which is used to model those proteins which have the same fold as proteins of known structures, but do not have homologous proteins with known structure.

• We used RaptorX server which predicts the structure based on protein threading method.

3D STRUCTURES OF HEMOLYSIN FROM RaptorX

Segment 1 (37-158) Segment 2 (159-1457)

3D STRUCTURES OF ACTIVATOR FROM RaptorX

POTRA_2

Activator Domain SP

Activator Domain Segment 1 (149-500)

POTRA_2 Domain Segment 2 (67-136)

Segment 3 (1-66)

POTRA DOMAIN POLYPEPTIDE-TRANSPORT-ASSOCIATED DOMAIN

• POTRA domains have similar structure comprising a three-stranded b sheet overlaid with a pair of antiparallel helices .

POTRA_2

Activator Domain SP

Kim et al., 2007. Structure and Function of an Essential Component of the Outer Membrane Protein Assembly Machine. SCIENCE VOL 317

E.coli POTRA Domain figure

Goal 4Identify and characterize the potential virulence factors in Haemophilus haemolyticus

Human Immune System

An Immune System (IS) is a system of biological structures and processes within an organism that protects again disease.

In order to function properly, IS must detect a wide variety of agents, from viruses to parasitic worm, and distinguish them from the organism’s own healthy tissue.

Pathogens can rapidly evolve and adapt to new environments to avoid detection and destruction by the immune system:

Phase variation

Phase variation

Phase variation is defined as the random switching of phenotype at frequencies that are much higher (sometimes >1%) than classical mutation rates.

Is a widespread source of intraespecific genotypic and phenotypic variation

Several different mechanisms are exploited by bacteria to switch gene and/or protein expression “on” or “off”

Combinatorial math:

A bacterium with just 20-phase variable loci can exist in 220 different states (more than a million)

Multiple defense mechanisms have evolved to recognize and neutralize pathogens:

Immune System

AdaptiveInnate Creates immunological memory after an initial response to a specific pathogen

Non-specific response to microorganism or toxins found in the cell

Microbes are identified by pattern recognition receptors, eg. LPS

Generic response

Does not confer long-lasting immunity against the pathogen.

1. Inflammatory response2. Activation of Complement System3. Antimicrobial peptides

Immune-Evasion target

Major component of innate IS

Complement System

Biochemical cascade that attacks the surfaces of foreign cells It can contain over 20 different proteins

Complement the killing of pathogens by antibodies:

- produces peptides that attract immune cells- opsonize (coat) the surface of a pathogen, marking it for the destruction

- increase vascular permeability

Host Immune Evasion

Microorganisms have developed many ways to evade complement actions:

• Trapping endogenous C1 inhibitor• Inactivating antibodies through capture of their FC regions• Mimicking structural regulators• Degradation crucial components of Complement System

How we did the analysis ?Virulence Factor Data Base Search of VF in Haemophilus genus

132 VF were retrieved

Upload matrix to MeV

Blastp against all the 25 genomes of Haemophilus

constraints:At least 40% identity

At least 70% of query coverage

Search in NCBI for all VF in Haemophilus genus

RefSeq protein sequences Data Base

Build a matrix with presence/absence +3 Presence-3 Absence

Heat Map was build HCL was generated

Results….

HCL: Hierarchical ClusterDistance Metric: Pearson correlationLinkage method: Average linkage clustering132 virulence factors analyzed25 samples: 19 NTHi 5 Hhae 1 THi

Cluster A : Uniform/regular pattern

Cluster B : heterogeneous/ irregular pattern

Cluster A : Uniform/regular pattern

• tad locus

• Type IV pili

• LPS biosynthesis

• tad locus

• tad locus• LPS biosynthesis

• Adherence

• Transferring-binding protein 1

• Exopolysaccharide

• Haemophilus iron transport locus

• Hemoglobing and hemoglobin-haptoglobin binding proteins

• Heme biosynthesis

• Cytolethal distending toxin

Cluster B : heterogeneous/ irregular pattern

Heme/hemopexin-binding complex

hxuAhxuBhxuCFepA Ferric enterobactin receptor, E. coli

BtucrfaCABC_ThptE

vitamin B12 receptor protein, E. coli1,5 Heptosyltransferase IATP binding cassette transporter familyLipopolysaccharidae heptosyltransferase I

Immunoglobulin A proteaseIgA1

Prevalence Ratio Analysis Patho vs Asymptomatic - Adherence

HAEMOAGGLUTINATING PILI (hifABCDE operon)

Function• Promote adherence to respiratory mucus

and human oropharyngeal epithelial cells• Facilitates colonization

Mechanism: Binding to the Anton antigen (An-Wj) common to buccal epithelial cells and erythrocytes

Prevalence Ratio analysis Patho vs Asymptomatic - AdherenceHAEMOAGGLUTINATING PILI (hifABCDE operon)

Role in virulence

• Expression of pili is a phase-variable phenomenon

• Variation in (TA) repeat units within the overlapping promoter region of hifA and hifB regulates the transcription of the gene

• 11 repeat units – reduced expression• 10 repeat units – maximal expression• 9 repeat units – transcriptional silencing

Found in M19501(5,4),AAZD00000000 (9),AAZE00000000(9),AAZJ00000000(5)

Pathogenic – Hi F3047 strain (9)

In M19107 – only hifA and hifB – split into two contigs –hence no info on TA repeats

Prevalence Ratio Analysis Patho vs Asymptomatic - Adherence

High Molecular Weight Protein 1/2

Function• Adhesins that mediate attachment to

human epithelial cells

Structure features• Autotransport protein• Secretion of these adhesins

HMW1A/HMW1B requires accessory proteins called HMW1B/HMW2B and HMW1C/HMW2C

Prevalence Ratio Analysis Patho vs Asymptomatic-LOS

lic3A

rfaC

Characteristics:  Biosynthesis pathway of LOS is producing a branched oligosaccharide attached to a lipid A via two 3-deoxy-D-manno-2-octulosonic acid (KDO) molecules.

 rfaC gene product adds the first heptose (Hep I) to KDO

rfaC mutants are shown to produce truncated LPS

rfaC is absent in all H.influenzae, but present in all H. haemolyticus

rfaC mutants also shown to decrease haemolytic activity and expression in E Coli

So, may be, retaining rfaC helps H. hae retain its haemolytic activity

siaA

lgtA

lex2B

Prevalence Ratio Analysis Patho vs Asymptomatic-LOS

lic3A

rfaC

Sialic Acid TransporterLic3A,SiaA or LsgB

Characteristics: Sialic acid is added as terminal nonreducing sugar to LOS – important for bacterial virulence.

These genes code for sialyltransferases which incorporates sialic acid into LOS

In the absence of this transporter, Hinf cannot survive when exposed to serum

Found to be absent in all H.haemolyticus

siaA

lgtA

lex2B

Prevalence Ratio Analysis Patho vs Asymptomatic-LOS

lic3A

rfaC

Phase variable glycosyl transferasesLex2B, LgtB

Characteristics:

Contributes to the significant intrastrain heterogeneity of lipopolysaccharide (LPS) composition in H. influenzae

And phase variable expression

Found to be absent in all H. haemolyticus

siaA

lgtA

lex2B

Prevalence Ratio Analysis Patho vs Asymptomatic- Immuno-evasion

•No difference in pattern observed in this case!

Prevalence Ratio Analysis Patho vs Asymptomatic- Iron acquisition

HxuABC

HxuA binds to heme-hemopexinHxuB releases HxuA from the cell surface into the mediumHxuC is involved in the transport of heme within the cell

Function• Using host heme-

hemopexin as the source of heme iron for growth

Absent in all H.haemolyticus

IgA1

IgA1

YadA is a potent serum resistance factor as it inhibits the classical pathway of complement, Yersinia adhesin A

Fba - Fibronectin-binding protein, streptococcus spp

Protein EmrsA, glmM, galU, galE, manA, manB

IgA1 – serine protease, cleaves IgA1

Protein E – adhesine protein, captures vitonecting (Vn), which prevents the formation of MAC

MAC (Membrane Attack Complex) it forms transmembrane channels, disrupting the phospholipid bilayer of target cells, leading

to cell lysis and death.

Is H. haemolyticus an opportunistic or pathogenic bacterium ?

Ambiental Opportunistic PathogenicInnate IS Adaptive IS

HuxABC

FepA

IgA1

Exopolysaccharides

ompP2

Protein E

YadA

fba

Btuc

rfaC/hptE

DISCUSSION

Hydrogenase-4 10-gene operon

• First identified in E. Coli in 1997• hyfABCDEFGHIJ - hyfR (transcriptional activator)• The proteins encoded by the hyf operon are proposed to

constitute a proton-translocating formate hydrogenlyase•  Hyf catalyzes dihydrogen production and ion transport when

the cells are grown at a starting pH of 7.5• This operon is silent in E. Coli – Hyd-3 is the active H2

evolving operon.

Hydrogenase and Virulence

• As per recent studies, Hyd-4 in Yersinia enterocolitica helps in gut colonization▫Using H2 produced during fermentation by intestinal

microflora• Also, hydrogenases facilitated respiratory hydrogen

use by Salmonella enterica and is considered essential for virulence

• So, understanding the expression and role of this hydrogenase operon could provide critical information for▫Characterization of Hhae▫Understanding a new mode of virulence in Hhae (May

be!)

ABC transporter system

•ATP-binding cassette (ABC) transporter system

•One of the largest protein families. •Found in all species and are evolutionarily

related. •Functionally diverse and have roles in a

wide range of important cellular functions.

Structural schema

• Bacterial genomes encode different numbers of ABC transporters, which correlate with their lifestyles, suggests that bacterial ABC transporters are likely to be necessary for growth and/or survival of the bacteria in their ecological niches

ABC Transporters and Virulence

• Virulence associated with uptake of nutrients▫ Polyamine, glutamine, sugar

• Virulence associated with uptake of metal ions▫ such as iron, zinc, and manganese

• Virulence associated with cell attachment• ABC transporter (outer membrane) proteins are sometimes

immunogenic too.• Based on the role of the ABC system in virulence, certain

components could be potential targets for developing vaccines too

• In our case, one of the closest homologs is the SalX gene from Pasteurella multocida– which is an ABC-type antimicrobial peptide transport system, ATPase component.

• Hence, characterizing this ABC transporter system could be insightful.