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Role of entF Gene in Iron Acquisition by
Brucella abortus 2308
By
Neeta Jain
Thesis submitted to the Faculty of the Virginia Polytechnic Institute and State University in
Partial fulfillment of the requirements for the degree of
Master of Science
in
Biomedical and Veterinary Sciences
Nammalwar Sriranganathan, Chairman
Stephen M. Boyle
Judy S. Riffle
Bernard S. Jortner
May 11th, 2009
Blacksburg, Virginia
Keywords: Brucella abortus, siderophores, colony forming units (CFU), entF, erythritol,
brucebactin
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Role of entF Gene in Iron Acquisition by
Brucella abortus 2308
Neeta Jain
Abstract
Brucella causes undulant fever in humans and uterine and systemic infection leading to
abortions in domestic animals and wild life. For the acquisition of iron in mammalian hosts,
species of Brucella are known to produce two siderophores, 2, 3-dihydroxy benzoic acid (2, 3-
DHBA) and brucebactin. Inability to synthesize of 2, 3-DHBA affects the ability of pathogen to
metabolize erythritol, replicate in trophoblast cells and cause abortion in pregnant ruminant host.
The entF gene has been implicated in the unresolved pathway allowing brucebactin biosynthesis
in Brucella. The research effort presented in this thesis tries to relate the role of entF in iron
acquisition and potential relation with erythritol metabolism by wild type B. abortus 2308. An
entF deletion mutant (BAN1) of B. abortus 2308, generated using cre-lox methodology was
found to be growth inhibited in iron minimal media compared to wild type strain. Growth
inhibition was further enhanced with the addition of an iron chelator or 0.1% erythritol.
Compared to wild type strain, no growth inhibition of BAN1 mutant was found in murine
J774A.1 macrophages, which suggests that Brucella could acquire iron inside mammalian cells.
The entF gene complemented mutant strains of BAN1 (BAN2A and BAN2B) were found to be
intermediate in their ability to grow in iron minimal media supplemented with 0.0.05%
erythritol, when compared to wild type and BAN1 strain. The results from the present thesis
demonstrate that entF gene plays an important role in iron acquisition and erythritol metabolism
by B. abortus 2308 under iron limiting conditions.
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Dedication
I dedicate this thesis to my family. Very special thanks for being the best part of my life.
To my late grandma and my parents who have been a source of encouragement and
inspiration to me throughout my life and for encouraging me to work hard and with full
honesty. To my brother, Anuj for supporting me in my determination to find a twilight of
hope in midst of difficulties. To my husband, Santosh for his patience, care, love, and
emotional support.
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Acknowledgments
There are a number of people whom I am indebted to for helping me write this thesis.
To God for bestowing the opportunity to work with the most wonderful advisor - Dr. N.
Sriranganathan (Dr. Nathan).
Dr Nathan, I am highly indebted to you for being so supportive and for helping me
determine my potential. Without you I could not have completed this work. Your teachings and
method of approach to scientific problems have helped me grow professionally. I will never
forget your teachings and will always remember, as taught by you, “to be ready to face challenges
as we are challenging Mother Nature”, “to not be afraid of repetition”, and “ to learn from
failure”. Your teachings made me vigilant and as a result I was able to extract meaningful
information and data from my experiments. You mean more than a mentor to me. Thanks a lot for
being so affectionate and caring.
Thank you Dr. Boyle for your guidance, teachings, and support. Without your help, I
would not have been able to write and present this work in an effective and productive manner. I
will always remember our insightful discussions in the CMMID corridors which were
instrumental in refining this work.
I am thankful to Dr. Riffle for teaching me more about chemistry. I appreciate you taking
a great interest in understanding the workings of the molecular biology of my study and providing
useful insights. I thank you for teaching me to always be ready and to continue to learn new
subjects even if they appear out of our own comfort zone.
I am thankful to Dr. Jortner who helped me stay on track and focused and in developing a
goal-oriented approach. Now, I understand the importance of planning and thinking about the
experiments as the chapters of a story book.
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I would like to thank Dr. Sheela Ramamoorthy for her guidance and support. I am very
grateful to Dr. Araceli Contreras for teaching me various techniques in BSL-3 and for bringing
out the much needed sincerity and punctuality in me. I am also grateful to Dr. M. Seleem for his
suggestions and support during my work.
I give very special thanks to Kay Carlson, my foster mother in the US. Without her
teachings and techniques, I would not have been able to complete this work. Her emotional
support played a key role during my thesis work as she always made me feel at home and at ease.
I am thankful to Mary Mainous for helping me with minimal media. I appreciate Nancy
Tenpenny and Alba Hall for their kind help. I am grateful to everyone in CMMID for keeping me
motivated. I thank all my colleagues Clifton, Deena, Gade, Lori and Parthiban, and for their help
during my thesis work and for providing meaningful suggestions while writing this work. Last but
not the least, I am also thankful to Ashish, Eva, Rajeev and for being great lab-mates. In the end,
I’m thankful to every person who has been directly and indirectly involved in enabling me to
complete my research.
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Table of Contents
Abstract ................................................................................................................. ii
Dedication ............................................................................................................ iii
Acknowledgments ................................................................................................ iv
Table of Contents .................................................................................................. vi
List of Figures: ................................................................................................... viii
List of Tables ........................................................................................................ ix
List of Abbreviations ..............................................................................................x
Chapter 1: Literature review ..................................................................................1
Iron: need and acquisition by intracellular pathogen Brucella spp. .........................1
Abstract ...................................................................................................................................1
Brucellosis and Brucella ..........................................................................................................2
Brucella: survival inside macrophage ......................................................................................3
Iron: Requirement of Host and Pathogen .................................................................................8
Regulation of iron in the host ..................................................................................................9
Bacterial strategies to steal iron from the host ........................................................................ 12
Brucella and Iron ................................................................................................................... 16
Iron acquisition by Brucella ................................................................................................... 20
Siderophores in Brucella ....................................................................................................... 21
Summary ............................................................................................................................... 25
Thesis introduction and objectives ........................................................................ 27
Chapter 2: ............................................................................................................. 29
Role of entF gene in the acquisition of iron by Brucella abortus 2308 ................. 29
Abstract ................................................................................................................................. 29
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Introduction ........................................................................................................................... 30
Material and Methods ............................................................................................................ 34
Bacterial strains, plasmids and oligonucleotides ............................................................................. 34
Culture media, chemicals and growth conditions ............................................................................ 34
Recombinant DNA methods .......................................................................................................... 35
Creation of deletion clone (BAN1) ................................................................................................. 35
Creation of complemented mutants BAN2A and BAN2B .............................................................. 38
Growth studies ............................................................................................................................... 42
In-vitro growth in iron minimal media…………………………………………………..42
In-vivo growth in macrophages…………………………………………………………..42
Results .................................................................................................................................. 43
Deletion and complementation of entF gene................................................................................... 43
Growth in minimal media .............................................................................................................. 47
Growth in the presence of iron chelator .......................................................................................... 49
Growth in iron minimal media supplemented with 0.1% erythritol ................................................. 51
Intracellular survival in J774A.1 macrophages ............................................................................... 54
Intracellular survival and growth in presence of iron chelator ......................................................... 54
Discussion ............................................................................................................................. 57
References............................................................................................................................. 68
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List of Figures:
Figure 1: Brucella survival inside macrophages. .........................................................................7
Figure 2: Regulation of iron inside macrophages. ...................................................................... 11
Figure 3: Common iron uptake systems in pathogenic bacteria. ................................................. 15
Figure 4: Diagram showing the erythritol metabolism pathway in B. abortus 2308.. .................. 19
Figure 5: Genetic organization of Brucella abortus 2308 entF-entCEBA-entD locus and the role
of known genes in the biosynthesis of 2, 3 dihydroxy benzoic acid (2, 3- DHBA).. ................... 24
Figure 6: Schematic diagram showing deletion cloning of entF gene using cre-lox methodology..
................................................................................................................................................. 37
Figure 7: Nucleotide sequence of the entF gene along with 300 base pair upstream, showing the
potential gene organization.. ...................................................................................................... 40
Figure 8: Schematic diagram showing the creation the two recombinant plasmids for
complementation of entF gene in BAN1 strain.. ........................................................................ 41
Figure 9: PCR results showing the difference in wild type, deletion mutant and complemented
strains.. ..................................................................................................................................... 45
Figure 10: Genetic complementation of erythritol-induced growth restriction of entF deletion
mutant (BAN1) by complemented clones, BAN2A and BAN2B. ............................................. 46
Figure 11: Comparative growth of entF deletion mutant B. abortus BAN1 and wild type B.
abortus 2308 in IMM.. .............................................................................................................. 48
Figure 12: Comparative growth of Brucella strains in presence of iron chelator EDDA.. ........... 50
Figure 13: Comparative growth of Brucella in presence of 0.1% erythritol. ............................... 52
Figure 14: Growth restriction of wild type B. abortus 2308 in the presence of erythritol.. .......... 53
Figure 15: Growth of wild type B. abortus and BAN1 in J774A.1 macrophages. ....................... 56
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List of Tables
Table 1. List of all the bacterial strains and plasmids ............................................................................. 65
Table 2. List of primers used; including the restriction site and the restriction enzyme ........................... 67
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List of Abbreviations
Amp Ampicillin
ATP Adenosine triphosphate
ADP Adenosine diphosphate
BCV Brucella containing vacuole
CFU Colony forming unit
Chl Chloromphenicol
DHBA Dihydroxy benzoic acid
DFA Deferroxime mesylate
DMEM Dulbeco's modified eagle's medium
EDDA Ethylenediamine-N.N'-diacetic acid
EDTA Ethylenediaminetetracetic acid
ER Endoplasmic reticulum
ery Erythritol
FeII Ferrous iron
FeIII Ferric iron
FeCl3 Ferric chloride
Fur Ferric iron uptake regulator
Gen Gentamicin
HPLC High performance liquid chromatography
IMM Iron minimal media
IL Interleukin
Kan Kanamycin
NRAMP Natural resistance associated macrophage proteins
OD Optical density
PCR Polymerase chain reaction
TLC Thin layer chromatography
TLR Toll like receptors
TSB Tryptic soy broth
TSA Tryptic soy agar
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Chapter 1: Literature review
Iron: need and acquisition by intracellular pathogen Brucella spp.
Abstract
Brucellosis caused by species of Brucella is a worldwide zoonosis affecting almost all
domestic animals, some aquatic species as well as human beings. Brucella being a facultative
intracellular pathogen like Mycobacterium spp. and Salmonella spp. has acquired ways to
survive inside the phagocytic cells. Besides their ability to prevent killing by phagocytic cells,
these bacteria are capable of acquiring essential nutrients from these cells. One such nutrient is
iron, which is an essential cofactor for many enzymes and is required for many metabolic
pathways. In order to avoid the possible toxicity and prevent microbial infections, the host tightly
regulates free iron internally. Host cellular transporters further reduce the availability of iron
inside phagocytic cells. To acquire iron from the host, pathogens have developed different
mechanisms, the most common one being the production of siderophores. The requirement of
siderophores for intracellular replication of Brucella is not very clear. There is some
experimental data that suggests that Brucella siderophores are somehow linked to erythritol
metabolism of the pathogen. Erythritol is an alcohol sugar that is found in abundance in ruminant
placental trophoblast cells. It has been shown that Brucella prefers erythritol to glucose and the
extensive replication in these trophoblasts leads to the pathogenesis of abortion due to B.
abortus. This may lead to the abortion storms in pregnant ruminants. A deletion mutant lacking
the siderophore, 2, 3- dihydroxy benzoic acid biosynthesis has been found to be incapable of
causing abortions, and is attenuated in the presence of erythritol. The role of the second Brucella
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siderophore, brucebactin still needs to be understood. This is a review of the current literature on
the siderophores of Brucella.
Brucellosis and Brucella
Brucellosis is one of the world’s most widespread zoonotic infections characterized by
abortion, infertility and chronic infections (31). The intracellular bacterial pathogen belonging to
genus Brucella infects almost all the species of animals and known to cause a febrile disease in
humans called “Undulant fever” or Malta fever” (138). Based on rRNA sequence comparison
Brucella is classified as α2- proteobacteria (108). Six species are currently known: B. abortus, B.
melitensis, B. suis, B. canis, B. neotomae, and B. ovis based on their biochemical finger print and
host preference for cattle, sheep and goat, pig, dog, desert rat and goat respectively (32).
The most pathogenic species worldwide is B. melitensis and is responsible for brucellosis
in caprines and ovines and cause severe infections in humans (28). Recently two Brucella
species have been isolated from marine mammals (B. ceti and B. pinnipedialis) and are reported
to be pathogenic to humans as well (155). More recently some other Brucella species have been
described and could join the group in near future (42, 146). The Centers for Disease Control and
Prevention (CDC) classifies B. abortus, B. melitensis and B. suis as “agents of bioterrorism” and
Bio-Safety-Level 3 (BSL3) organisms due to their ability to be readily spread through mucosal
surfaces; moreover they can be made drug resistant and used to infect humans (59). The
infectious dose for human is very low and lies between 10-100 organisms (115) and can be
transmitted through direct contact with tissues from infected animals, ingestion of infected meat,
milk and its products.
Brucellae are Gram-negative, coccobacilli and classified as facultative intracellular
bacterial pathogens (23) that predominantly replicate in two cell types, macrophages and
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placental trophoblasts (6). Other then these two major cell types, the pathogen can also infect
other cell types like NIH 3T3 (murine fibroblasts cells), MDBK (Madin-Darby bovine kidney
cells), BHK (baby hamster kidney cells), HeLA (human epitheloid cells) and Vero (green
monkey kidney cells) (122). The Brucella genome is constituted by two chromosomes, 2.05 and
1.15 Mb, and the wild type Brucella does not contain any naturally occurring plasmid (103).
Brucella does not appear to have the classical virulence factors like exotoxins, cytolysins,
capsules, pili, fimbriae or virulence plasmids (107). However, the species does have genes that
are important for the host-pathogen interaction, and produces factors that have an impact on
phagocytosis, phagolysosomal fusion, cytokine secretion and apoptosis (81). Since it is closely
related to nonpathogenic the soil bacterium Ochrobactrum (161), it will be interesting to learn
how Brucella has acquired some of these pathogenic mechanisms that allow survival inside the
cells of the body’s immune system. The other aspect that needs extensive studies is to understand
how genetically similar species of Brucella have such distinct preferred hosts. Brucella causes a
chronic persistent infection in certain hosts and is easily transmitted to others of same host
species (67). Also noteworthy is the observation that Brucella prefers erythritol, a four carbon
sugar, as a carbon source over glucose (154). This fact is important as erythritol is abundant in
the ruminant placental trophoblast which is the site of predilection for Brucella infection. Rapid
multiplication of pathogens in trophoblast lead to the induction of abortions (168). Why Brucella
prefers erythritol and what are the other factors that contribute to the pathogenesis and
transmission of infection from one cell type to another in the host still needs to be resolved.
Brucella: survival inside macrophage
As a facultative intracellular pathogen, Brucella survives and replicates inside both
professional (macrophages and monocytes) and non-professional (epithelial cells and fibroblasts)
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phagocytic cells (93). Apart from tropism for the reproductive organs, it can survive inside a
variety of cell types at different stages of infection. After entering the host body the pathogen
does not cause neutrophilia and induces low levels of TNF-α, IL-1β and IL-6 production (10).
Thus it has the ability to escape toll-like receptors (TLR4 and TLR5) recognition and signaling
(160). After escaping the initial epithelial cells the pathogen enters the macrophages through
special structures known as lipid rafts or microdomains; Brucella LPS (lipopolysaccharide) is
essential for this interaction (111). Further the pathogen resists the acidic environment inside the
phagosomes and uses the low pH as a signal for the expression of virulence factors necessary for
intracellular proliferation (123). Inside the phagosomes Brucella induces changes on the surface
of phagosome (P) and blocks fusion with lysosomes (L) (110) (Figure 1). Although the majority
of the Brucella are killed (25), the ones that survive are those that avoid P-L fusion. This
inhibition is shown to be somehow related to the synthesis of cyclic β 1-2-glucans synthesized by
Brucella (8). After preventing P-L fusion the Brucella containing vacuole (BCV) migrates
towards endoplasmic reticulum (ER). Fusion between the Brucella containing vacuole (BCV)
and endoplasmic reticulum depends on the expression of type IV secretion system encoded by
virB operon (17). BCVs associated with ER acquire membranes (121) and essential nutrients
from nearby ER and survive and replicate inside the vacuole termed as “replisome” (25). Other
factors that are found to play a role in virulence of Brucella include: Cu/Zn superoxide-
dismutase (SodC) (64), catalase (85), base excision repair (BER), urease, BvrS/BvrR two-
component regulatory system, alkyl hyperoxide reductase, cytochrome oxidase, nitric oxide
reductase, Brucella virulence factor A (BvfA) and phophotidylcholine (150). Separate studies
have shown the necessity of these factors, but the whole cascade of gene expression and the role
of their products in virulence are not clear.
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Moreover, studies are needed to understand the membrane properties and internal
environment of BCV. It has been known that Brucella remains in BCV, an early endosomal
compartment, soon after its entry into the cell. The membrane of the BCV has been found to
have early endosomal marker (transferrin receptors), GTP binding protein (Rab5) and early
endosomal antigen (EEIA) (47). It has been shown that the BCV does not fuse with late
endosomes or lysosomes. But confocal microscopy shows that BCV interacts with late
endosomes as well as with the lysosomes and transiently acquire markers like CD63, RB7 and
RILP (158). These results explain the presence of lysosomal associated membrane protein
(LAMP1) on BCV as well as the acquisition of acidic environment for the expression of virB.
However the question of how Brucella survives after BCV interacts with lysosome still remains.
Finally, BCV had been shown to acquire endoplasmic markers like calreticulin and calnexin and
become associated with endoplasmic reticulum (ER); this has been confirmed by the presence of
luminal ER marker glucose 6-phosphatase (25, 58). After establishing this niche, the pathogen
multiplies inside replisomes. However, not much is known about the internal environment of
replisomes. It has been predicted that Brucella survives in a scarcity of nutrients (134) but how
the pathogen acquires nutrients from host needs to be examined. One such essential nutrient for
the intracellular survival and replication is iron that is tightly bound in host tissues and not
readily available inside the vacuoles containing Brucella. Therefore a study of iron metabolism
in Brucella would help us to understand the activity of this pathogen inside the vacuoles and
thereby help discern pathogenesis. Not much is known about the intracellular trafficking of
Brucella in trophoblast but it has been shown that pathogen replicates in intracellular
compartments associated with ER in trophoblast as well (6, 100). The only fact known about the
trophoblast related to Brucella is the abundance of erythritol and the pathogen’s preference for
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the sugar (154). However, in trophoblasts the function of the BCV in trophoblasts needs to be
more completely elucidated.
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Figure 1: Brucella survival inside macrophages: The pathogens enter through lipid rafts and then
prevent phagosomal-lysosomal fusion and migrate towards endoplasmic reticulum (ER). After
acquiring membranes from ER, Brucella replicates inside special niche, replisomes.
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Iron: Requirement of Host and Pathogen
Iron is required for survival and growth by almost all the prokaryotes and eukaryotes
except Borrelia burgdorferi (124) and lactic acid bacteria like Lactobacillus plantarum (166)
where manganese and cobalt replace iron. Iron concentration in vertebrate serum is estimated to
be 10-24
M (131) and the cytoplasmic iron concentration required for the bacterial growth is 10-6
M (7). The importance of iron lies in the fact that it serves as the catalytic center of enzymes for
redox reaction (162). These enzymes are involved in cellular process like electron transport,
oxygen transport, synthesis of DNA, amino acids and nucleotides and peroxide reaction.
Moreover in the host the iron has been associated with the innate immunity as it is needed for
respiratory burst and inducible nitric oxide synthase (iNOS) mediated reactive nitrogen
intermediates (RNI) production (11, 30, 144). Iron availability also affects the adaptive immune
responses. For example, iron deficiency down regulates the T-cell response in several
experimental models (71, 90).
The importance of iron for pathogens can be estimated from the fact that ovotransferrin
present in egg white suppresses growth of wide range of microbes because of its capability to
chelate iron (143). It has been shown that iron is necessary for the pathogenicity of bacteria,
viruses and fungi. Iron is crucial for microbial replication, electron transport, glycolysis, DNA
synthesis and defense against reactive oxygen intermediates (ROI) (165). Iron overload in the
host is believed to make them more susceptible to the pathogens: more iron in the natural diet of
African adults makes them more susceptible for tuberculosis (61). Human Immunodeficiency
Virus (HIV) infection is associated with the disturbances in host iron metabolism leading to
enhanced deposition of iron in bone marrow and increased opportunistic infections (43). A
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hereditary disease condition known as haemochromatosis (tissue iron overload) caused by the
mutation in the HFE (haemochromatosis) gene in humans is associated with increased incidences
of salmonellosis and yersiniosis (105, 120).
Regulation of iron in the host
Iron is one of the most abundant elements on earth, but due to the aerobic atmosphere
of our planet it is converted and available as oxyhydroxide polymers (112). Moreover, the
bioavailability of this molecule is restricted by its low solubility in water and the tendency to
make complexes with a great number of organic molecules (63). The oxidised ferric (FeIII) form
is insoluble while the reduced ferrous (FeII) form participates in Fenton reaction generating
hydroxyl free radicals, which in turn further oxidizes and break apart organic molecules, and
thus are toxic to the cells (162). For these reasons iron homeostasis is strictly regulated and
practically very little free iron exists in the body. At physiological pH (~7.0) iron remains in the
ferric state while in acidic environments it is in the ferrous form. Nearly 80% of bodily iron is
found in hemoglobin within erythrocytes, when senescent are phagocytosed by macrophages and
iron is recycled and exported to plasma (153) (Figure 2). In the body iron is found either in
bound form (intracellularly as heme, iron-sulphur proteins or ferritins) and very little is
conjugated to proteins like lactoferrin (secretions and body surface), transferrin (plasma, lymph,
cerebrospinal fluid, serum) (119). Lactoferrin and transferrin have high affinities for the ferric
compared to the ferrous form of iron. Hepcidins, which are peptides constituting of 20-25 amino
acids, are trimmed from larger precursor proteins in the liver, play important role in regulating
the iron homeostasis (62). Moreover the regulatory process to decrease the iron level in the body
is activated during the time of infection. For intracellular pathogens the iron restriction is
stronger as interferon-gamma (IFN-γ) activated macrophages reduce their transferrin binding
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receptors (75, 167). Recently discovered natural resistance associated macrophage proteins
(Nramp1 and Nramp2) further decrease the iron inside the phagosome or late endosomes by
mediating the active efflux from the site (75, 173) (Figure 2). Even with so much tight
regulation of iron availability in the host, the pathogens have developed ways to acquire iron and
use it for their survival and growth. However, iron deficiency is detrimental to the host and thus
the host cannot reduce the iron in the body below certain level without affecting itself.
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Figure 2: Regulation of iron inside macrophages. Macrophages acquire iron by scavenging
transferrin and erythrocytes. Iron is released in the cytoplasm and remains in bound form with
ferritin. Proteins like Nramp1 and Nramp2 sequester iron from the late endosomal compartments
to restrict the growth of pathogens.
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Bacterial strategies to steal iron from the host
There are mainly two ways in which pathogens acquire iron from the host: The direct
method, which involves the destruction of the iron containing compounds in the host or the
synthesis of surface receptors on their cell membrane to bind these compounds (Figure 3). The
direct sources include free iron, transferrin, lactoferrin, ferritins, heme source like hemoglobin,
haptoglobin-hemoglobin, hemopexin, albumin and some other iron conjugated proteins and
glycoproteins (162). Most of the hemolytic bacteria that reside in the blood stream breakdown
the iron containing compounds or obtain iron by the cleavage of hemoglobin (129).
Microorganisms that acquire iron in this way include Yersinia, Shigella, Vibrio, Escherichia and
Haemophilus (12, 102). Y. pestis and some other enterobacteria have been shown to have a heme
uptake system and may use the entire hemoglobin complex as the source of iron (135). In other
cases pathogens have receptors on their surface to bind to lactoferrin and transferrin including
Neisseria (33), Haemophilus influenzae (148) and Helicobacter pylori (49). This type of iron
uptake needs the removal of iron at the external surface from lactoferrin and transferrin as these
proteins are too large to pass through the bacterial cell envelop (89) (Figure 3). Receptor
mediated iron acquisition is highly species specific. For example, Neisseria meningitis can
develop receptors to bind transferrin in humans but not in non-human primates (73). Likewise,
Actinobacillus pleuropnemoniae can only cause disease in hogs as the pathogen has receptors to
bind hog transferrin (72). Other less efficient and specific strategies are seen in intracellular
pathogen Listeria monocytogenes and involves the production of soluble reductase that can
reduce and release iron from transferrin (34). Corynebacterium diptheriae uses a heme
oxygenase to degrade heme (145). So far the only organism that showed the potential to use
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holoferritin as an iron source is Neisseria meningitides (91). As seen in the cases of intracellular
pathogens the main source of direct iron is ferritin and is readily available for the bacteria living
in cytoplasm. Alternatively, ferric dicitrate is another source of iron for intracellular pathogens as
it is an important transitional chelator of internalized iron (114). This system involves a TonB-
dependent transporter FecA encoded by fec operon and is negatively regulated by Fur in E. coli
(125) and Bacillus spp (77). Intracellular pathogens like Salmonella and Mycobacterium encode
host Nramp homologues, DmntH (97) and Mramp (2) respectively which can mimic the natural
host iron binding moieties and bind to iron.
The indirect method involves the production and release of iron chelators called
“siderophores” (Figure 3). The importance of siderophores for iron acquisition has been shown
by the loss of virulence in siderophore deficient mutants of E. coli in mice (170) and Vibrio
anguillarum in fish (36). Siderophore production is confined to bacteria and fungi and is not
present in plants or animals (112). Siderophores are low molecular weight molecules and have
high affinity for FeIII (37). Depending on the kind of oxygen moiety coordinating the FeIII,
siderophores are of three types: catecholates, hydroxymates and carboxylate (104). Mixed type
siderophores are also common with two groups in the single molecule. E. coli, Salmonella
enterica, Shigella dysenteriae and many other Gram negative bacteria possess a 24 kb gene
cluster, encoding almost thirteen proteins which are required for the synthesis and transport of
the siderophore “enterobactin”(131). The basic substrate for the synthesis is chorismate which is
converted to 2, 3-dihydroxy benzoic acid (2, 3-DHBA) with the action of EntC, EntB, EntA and
EntD proteins (137). 2, 3- DHBA and serine act as substrate for the synthesis of enterobactin (2,
3-dihydroxybenzoylserine trilactone) and the conversion requires the action of EntE, EntB and
EntF proteins (66). Most bacteria make two kinds of siderophores. Because enterobactin is not
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very effective inside the animals due to its hydrophobicity, most of the enteric bacteria make
another siderophore, aerobactin which is much more efficient in-vivo (26). Mycobacterium,
Nocardiae and Rhodococci are capable of producing membrane associated mycobactin and
extracellular siderophores, carboxymycobactin and exochelin (130). Pseudomonades produce
fluorescent siderophores known as pseudobactins and pyoverdins (21). Siderophore detection
can be accomplished after growing organism in iron deficient media or by adding strong iron
chelators to the media that will make iron unavailable, and force bacteria to produce siderophore.
Siderophore detection in the supernatants could be achieved by chemical assays like chrome
azurol S assay (149) or techniques like thin layer chromatography (TLC) (174) and high
performance liquid chromatography (HPLC) (88). Siderophore are thermodynamically stable
molecules with affinity for other metal ions as well like gallium. Siderophores have been also
viewed as the tool for drug delivery against some complex diseases like tuberculosis (106).
Iron regulation in bacteria is equally important to iron acquisition as excess
intracellular iron can damage the bacterial cell. Most bacteria posses ferric iron uptake regulator
(Fur) or a transcriptional repressor with fur like activity and regulate the biosynthesis and uptake
of siderophores (50). To regulate the siderophore biosynthesis another type of regulation is
present that uses the end product of the siderophore as the coactivator for the induction of
siderophore biosynthesis genes. These activators are AraC like present in many bacteria like
Bordetella spp. (13) and Pseudomonas aeruginosa (79). Thus both host and pathogen have
their own strategies to acquire iron and also to regulate its intracellular concentration since
this metal is required and regulated by both.
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Figure 3: Common iron uptake systems in pathogenic bacteria. The direct mechanisms include
receptors mediated uptake involving the uptake of heme and iron binding proteins, transferrin.
Indirect way to acquire iron is through the biosynthesis of siderophore that binds to iron and
bring it to the bacterial cell.
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Brucella and Iron
Brucella also needs iron for its survival and growth (52, 163). There is not enough data to
prove that the iron is required for Brucella virulence and infectivity, other than the need of iron
for electron transport, glycolysis and DNA replication. There is a direct or indirect relation
between iron and Brucella virulence factors such as lipopolysaccharide (136), superoxide
dismutase (57, 175), catalase (45) and urease (78). Brucella cobG gene encodes an enzyme,
involved in the biosynthesis of cobalamin (vitamin B12), which contains a Fe-S center (147).
Brucella has different and poorly understood systems to acquire iron from the host. All of them
are tightly regulated as excessive iron can enhance the chances of oxidative damage, which is the
primary mechanism employed by phagocytes to kill Brucella. Compared to mutants of the
virulence factors such as diptheria toxin (164) and membrane protein in Vibrio (69), no Brucella
mutants have been described, which are attenuated under iron deficient conditions. The only
deletion mutant that showed evidence of changes from wild type was a siderophore mutant
(BHB1), which could not produce 2, 3- DHBA and did not cause abortions in pregnant
ruminants compared to wild type (16). But lack of siderophores did not affect the infectivity in
case of mice (15). This effect could be either because mice are not definitive host for Brucella or
that siderophores are needed for iron acquisition only in case of pregnant animals.
There is an unresolved relationship between availability of iron and erythritol metabolism
by Brucella (14). One of the major breakthroughs in Brucella research came when chemical
basis of Brucella pathogenicity was related to a four carbon alcohol sugar, erythritol, by Smith et
al. (154). Erythritol is in abundance in trophoblast cells and fetal tissue and was regarded as the
reason for the predication of Brucella for these sites (169). Later, the whole operon (ery)
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containing ery A,B,C and D (Figure 4) involved in erythritol metabolism was identified (139).
The first step in erythritol metabolism requires activation of erythritol by phosphorylation with
ATP (156). Thus energy is first required to metabolize erythritol. Since then, several ery
deletion mutants including the vaccine strain B-19 (S-19), that has a partial deletion of the gene
encoding the erythritol degradation enzyme, D-erythrulose 1-phosphate dehydrogenase (141,
157), has been studied.
All the ery deletion mutants and S-19 in particular were found to be sensitive to
erythritol and this inhibition was attributed to the decreased levels of ATP as well as the
accumulation of erythrulose 1-phosphate which could be toxic to Brucella (20, 156). The eryC
deletion mutant was found to be attenuated in its ability to grow inside macrophages as well but
there was no satisfactory explanation given (22). The only explanation could be the scarcity of
available iron inside macrophage, which is needed for an enzyme, 3-keto-L-erythrose 4-
phosphate dehydrogenase, involved in erythritol catabolism (156). However, this association
still needs to be proved experimentally
Another interesting result relating erythritol metabolism and iron acquisition was
observed when the growth of a siderophore mutant was compared with wild type strain in
trophoblast in the presence of 0.1% erythritol (116). The deletion mutant was attenuated
compared to wild type strain and the lack of siderophore-mediated iron acquisition in mutant
strain was considered as the reason.
The above two observations need further confirmations as Brucella can acquire iron
through alternative routes as well (98). All these iron acquisition pathways are tightly regulated
and are poorly understood. But evidence has shown the potential relationship between erythritol
metabolism and need of iron in Brucella spp.
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Further, understanding the need for the acquisition of iron by Brucella could help us to
understand two things, first how the pathogen acquires important nutrients inside the BCVs and
second, how the availability of these nutrients affects the pathogenicity of the organism.
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Figure 4: Diagram showing the erythritol metabolism pathway in B. abortus 2308. Also,
included are the enzymes involved in the conversion of erythritol to dihydroxy acetone
phosphate including 3-keto-erythrose 4- PO4 dehydrogenase, an iron coupled enzyme.
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Iron acquisition by Brucella
Other than siderophores Brucella can use free iron and heme as sources of iron. Although
heme uptake is not essential for Brucella growth in culture media, its utilization could be an
effective way to acquire iron. Macrophages and trophoblasts, the cells in which Brucella
survives and replicates, phagocytose and degrade erythrocytes and thus liberate heme (6, 18)
(Figure 2). B. melitensis 16 M and B .suis 1190 genomes have open reading frames (ORFs)
encoding receptors similar to ShuA in Shigella dysenteriae (172), and ChuA in case of E. coli
(159) which are responsible for the transport of heme across the membranes. The gene
homologue is named BhuA (Brucella heme uptake). BhuA is necessary to maintain a chronic
infection in mice due to B. abortus (117). Recently an outer membrane protein Omp31 has been
shown as the heme binding protein in Brucella similar to Bartonella (46). Heme itself is required
for many physiological process, it acts as prosthetic group for hydroxylation, electron transport
and sensing the diatomic gases (113). Unlike Bartonella, Brucella can synthesize heme from the
precursor δ-aminolaevulinic acid through six steps to form protoporphyrin IX which is finally
conjugated to ferrous ion catalyzed by the enzyme ferrochelatase (HemH) to give the heme
molecule. The absence of HemH affects the virulence of B. abortus and its intracellular survival
(3). During the iron scarce conditions, synthesis of heme is shown to be regulated by irr gene
similar to Bradyrhizbium japonicum and Rhizobium leguminosarum (98). Irr belongs to the Fur
(ferric uptake regulator) family that are involved in metal dependent regulations in bacteria (50)
and the irr gene is present in α-2 group of proteobacteria only.
Other potential sources of iron for Brucella could be ferric dicitrate as citrate binds to
the iron in the cytoplasm. But so far no gene for FecA analogue has been identified in Brucella.
As mentioned earlier iron remains in ferrous form (FeII) at acidic pH, thus there are chances that
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Brucella could use ferrous iron inside the phagosome that has lower pH (87) during early
infection. There is very little information in the Brucella literature regarding the last two iron
sources and this remains to be explored experimentally.
Siderophores in Brucella
Similar to most other bacteria , Brucella also produces two kinds of siderophores, 2, 3
dihydroxy benzoic acid (2, 3-DHBA) (96) and brucebactin (70). But unlike many other
pathogens experimental evidence shows that Brucella is unable to use more complex
polycatechols like enterochelin or hydroxamate siderophores (96). As described earlier for
enteric bacteria, Brucella possess an operon for the biosynthesis of 2, 3-DHBA. The operon is
comprised of four genes entC, E, B, and A which are also known as dhbC, E, B, and A (16)
(Figure 5). The entE gene is a homologue of the E. coli entE, a gene encoding an enzyme
involved in the conversion of 2, 3-DHBA into the more complex catechol siderophore
enterochelin (65). A homologue of E. coli entD (entD in Brucella) and Vibrio cholerae vibH
(known as entF in Brucella) has been located in the flanking regions of entCEBA operon (16)
(Figure 5). The importance of siderophores for iron acquisition in Brucella has been tested by
creating siderophore mutants. It has been shown that the isogenic dhbC deletion mutant of a
virulent B. abortus 2308 failed to produce 2,3-DHBA, but it did not affect the replication of the
mutant in cultured naïve and IFN-γ activated peritoneal macrophages from BALB/c mice when
compared to wild type Brucella (15). Moreover, the mutation had only a minimal effect on the
intracellular survival, when compared to the wild type strain. Establishment of chronic infection
which is characteristic of Brucella spp was barely affected for the dhbC mutant compared to wild
type strain in BALB/c mice. So it was thought that siderophores are not a major method of the
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iron acquisition in case of Brucella. However the same group of scientists later showed that B.
abortus dhbC mutant behaved very differently in case of pregnant animals (14). When compared
to wild type strain, the mutant was remarkably attenuated relative to the induction of abortions.
A possible clue for this was gained after in-vitro studies with the mutant. When the mutant was
grown in iron limiting media in which the erythritol was the source of carbon, the strain showed
slow and restricted growth compared to wild type. This restriction was not present when mutant
was grown in iron limiting media with sources of carbon other than erythritol. This observation
suggests that Brucella needs iron and may thus require siderophores when it has to utilize
erythritol as a carbon source such as that is found in the placental trophoblasts.
Although it has been reported that Brucella produces a second siderophore, brucebactin,
along with DHBA, this siderophore has never been characterized. But later through thin layer
chromatography to screen for siderophores, a Tn5 mutant of B. abortus 2308 was compared to
wild type grown in iron deficient media (70). The mutant (BAM41) of B. abortus 2308 could not
make the second catechol in response to the iron deprivation, but overproduced 2, 3-DHBA
under the same growth condition. The site of the Tn5 insertion in the BAM41 strain is within the
gene (entF) proximal to the dhbCEBA but was predicted to be transcribed in the opposite
direction (Figure 5). This gene is a homologue of vibH, a gene involved in the conversion of 2, 3
DHBA to a more complex siderophore, vibriobactin, in V. cholerae (82). When tested for the
growth in macrophages the BAM41 mutant did not show any difference compared to wild type.
Expression of siderophore genes and siderophore synthesis has been shown to be
positively regulated by irr, the same gene that negatively regulates the heme biosynthesis (99).
Using an electrophoretic mobility shift assay, the irr encoded protein was shown to bind to the
upstream region of operon involved in the biosynthesis of the catecholic siderophore in Brucella.
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In one case the mutation in irr resulted in decreased synthesis of siderophores, both DHBA and
brucebactin. In contrast the mutant showed higher levels of heme, catalase activity and resistance
to hydrogen peroxide than the wild type strain. Similar to other siderophore mutants, the irr
mutant did not show any difference in it ability for intracellular replication and pathogenicity in
mouse model. Another study shows that similar to Bordetella bronchiseptica and Sinorhizobium
meliloti, Brucella AraC like transcriptional regulator (DhbR) regulates the genes for siderophore
synthesis and transport (4).
The mechanisms by which siderophores enter the cell membrane of Brucella have not
been extensively investigated. Non-induction of any outer membrane protein expression under
low iron conditions suggested that DHBA uses a receptor independent pathway or some
unknown receptors (96). But recently through the transposon mutagenesis it has been shown that
the Ton protein complex and an ABC transporter are involved in the transport of FeIII-DHBA in
Brucella (40). Siderophores appear to be important for iron acquisition in Brucella, but none of
the siderophore mutants were attenuated with regard to their ability to survive intracellularly or
in their infection profile in mice. The only difference has been there with respect to infection
leading to abortion in pregnant animals. So, one can hypothesize that siderophores are essential
during erythritol metabolism but comparative studies using the siderophore mutants under
different conditions are needed to accurately test this hypothesis.
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Brucebactin
entC entE entB entA entDentF
TATA BOX
Orientation for transcription
entCEBAoperon
Chorismate
Isochorismate synthase
Isochorismate
Isochromatase
di DHBA
Dehydrogenase
2,3-DHBA
EntD, EntE, EntF
?
Figure 5: Genetic organization of Brucella abortus 2308 entF-entCEBA-entD locus and the role of
known genes in the biosynthesis of 2, 3 dihydroxy benzoic acid (2, 3- DHBA). Also, showing the
orientation of transcription for different genes. Pathway for the biosynthesis of brucebactin is
still unresolved.
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Summary
Understanding the pathogenesis of bacterial diseases opens novel portals to develop
control measures of infection through vaccine and drug therapy. Essential factors that are needed
for the survival and replication of the pathogen could be targeted to control the pathogen. But in
case of Brucella spp., understanding the cascade of pathogenic events has been a challenge for
more than a century.
Brucellosis imposes a huge economic burden on the developing world and has a potential
to be used as a biothreat. In fact, B. suis was one of the first weaponised pathogens developed by
the U.S. military (118). Brucella has shown the capability to use cells of immune systems as
their replicative niche. This in turn allows the pathogen to evade the immune system and resist
the killing mechanism. But how Brucella accomplishes all this is still not completely understood.
Looking at some of the simple mechanisms such as how this pathogen acquires nutrients from
the host could help to understand the host-pathogen relationship.
Essential nutrients like iron are absolutely necessary for basic life process but are very
tightly regulated in pathogen and host. Controlling the free iron in the host is a way to control
pathogen. But bacterial pathogens have acquired ways to obtain iron from host. The most
common way is by producing natural iron chelators, siderophores. Brucella also produces two
siderophores, 2, 3- dihydroxy benzoic acid and brucebactin. The interest in siderophore role of
iron acquisition in Brucella came from the fact that blocking the siderophore (2, 3- DHBA)
mediated iron acquisition affects the normal pathogenesis in pregnant hosts. Limitations in the
siderophore mediated iron acquisition causes growth inhibition of pathogen in the presence of
erythritol.
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Erythritol is a four carbon alcohol sugar and has been associated with the virulence of
Brucella and its predilection for the trophoblast cells. It is paradoxical that the lack of
siderophores does not make any difference towards the chronic infection in mice and survival
inside the macrophages, where iron is not readily available. Brucebactin, which has been
discovered through a Tn5 insertion in entF gene in B. abortus 2308, is predicted as stronger
siderophore compared to 2, 3- DHBA. But there has been no relation established so far between
brucebactin biosynthesis and erythritol metabolism by B. abortus 2308. Thus the focus of this
thesis is to determine the relationship between entF gene, iron acquisition and erythritol
metabolism in Brucella.
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Thesis introduction and objectives
Brucella spp are Gram-negative facultative aerobic intracellular bacteria named after the
discoverer Sir David Bruce (68). On the basis of host preference, pathogenicity, metabolism and
genotyping data the genus has been divided into 6 species and 17 biovars, as revised in 2003
(67). Currently six recognized species are B. melitensis, B. abortus, B. canis, B. suis, B.
neotomae and B. ovis. With the discovery of new marine Brucella species (B. ceti and B.
pinnipedialis) the genus will expand further in near future (29, 41, 55). Brucella causes
Brucellosis in almost all domestic species of animals and Undulant or Malta fever in humans
(31). Although the genome sequences of at least 10 Brucella species are available (48, 76, 118)
the genes that are involved in virulence or pathogenesis are not fully understood. Moreover it is
not clear why different species of this genus with high homology of their genomes (19, 60) have
different host preferences.
B. abortus 2308 primarily affects cattle and mainly infects two host cell types:
macrophages and trophoblasts (5, 100). It causes abortions in pregnant ruminants because of its
affinity for a four carbon sugar called erythritol, which is abundant in trophoblasts of the
placenta (154) . Recently published genomic comparison between vaccine strain S19 and
virulent strain B. abortus has shown that there are 24 genes that are different between them. They
could be involved in pathogenesis, but experimental confirmation of this is not available (35).
The literature review on Brucella siderophores shows that the siderophore lacking mutant does
not cause abortion in pregnant ruminant hosts (14). The entC deletion mutant of B. abortus 2308,
which could not produce siderophores, was found to be attenuated when grown in the presence
of erythritol in culture media as well as in macrophages and trophoblasts under iron limiting
conditions compared to wild type strain (116).
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Siderophores are iron chelators, produced by almost all pathogenic bacterial species
under iron limiting conditions (130). These molecules are capable of sequestering iron from iron
binding proteins such as transferrin and lactoferrin of the host (104). Brucella species survive
and replicates inside macrophages, where essential nutrients like iron are limited (87, 134). Thus
the study of factors that contribute to its intracellular survival and replication are essential to
understand the host-pathogen interaction. Brucella makes two types of siderophores, 2,3 –
dihydroxy benzoic acid (96) and brucebactin (70). The relation between erythritol metabolism
and Brucella siderophore has been shown for the simple siderophore 2, 3-DHBA, but no such
study has been reported for the other complex siderophore “brucebactin.”
The purpose of the present study was to determine the role of entF gene in the acquisition of iron
from its environment by B. abortus 2308. The entF gene was previously found to be involved in
the biosynthesis of brucebactin (70). The following are the specific objectives for the thesis
research:
1) Determine the role of entF gene in iron acquisition by B. abortus 2308 under in vitro as
well as in cell cultures.
2) Determine the effect of deletion of the entF gene on the metabolism of erythritol by B.
abortus 2308
3) Determine the effect of complementation of deleted entF gene in B. abortus 2308.
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Chapter 2:
Role of entF gene in the acquisition of iron by Brucella abortus 2308
Abstract
Almost all pathogenic bacteria require iron as an essential nutrient. As free iron in body
cells and body fluid of the host are highly regulated, pathogens have developed ways to acquire
iron from host. Enterobactin is a low molecular weight iron chelating molecule (siderophore)
produced by E. coli. The product of four genes, entB entD, entE and entF, are required to
assemble this cyclic trimer by a mechanism similar to peptide antibiotic synthesis. 2, 3-
dihydroxybenzoic acid (2, 3-DHBA) is the precursor used in the biosynthesis of enterobactin and
also acts as a siderophore for Brucella. Previous studies in Brucella have shown that 2, 3- DHBA
is a potential siderophore and is associated with erythritol utilization. It is also associated with
wild type virulence in pregnant ruminants leading to abortion. Brucella has been shown to
produce another siderophore called “brucebactin” through an unknown mechanism. Similar to E.
coli, Brucella also possesses entD, entE, entA and entB genes, whose specific roles are still
unknown. The entF gene in Brucella is homologous with vibH gene of Vibrio cholerae which is
involved in vibriobactin biosynthesis. Employing the cre-lox methodology, a ∆entF deletion
mutant (BAN1) was created from wild type B. abortus 2308. Results of polymerase chain
reaction (PCR) show that there is a deletion of 497 base pairs in entF gene. The entF deletion
mutant showed significant growth attenuation in iron minimal media in comparison to the wild
type strain. Attenuation was enhanced in the presence of chemical iron chelator and erythritol. In
contrast to iron minimal media, in J774A.1 macrophages, there was no significant difference in
survival and growth rate of the BAN1 mutant compared to the wild type strain. Using a software
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programme, a promoter region was detected upstream of the entF gene. To complement the
deletion and to analyze the effect of homologous (entF promoter) compared to heterologous
(groE) Brucella promoter, the entF gene was cloned in the expression vector and transformed
into the deletion mutant. The entF gene complemented the entF deletion mutant and partially
restored the growth in iron minima media supplemented with 0.05% erythritol. These results
suggest a role of the entF gene in iron acquisition by Brucella.
Introduction
Iron is the second most abundant metal on earth (27) and throughout evolution most
organisms have evolved or acquired iron dependent enzymes that are involved in the essential
life processes including electron transport and glycolysis (162). Although iron is abundant in
the environment, it is not readily available inside the infected or uninfected host (119). The host
limits the availability of free iron to prevent either oxidative damage to itself or replication of
pathogens. Most of the iron in the host is present in the form of hemoglobin in red blood cells
and the rest is in bound form including proteins such as transferrin, lactoferrin and ferritin (119).
To get iron from iron binding proteins, pathogens have developed special mechanisms. A
common mechanism is the production of strong iron chelator called siderophores (129). These
are low molecular weight molecules with high affinity for FeIII (112). Macrophages reduce the
cytoplasmic iron through proteins such as natural resistance associated macrophage protein
(Nramp1 and Nramp2) (74) (Figure 2). Thus limitation of iron is more notable for intracellular
pathogens such as Brucella spp. and Mycobacterium spp. replicating in macrophages.
Mycobacterium has been shown to produce the Nramp family of proteins that help in the
acquisition of iron by the pathogen (2) but no such protein has been shown yet in case of
Brucella.
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Brucella spp are a Gram-negative facultative intracellular pathogens. Six species has
been identified so far namely B. abortus, B. melitensis, B. suis, B. canis, B. neotomae, and B. ovis
that preferentially infect cattle, sheep, pigs, dog, wild rats and goats respectively (31). Of these
B. abortus, B. melitensis and B. suis are highly zoonotic in nature and can be transmitted to
humans (127). Two Brucella species have been isolated from marine mammals (155).
Brucellosis has been controlled in much of the developed world using surveillance, test and
slaughter of sero-positive animals and calfhood vaccination. But the disease is still endemic in
many developing countries and recently it is re-emerging as a significant travel-related disease
(101).
Brucella is a bacterial pathogen with no classic virulence factors like LPS, endotoxins,
exotoxins, fimbriae, capsules, plasmids, lysogenic phage, etc., but is capable of causing acute
and chronic disease (107). Brucella resists the killing inside macrophages by preventing
phagosomal- lysosomal fusion (Figure 1) through a mechanism which is not well understood
(81). Later, the pathogen reaches its replicative niche, Brucella containing vacuoles (BCVs) and
“replisomes”, and acquire membranes and nutrients from endoplasmic reticulum. The pathogen
is capable of surviving and replicating inside these vacuoles under nutrient deficient conditions
(134). There are several factors that have been demonstrated to be involved in Brucella
pathogenicity, but the whole mechanism is not well described or understood. The environment
inside BCVs is not completely known and mechanisms through which pathogen acquires all the
essential nutrients inside these vacuoles need to be studied in more detail.
B. abortus 2308 primarily affects cattle and is responsible for abortion storms in herd. A
four carbon sugar, erythritol, which is abundant in bovine trophoblast epithelial cells of the
placenta is believed to be the reason behind the nidation of this bacterium (154). This can be
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further substantiated by the fact that vaccine B. abortus strain S19 (B19), which lacks the fully
functional ery operon (Figure 4) that expresses the proteins required for erythritol metabolism,
does not cause abortion in cattle (35, 141). At the time of pregnancy this pathogen appears to
migrate to the trophoblast epithelial cells from the maternal circulation and rapidly multiplies
there (100). By the time of third trimester, the pathogen replicates to such an extent that it causes
placental lesions leading to abortions. The other factors that may contribute to the placental
damage and abortion by this pathogen are not clearly understood. One such factor could be the
siderophores of Brucella (14) . Most of the studies related to Brucella siderophores have been
associated with 2, 3-dihydroxy benzoic acid (2, 3-DHBA) which for years was considered to be
the only siderophore produced by Brucella. It has been shown that deletion of the entC gene
involved in 2,3-DHBA biosynthesis has no effect on Brucella’s ability to grow in iron minimal
media or inside macrophages (15). But the entC deletion mutant was found to be attenuated in
presence of erythritol and most importantly was unable to cause abortions in pregnant bovines.
The relation between siderophore biosynthesis and erythritol utilization has been implicated as
the reason for the attenuation but is poorly understood. Brucella has been shown to produce
another siderophore named brucebactin (70). Brucebactin was discovered while studying an entF
deletion mutant as the result of defining a transposon insertion. The mutant was unable to
produce a stronger iron chelator, brucebactin, whose deficiency could not be compensated by 2,
3- DHBA. Whether or not brucebactin also has a role in erythritol metabolism has not been
studied. The only gene that has been shown to be involved in brucebactin biosynthesis is entF.
This 1410 base pair gene does not show significant homology with E. coli entF that is involved
in the biosynthesis of catechol siderophore, enterobactin. But the protein translated from entF
has significant homology with vibH, a gene involved in biosynthesis of vibriobactin, a
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siderophore by Vibrio spp. (82). VibH is a nonribosomal peptide synthetase that utilizes its
condensation domain to fuse 2, 3-DHBA to the polyamine norspermidine during the biosynthesis
of vibriobactin in V. cholerae (83). Also, the functional domain required for the amide synthase
activity of V. cholerae VibH is highly conserved in B. abortus homologue (16). Inspite of having
these similarities with V. cholerae vibH the specific role of Brucella entF still needs to be
studied.
Another interesting aspect of entF gene is the orientation of transcription. The gene
transcribes in opposite direction to the four other genes in the siderophore operon (entE,C,B,A,)
described for B. abortus 2308 (Figure 5). The role of entF gene is not clearly understood as there
are contradictory reports on the expression of this gene during iron limiting conditions (16, 70).
Moreover, the brucebactin biosynthesis pathway is not known. Therefore, the primary aim of the
present research effort was to study the role of entF gene in growth of Brucella under iron
limiting conditions and in the presence of erythritol.
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Material and Methods
Bacterial strains, plasmids and oligonucleotides
All bacterial strains and plasmids used in this study and their sources are described in
Table 1. The different oligonucleotides used as primers for polymerase chain reactions (PCR)
with their respective restriction sites and enzymes are described in Table 2
Culture media, chemicals and growth conditions
Bacteria were grown in trypic soy broth (TSB) or on tryptic soy agar (TSA) plates (Difco
Laboratories, Detroit, MI) for 3-4 days at 37°C in an atmosphere containing 5% CO2.
Appropriate antibiotics, gentamicin (50µg/ml), ampicillin (100µg/ml), kanamycin (50-100µg/ml)
and chlorompheniol (30µg/ml) (Sigma- Aldrich Inc, Saint Louis, MO) were added to the media,
while screening the mutants and complemented mutants. All chemicals,
ethylenediaminetetraacetic acid (EDTA), ethylenediamine-N.N’- diacetic acid (EDDA), iron
chloride III (FeCl3), deferroxime mesylate (DFA), meso-erythritol, TritonX-100 were bought
from Sigma-Aldrich Inc (Saint Louis, MO). Macrophage culture media, Dulbecco’s Modified
Eagle’s medium (DMEM) and fetal bovine serum were also bought from Sigma-Aldrich Inc
(Saint Louis, MO).
To study the growth in iron limiting conditions iron minimal media (IMM) was prepared
as described by Lopez Goni et al. (96). All glass ware were was with EDTA to chelate iron and
then rinsed three times with double distilled water (53). Iron chloride III was added to IMM as
the positive control media at the concentration of 50µM (14). Iron chelator EDDA was added at
the concentration of 15µM. This concentration was found to arrest the growth of entF deletion
mutant of B. abortus 2308 in iron limiting conditions (70). Before adding EDDA it was first
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deferrated using HCl and acetone, as previously described (133). Meso erythritol was added at
the concentration of 0.1% to IMM. This concentration has been found to show the difference in
the growth of 2,3 DHBA Brucella mutant compared to wild type (14). Deferoxymine mesylate
(DFA) was added to 15µM concentration, 48 hours prior to the infecting the J774A.1
macrophages with B. abortus 2308 as previously described (109).
Recombinant DNA methods
DNA ligations, restriction endonuclease digestions and agarose gel electrophoresis were
performed according to the standard techniques described by Sambrook (1989). The polymerase
chain reactions were performed using Platinum PCR Supermix High Fidelity (Invitrogen
Corporation, Grand Island, NY) and a Gradient Mastercycler (Eppendorf). Oligonucleotides
were purchased from Sigma-Genosys (Sigma-Aldrich Inc, Saint Louis, MO). Restriction
enzymes and ligation reaction kit were purchased from Promega (Madison, Wisconsin).
Commercial kits were used for DNA extraction (QIAZEN DNA extraction kit), plasmid
extraction (QIA prep Spin Miniprep Kit, QIAZEN) and gel extraction and to clean PCR products
(QIAZEN PCR cleanup kit).
Creation of deletion clone (BAN1)
A 497 base pairs segment was deleted from the middle of entF gene in B. abortus 2308
using Cre-Lox methodology (Figure 6). Briefly, a 903 bp fragment (entF1) containing 307 bp
upstream of the gene and 596 bp from the start of gene was amplified from B. abortus 2308
genome using specific primers (Table 2) and digested using EcoR1 and BamH1 restriction
enzymes. Similarly, a 540 bp fragment (entF2) that included 317 bp before the C-terminal end of
gene and 223 bp downstream of the gene was amplified and restriction digested using BamH1
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and Sph1. These two segments were ligated into plasmid pGEM3Z at the BamH1 site between
EcoR1 and Sph1 multiple cloning sites and ligated at BamH1 cut flanking regions. A 1kb
fragment containing antibiotic (gentamicin) resistant cassette flanked by loxP sites on both sides,
was obtained from pUCGmLox vector using BamH1 restriction enzyme digestion. This cassette
was cloned into the recombinant pGEM3Z plasmid in between two amplified fragments at the
BamH1 site. Recombinant vector (pGmloxF12) was electroporated in B. abortus 2308. Through
the homologous recombination the GmLox cassette flanked by the amplified DNA fragments
replaced the native gene and thus removed 497 bp sequence from the gene. Colonies were
screened for gentamicin resistance and ampicillin susceptibility to ensure a double cross over
event which was then confirmed by PCR using gene specific primers, i.e. entF forward and
reverse (Table 2). Further, to remove the antibiotic resistant gene from the mutated clone,
plasmid pCM158 carrying kanamycin resistance that expresses the Cre enzyme was
electroporated into the mutant clone. After three passages colonies were screened for kanamycin
and gentamicin susceptibility and the deletion of 497 base pair of entF gene was confirmed by
PCR.
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A B
C
BAN1 mutant genome
Figure 6 Schematic diagram showing deletion cloning of entF gene using cre-lox methodology.
A) Recombinant pGEM3Z having entF1 and entF2 regions and GmLox cassette, B) Showing the
double homologous recombination between Brucella abortus 2308 genome and recombinant
plasmid sites resulting in the deletion of 497 bp of entF gene with GmLox cassette, C) Deletion
of gentamicin resistance gene between two lox sites by the activity of Cre enzyme generating the
deletion mutant BAN1. (USR= upstream region, DSR= downstream region, accC1= gentamicin
resistance gene, loxP= DNA sequence for cre enzyme activity)
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Creation of complemented mutants BAN2A and BAN2B
To create the vector for complementing the entF deletion gene in BAN1 mutant two
strategies were used (Figure 8). Plasmid pNSGroE was used as the expression vector (152). In
the first strategy the entF gene was amplified using entF forward and reverse primers and was
cloned in to the vector between BamH1 and Xba1 restriction sites. This recombinant plasmid
(pNSGroentF) was electroporated into BAN1 mutant and colonies were screened for
chloromphenicol resistance. This clone was named as BAN2A; the presence of complete gene in
the plasmid was confirmed by extracting the plasmid and genomic DNA from BAN2A, these
were amplified by PCR using entF forward and reverse primers.
Using a automated genome annotation software ( FgenesB module, Softberry Inc., Mount
Kisco, NY) the upstream region of entF gene was checked and a potential promoter region
(Figure 7) was identified (-10, AAGTATTAT and -35, TTGCCG positions). As a promoter
region was detected in upstream region of entF., It was thought that the groE promotor could
possibly interfere with regulation and expression of entF gene, so we devised the following
cloning strategy to include entF promotor along with the entF gene. In the second strategy the
groE promoter was excised from pNSGroE using Sal1 and Xba1 enzymes. Then the entF gene
along with 300 bp upstream was amplified from B. abortus 2308 genomic DNA using primers
entF/P forward plus reverse. The amplicon was ligated in between Sal1 and Xba1 restriction
sites of the plasmid. For this cloning effort the reverse primer was designed in such a way that a
6x His tag occurred before the stop codon (entF/PR, Table 2). These two strategies were used to
determine the difference in expression of entF gene with its own promoter versus a groE
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promoter. The second recombinant plasmid was electroporated into the BAN1 mutant to create
BAN2B strain. Screening and confirmation of BAN2B was done similarly as BAN2A.
BAN2A and BAN2B were tested in iron minimal media supplemented with 0.05% erythritol for
the phenotypic complementation to wild type under similar growth conditions.
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.
Figure 7: Nucleotide sequence of entF gene along with 300 base pair upstream, showing the
potential gene organization. This includes 1410 base pair of entF gene (blue color). The
sequence predicted as -10 and -35 regions (red color) of potential promoter are marked.
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Figure 8: Schematic diagram showing the creation the two recombinant plasmids for
complementation of entF gene in BAN1 strain. Recombinant plasmid pNSGrentF was created by
ligating the ampilified entF gene sequence from B. abortus 2308 into pNSGroE expression
vector between the BamHI and XbaI restriction sites (A). To create recombinant plasmid
pNSentF/P, first groE promoter was dropped from plasmid pNSGroE using enzymes SalI and
XbaI (B). The amplified entF including the 300 bp upstream was ligated between these
restriction sites (C).
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Growth studies
In vitro growth in iron minimal media
Cultures were started at a concentration of 106 bacteria /ml. All supplements were filter
sterilized (FeCl3, erythritol and EDDA) and added 48 hours prior to start of the growth. After the
start of growth, 200µL of culture was sampled in triplicate and 10 fold serial dilutions were made
in phosphate buffer saline and plated on TSA plates to determine the colony forming units
(CFUs) after the incubation for 2-3 days. All the cultures were maintained and monitored for 10
days.
In vivo growth in macrophages
Approximately 1 x 106 J774A.1 cells were seeded per well in a 24 well plate (Corning
Incorporated) 24 hrs prior to infection. The cells were grown in Dulbecco’s Modified Eagle’s
medium (DMEM) supplemented with 10% heat inactivated fetal calf serum and a humidified 5%
CO2 atmosphere at 37°C. 18-24 hrs later, the J774A.1 cells were infected at a MOI of 100:1
(100 Brucella per macrophage) and incubated for an hour to allow the phagocytosis and
internalization of bacteria. After one hr of incubation (which was considered as the 0 hour time
point for all the experiments), the cell culture media was removed and wells were washed three
times with DMEM media containing 50 µg/ml gentamicin to wash off all the extracellular
bacteria. Fresh DMEM containing 50µg/ml gentamicin and 10% FBS was added for the further
incubation. As needed deferroxime mesylate (DFA), was added to the culture media 48 hrs to the
final concentration of 30µM before infecting the J774A.1 cells. At 0, 24 and 48 hours post
infection, media was removed from the wells (three wells per time point) and wells were washed
twice with PBS to remove the traces of media and gentamicin. The cells were then lysed using
1ml of 0.1% TritonX-100 and left for 20 minutes to allow complete lysis of cells. Lysates were
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collected and serial dilutions were prepared in PBS and plated on TSA plates to determine the
colony forming units after incubating the plates for 3-4 days.
Results
Deletion and complementation of entF gene
PCR results using Brucella genomic DNA and BAN1 (mutant strain) DNA show clearly
the deletion of 497 bp within the entF gene in the mutant strain BAN1 (Figure 9). Left side of the
DNA ladder (Lane 6) in the gel picture shows the PCR results using entF forward and reverse
primers while right side shows amplicons obtained with entF/P (amplify entF gene along with its
promoter) forward and reverse primers were used.
Amplicons obtained by using the genomic DNA from the wild type strains shows
products of 1410 bp (Figure 9, Lane 4) when entF forward and reverse primers were used.
Amplicon of 1710 bp (Figure 9, Lane 8) was obtained when entF/P forward and reverse primers
were used to amplify genomic DNA from wild type strain.
In contrast to wild type strain, when BAN1 genomic DNA was used as template, which
has a deletion of 497 bp fragment, while 34 bp were added as the loxP sites remained after the
removal of antibiotic resistant cassette, shows the amplicons of 947 bp (Figure 9, Lane 3) and
1247 bp (Figure 9, Lane 9) while using entF (forward and reverse) and entF/P (forward and
reverse) primers respectively.
Complemented entF mutant (BAN2A and BAN2B) show same product as BAN1 mutant
when genomic DNA from the strains were amplified (Figure 9, Lane 2 & 10); in contrast when
the plasmid DNA (pNSGroentF or pNSentF/P) was used as the template, the resultant amplicons
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were of the same size as the wild type (Figure 9, Lane 1 & 11) showing the genetic
complementation of the deleted gene in complemented mutants through the plasmids.
Both the plasmids used for the complementation could partially restore the growth
restriction of mutant only up to 48 hours of growth in minimal media supplemented with 0.1%
erythritol (Figure 10). Plasmid from both the complemented clones was extracted and analyzed
through commercial nucleotide sequencing (VBI, Virginia Tech, Blacksburg) to ensure the
absence of any possible mutation in the cloned sequence.
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Figure 9: PCR results showing the difference in wild type, deletion mutant and
complemented strains. Lane 1-4 shows the amplicon obtained when entF forward and
reverse primers were used. While lane 8-11 represents the amplicons generated when the
entF/P forward and reverse primers were used.
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**
*
0
1
2
3
4
5
6
7
8
CFU
(Lo
g10
)
2308 BAN1 BAN2A BAN2B
Figure 10: Genetic complementation of erythritol-induced growth restriction of entF deletion
mutant,BAN1 by complemented clones, BAN2A and BAN2B. Growth was evaluated at 48 hour
of growth in iron minimal media supplemented with 0.05% erythritol. (* represents p value ≤
0.001 relative to BAN1 as the control).
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Growth in minimal media
Brucella specific iron minimal media contains glycerol as the sole carbon source and
defined amounts of other essential nutrients (96). To extract the iron, liquid media is treated with
8-hydroxy quinolone and metal-oxime chelates are removed by using choloroform. The
concentration of iron in minimal media (IMM) was determined at Toxicology laboratory
(VMRCVM, Blacksburg, VA, 24061) using atomic absorption spectrophotometry (flame
method) and was found to be less than 0.1 ppm. Starting with 106 cfu/ ml of culture, the growth
of BAN1 mutant was significantly slower compared to wild type strain in IMM (Figure 11.A).
The wild type strain grew to 108 cfu/ml of culture on the 5
th day but mutant did not reach the
same concentration till day 10. A significant difference in the rate of growth was detected as
soon as 24 hours after the start of cultures. A stationary phase in B. abortus 2308 was observed
between 144 and 168 hours of growth and a decline phase started after 196 hours of growth.
Compared to wild type, the BAN1 mutant had a longer lag phase and slow growth rate. The
slower growth rate of the BAN1 mutant strain was restored by the addition 50µM iron chloride
(Figure 11.B). Both wild type and mutant strains grew similarly in media supplemented with iron
over the period of 10 days. The decline phase started soon after reaching the concentration of 109
cfu/ml (at 96 hours) in case of both strains in the presence of iron supplemented IMM.
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Figure 11: Comparative growth of entF deletion mutant B. abortus BAN1 and wild type
B.abortus 2308 in IMM. A) Growth IMM over a period of 240 hours showing the slower growth
of BAN1 compared to wild type B. abortus 2308. B) Growth after the supplementation with
50µM FeCl3 showing the similar growth patterns of both mutant and wild type strain. Statistical
significance was calculated (P < 0.001) by t test analysis by using replicate samples for each
culture condition (*represents p value ≤ 0.001).
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Growth in the presence of iron chelator
Adding chemical iron chelator to IMM should further restrict the availability of iron to
pathogens. In the presence of 15µM EDDA that bound the traces of iron (≤0.1 ppm) present in
IMM, growth of BAN1 mutant was severely restricted (Figure 12A). The mutant strain was able
to survive but appeared not to multiply. The wild type strain multiplied up to 144 hrs and
exhibited a sharp decline phase after 192 hours. Supplementation of iron in the form of 50µM
FeCl3 completely restored the growth pattern of the BAN1mutant to that of wild type B. abortus
2308 (Figure. 12B). These experiments were performed three times and each time samples were
taken in triplicates. Iron chelator was added 24 hrs prior to the start of experiment to the media to
ensure the binding of all available iron in the media. Increased amount of EDDA restricted the
growth of wild type strain as well (data not shown).
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Figure 12: Comparative growth of Brucella strains in presence of iron chelator EDDA. A)
Growth in presence of 15µM EDDA added to IMM showing the marked growth attenuation of
mutant strain compared to wild type strain. B) Growth of mutant and wild type strains in the
presence of 15µM EDDA and 50µM FeCl3 showing the restoration of growth pattern. Statistical
significance was calculated (P < 0.001) by t test analysis by using replicate samples for each
culture condition (*represents p value ≤ 0.001).
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Growth in iron minimal media supplemented with 0.1% erythritol
Erythritol metabolism and iron acquisition are known to be related through unknown
mechanisms (14). When grown in the presence of 0.1% erythritol in IMM (Figure 13A), the
BAN1 mutant was unable to grow and appeared to die after 48 hours. Wild type Brucella also
had a longer lag phase in the presence of erythritol (7.5 cfu/ml of culture) and the stationary
phase cfu were less compared to cfu in minimal media without erythritol (8.2 cfu/ml of culture).
This suggests that the requirement of iron is much more significant in the presence of erythritol.
Wild type B. abortus was able to grow while the BAN1 mutant did not show any growth in the
presence of erythritol. To rule out the possibility of erythritol toxicity to the BAN1 mutant,
growth was tested in the presence of FeCl3 added at the concentration of 50µM to the IMM
containing erythritol (Figure 13B). Both wild type and mutant strains grew similarly and implies
that there was no toxic effect due to erythritol but much more iron is needed in presence of
erythritol. Increasing the amount of erythritol to 0.5% restricted the growth of wild type strain as
well; this limitation was restored by the addition of 100 µM FeCl3 (Figure 14), even though not
perfectly.
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Figure 13: Comparative growth of Brucella in presence of 0.1% erythritol. A) Growth of mutant
vs wild type B.abortus 2308 in the presence of 0.1% meso-erythritol supplemented in iron
minimal media showing the attenuation of wild type strain while arresting the mutant strain in
lag phase of growth. B) Growth curve showing the effect of supplementation of 50µM FeCl3 on
the growth attenuation due to erythritol of wild type as well the BAN1 mutant strain. Statistical
significance was calculated (P < 0.001) by t test analysis by using replicate samples for each
culture condition (*represents p value ≤ 0.001)
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Figure 14: Growth restriction of wild type B. abortus 2308 in the presence of erythritol.
Significant differences in the growth of B. abortus 2308 in the presence of 0.5% erythritol
compared to 0.1% erythritol; 100 µM FeCl3 restored the restriction in growth. (*represents p
value ≤ 0.001).
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Intracellular survival in J774A.1 macrophages
Phagocytosis of Brucella by macrophages occurs in first 15-30 min of infection of
macrophages (9, 25). To study the intracellular survival and growth after incubating for an hour
the extracellular pathogens are removed by changing the media. Further, to restrict the
extracellular growth of the pathogen, 50µg/ml gentamicin was added to the media to kill any
Brucella released from the macrophages. This much amount of gentamicin is known not to
affect the intracellular growth and survival of pathogens (80, 142). By restricting the growth of
extracellular Brucella, their survival and growth inside macrophages can be studied after initial
phagocytosis. No significant difference was found in the intracellular survival and replication of
the BAN1 mutant compared to wild type B.abortus in J774A.1 macrophages at 24 and 48 hours
respectively (Figure 15A). Similarly, no significance difference was found in survival and
growth of two strains when the media used to grow the macrophages was supplemented with
0.1% erythritol (Figure 15B). This observation suggests that there should be enough available
iron in macrophage culture media (DMEM) to support the growth of both wild type as well as
mutant strains inside the macrophages.
Intracellular survival and growth in presence of iron chelator
Restriction of freely available iron present in DMEM by iron chelator DFA (30µm) did
not have any effect on the survival and growth patterns of the BAN1 mutant compared to wild
type strain (Figure 15C). Macrophage cells were grown in the media containing DFA for 48
hours and then were infected in order to chelate iron both extracellularly and intracellularly.
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When the amount of DFA in macrophage culture media was increased to 45µM and 60µM, we
found macrophages were unable to grow (data not shown).
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Figure 15: Growth of wild type B. abortus and BAN1 in J774A.1 macrophages. Survival and
replication of wild type and mutant strain in J774A.1 macrophages over a period of 48 hours in
the presence of glucose as the only carbon source (A), in the presence of 0.1% erythritol (B) and
after the addition of 30µM DFA to chelate iron in the media (C). No significant differences
between wild type and mutant strain were observed.
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Discussion
The role of natural iron chelator i.e. a siderophore is not well understood in case of
intracellular pathogen Brucella. Unlike Mycobacterium (44), siderophore mutants of wild type B.
abortus 2308 do not lose their ability to survive and replicate inside macrophages (15, 70).
Brucella siderophore research began with the discovery of 2, 3-dihydroxy benzoic acid (2, 3-
DHBA) as Brucella siderophore (96). Through extensive studies it was found that Brucella does
not need 2,3-DHBA for its survival in-vivo experiments in mice (15). The relationship changed
with the finding that a siderophore is extremely important to acquire iron in the presence of
erythritol (14). The mutant lacking the ability to synthesize of 2, 3-DHBA could not cause
abortions in the pregnant ruminant host. Later, through a Tn5 insertion (transposon mutagenesis)
Gonzalez Carrero et al (70) found that Brucella also produces another siderophore named
“brucebactin”. The entF gene which is upstream to the entABCD operon (Figure 5) was found to
be interrupted in the mutant unable to synthesize brucebactin and thus a possible role of the gene
in the biosynthesis of brucebactin was predicted (70). But its role was disputed when Bellaire et
al (16) did not find any transcript of the gene under iron limiting conditions and suggested that
Brucella synthesizes brucebactin during late stages of growth and that too at much lower levels
(16). The work presented here suggests that entF has a significant role in iron acquisition by B.
abortus 2308.
The deletion mutant (BAN1) created using cre-lox methodology grew slowly compared
to wild type strain in IMM (Figure 11A). Addition of 50µM FeCl3 relieved the growth restriction
(Figure 11B) suggesting that only iron was the limiting factor in the media. These results do not
agree with the previous publications (14, 70) where no significant differences were found in the
growth of siderophore mutants compared to wild type strain in iron minimal media. There could
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be two reasons for the different findings. First, all the glassware in this study were thoroughly
rinsed with EDTA solution (24, 53, 95), to chelate traces of metallic ions and then rinsed three
times with double distill water before use.
Secondly, the growth and survival was determined by determining the actual colony
forming units (CFUs) instead of optical density (OD) used by other scientists to compare the
growth of siderophore mutants to the wild type strain (14, 70). OD values are based on the
absorbance/scattering of a particular wavelength of light passing through a solution/suspension
and thus cannot differentiate between dead and live bacteria (1). Based on Beer’s law, OD values
of different bacteria could be comparable at certain concentrations where the cell density is same
and has same absorbance and thus independent of cell size (86)
Cell density is influenced by variety of factors including bacterial species (39). A mutant
could be different in its morphology compared to wild type and thus the density of two might
vary and thus growth comparisons based on OD values might not be accurate. Under heat stress
Sinorhizobium spp. exhibit changes in its morphology (128). When grown under iron deprivation
Campylobacter jejuni shows changes in morphology which has an effect on the cell density (54).
Iron deficiency may directly affect cell growth and division and have found to result in elongated
forms of the pathogen growing under iron limiting conditions (171). The differences in the
morphology of the wild type and mutant strain should be studied to confirm if the deletion of
entF gene brings any changes to the bacterial cell size or shape.
Moreover, the accuracy of OD measurement is restricted to a minimum to 107
cfu/ml of
culture, thus it requires a high concentration of bacteria (38). Concentrations of bacteria below
this value might not be detected (39). The linear relationship between OD and cfu at high cell
concentration is not strictly observed (56). Thus, there are many factors that restrict the use of
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OD values to measure the growth of two different strains of bacteria. Estimating cfu is time
consuming and labor intensive method compared to OD measurements. But it is the true estimate
of the live bacteria in the broth culture specially under stressed growth conditions (132) when
growth is slow and chances of morphological changes are high. This reason needs to be further
confirmed by standardizing the cfu vs OD values and then determining the differences, if any.
Under scarcity of iron, the wild type strain itself could not reach a concentration of 109
cfu/ml in iron minimal media (Figure 11A) and also had a slower growth rate compared to its
own growth in IMM supplemented with iron (Figure 11B). This confirms the importance of iron
for the growth of B. abortus 2308 as shown by other workers (52, 116, 163).
If a gene affects the growth of a bacterium under iron limiting conditions, it might be
involved in acquisition, transport or metabolism within the iron pathway of a bacterium. It has
been shown by Gonzalez Carrero et al (70) that transposon insertional inactivation in entF gene
of B. abortus 2308 resulted in its inability to synthesize brucebactin. The absence of brucebactin
was confirmed by thin layer chromatography (TLC). In our study, addition of EDDA to IMM
greatly restricted the growth of the BAN1 mutant compared to its own growth in IMM. The
BAN1 mutant was able to survive in the presence of EDDA in IMM but could not multiply over
a period of ten days. Thus the role of the entF gene depends on the degree of iron restriction in
the growth media. This might suggest a significant role for entF gene in iron acquisition
compared to iron metabolism. From the data in Figure 12A it can be seen that there was no effect
of the addition of EDDA on the growth of wild type strain in IMM. This indicates a stronger iron
acquisition system in the wild type compared to deletion mutant, BAN1, as shown by other
authors (70). Comparing the growth of BAN1 mutant strain in the IMM with and without EDDA
(Figures 11A and 12A), it appears that the role of entF gene is more important, when iron is
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strongly bound to other iron binding proteins or iron chelators. This finding agrees with the
observation by Gonazalez Carrero et al (70), who suggested that brucebactin is a stronger
chelating agent than 2, 3 DHBA. Thus Brucella appears to produces brucebactin at later stages of
growth and under highly restricted levels of iron in the growth medium (70). The best way to
confirm the role of entF gene in the siderophore (brucebactin) biosynthesis is by showing the
absence of brucebactin in BAN1 cultures. Thin layer chromatography (TLC) (70) and high
performance liquid chromatography (HPLC) (88) could be used. Our efforts to detect
brucebactin through HPLC were unsuccessful as the structure and property of brucebactin are
still unknown and there is no standard positive control to compare and detect brucebactin.
Growth of the mutant and wild type strain in IMM supplemented with 0.1% erythritol
(Figure 13A) clearly suggest that much more iron is needed for the efficient metabolism of
erythritol. Brucella vaccine strain S19 was found to have a deletion in an erythritol catabolic
gene and was susceptible to erythritol (141). However, erythritol tolerant mutants arise from
sensitive cultures of S19. Sangari et al (140) found that these resistant mutants do not grow in the
presence of glycerol as carbon source which was consistent with the notion that glycerol
transporter is also an erythritol transporter (51). For unknown reasons Brucella has been shown
to prefer erythritol over most common easily metabolized carbon sources like glucose and
glycerol (84, 154).
Sperry and Robertson (156) found that erythritol kinase activity is 8 times stronger than
glucokinase in B. abortus. Furthermore, if there is any obstruction in the other metabolic steps,
the bacterium faces a rapid drop in ATP concentration and starts to die (157). The only link that
directly connects erythritol catabolism and iron is an enzyme 3-keto-L-erythrose 4-phosphate
dehydrogenase (Figure 4) which is iron linked and is crucial for the erythritol catabolism.
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Comparing the growth of wild type strain in Figures 11A and 13A, it is clear that wild type strain
could not reach the stationary phase density in the presence of erythritol as it reached in IMM
without erythritol. This suggests that much more iron is needed in the presence of erythritol
possibly because of the involvement of an iron linked enzyme in the erythritol metabolism. This
might also explains the death of BAN1 mutant, which is deficient in acquiring iron and unable to
catabolise erythritol efficiently. Lack of the entF gene restricts the ability of the mutant to
acquire iron thus resulting in a scarcity of iron inside bacterial cytoplasm that leads to enzyme
malfunction, required to carry on the erythritol catabolism. Figure 13A shows the gradual death
of mutant strain in the presence of 0.1% erythritol in IMM. To rule out the possibility of any
toxic effect of erythritol, further supplementation of 50µM FeCl3, reversed the inhibition in
growth. This suggests that much more iron is needed by the pathogen in the presence of
erythritol and agrees with the results from others (14). At a higher percentage of erythritol
(0.5%), growth of wild type strain itself was attenuated and could be restored by the addition of
100µM FeCl3 (Figure 14).
Based on enzymes components of the erythritol metabolic pathway in Brucella, the first
step involves the phosphorylation of erythritol via an ATP dependent kinase (156). Thus the
pathogen needs to invest energy first before it can metabolize the substrate and gain energy. This
may slow down the growth process and results in a longer lag phase of growth of wild type strain
in the presence of erythritol in IMM. Moreover, it has also been found to be involved in
regulation of gene expression (94), thus it might be possible that iron is somehow involved in the
regulation of the operon encoding the enzymes for erythritol metabolism. These possibilities
need to be explored experimentally.
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Unlike the IMM cultures, the mutant did not show any attenuation inside J774A.1
macrophages. These findings corroborates with previous results by Gonzalez Carrero et al (70).
The same group of scientists showed the entF deletion mutants make 2, 3-DHBA and the amount
synthesized in iron limited condition is more than in the wild type strain (70). The results
reported here do not agree with the results presented by Parent et al (116) who compared the
growth and survival of 2, 3-DHBA mutant with the wild type strain in J774A.1 macrophage. In
their study a lesser number of mutants were able to survive the killing by macrophages after 48
hrs; however the surviving bacteria grew at the similar rate as wild type strain. The lack of
attenuation of the BAN1 mutant inside macrophages could be because of two reasons; first, there
is sufficient iron inside the macrophages as they were grown in levels of fetal bovine serum
(10%). Second, the BAN1 mutant should be able to synthesize 2, 3-DHBA, also shown by
Gonzalez-Carrero et al. (70), thus it can acquire iron inside macrophages.
It was predicted that brucebactin is a stronger siderophore than 2, 3-DHBA and thus
required to chelate iron when it is tightly regulated (70). To test this 30µM deferroxime mesylate
(DFA), an iron chelating agent, was added to the in-vitro macrophage cultures. There was no
effect on the growth of the mutant under these conditions. Increasing the concentration of DFA
inhibited the growth of macrophages and thus prevented any conclusions. Again these results do
not agree with those reported by Parent et al (116) who showed the differences not only in
survival but also growth of 2, 3-DHBA mutant compared to wild type. The reasons for these
differences could be the presence of enough iron in the media or efficiency of 2, 3-DHBA to act
as siderophore under these conditions.
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There was no significant effect of adding 0.1% erythritol to DMEM media as was seen in
the case of broth cultures. The possible reasons could be ample amounts of free iron in DMEM
media used for the cultures and mutant can produce 2, 3 DHBA that serves as iron chelator.
From the Brucella genome map (Figure 5) it is clear that the entF gene is located next to
the operon involved in the biosynthesis of Brucella siderophores. The upstream entF gene
transcribes in the opposite direction and for the first time using FgenesB module (Softberry Inc.,
Mount Kisco, NY) the presence of a promoter region (-35 and -10 regions, Figure 7) upstream
of entF was observed. Phenotypic complementation of the deleted gene in complemented
mutant BAN2B (Figure 10) shows that the identified promoter is functional in B. abortus 2308.
Nonetheless, additional studies need to be performed to further confirm the functionality of the
promoter. Such studies could include the transcriptional fusion of reporter gene like β-
galactosidase to the promoter and examining the expression levels of the reporter gene in iron
minimal media with and without erythritol. Complementation was better achieved using groE
promoter (BAN2A) instead of predicted entF promoter. This observation agrees with the
previous findings showing better expression of Brucella genes under groE promoter in B.
abortus 2308 compared to other promoters (151).
The phenotypic complementation could not sustain growth of BAN2A and BAN2B in
IMM supplemented with 0.05% erythritol for an extended period of time. After 48 hours both the
complemented mutants were growing slower than BAN1 mutant strain. pNSGroE plasmid is
known to be eliminated by the bacteria in vivo (in mice and other animals) and under minimal
medium growth conditions (personnel communication, Dr. M. Seleem, ICTAS, Virginia Tech).
The plasmid used in the present study is a medium copy number plasmid and thus the cloned
gene might exhibit negative feedback on its own expression after a period of time. In case of
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gene regulation studies of Brucella siderophore mutants it has been seen that single copy number
plasmids (like pMR10) work much better than multicopy plasmids (personal communication, Dr.
M. Roop, East Carolina University). These possibility needs to be explored by testing further by
testing the complemented mutant (BAN2A and BAN2B) at each step of growth i.e. if the
complemented strain still carry the plasmid.
In case of Brucella, siderophore mutants do not show any difference in their
pathogenicity in mice (15). The only differences were found when some of them were tested in
pregnant ruminants (14, 16) and this approach was beyond the scope of the present thesis.
In conclusion, studies presented in this thesis indicated for the first time the role of entF
gene in iron acquisition and especially in the presence of erythritol. Also, a functional promoter
element for the entF gene was demonstrated. There is a correlation between erythritol
metabolism and need for more iron by the pathogen. The future research efforts should address
definition of the pathway for the biosynthesis of siderophores in Brucella. These efforts should
also attempt to purify and characterize brucebactin as well as to establish a relationship between
erythritol metabolism and siderophore biosynthesis. This could be done by creating mutants that
lack both siderophore biosynthesis and erythritol catabolism genes and then testing them for their
ability to grow under iron limiting conditions.
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65
Table 1. List of all the bacterial strains and plasmids used in the present study
Bacterial strain Characteristic References
Brucella abortus 2308 Virulent wild type Brucella abortus strain (31)
E.coli Mach 1 Chemically competent cells, endA1, recA1, tonA, panD Invitrogen
B. abortus 2308 ∆entF
(BAN1)
Brucella abortus 2308 deletion mutant
Contain deletion of 497 bp in entF gene
This study
B. abortus ∆entF pNSGrentF
(BAN2A)
Complemented mutant strain. BAN1 containing pNSGrentF
plasmid
Chloromphenicol resistance
This study
B. abortus ∆entF pNSentF/P
(BAN2B)
Complemented mutant strain. BAN1 containing pNSentF/P
plasmid
Chloromphenicol resistance
This study
Plasmid Characteristic Reference
pGEM3Z Cloning vector
Ampicillin resistance
Promega
pUCGmlox pUC18-based vector containing the loxP sites flanked aacC1 gene
Ampicillin and gentamicin resistant
(126)
pGmLoxF12 PGEM3Z vector with entF1 and entF2 regions and Gmlox cassette
Ampicillin and gentamicin resistance
This study
pCM158 “Cre” enzyme expression plasmid
Kanamycin resistant
(92)
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66
pNSGroE Expression vector
Chloromphenicol resistance
(152)
pNSGrentF Expression vector containing entF gene
Chloromphenicol resistance
This study
pNSentF/P entF gene along with 300 base pairs upstream that contains
the promoter sequence for the gene
Chloromphenicol resistance
This study
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67
Table 2. List of Primers used in the present study. Also showing the restriction site and the
restriction enzyme
Primer name Product Oligonucleotide sequence
entF1 forward
entF1 reverse
emf 903bp
5’ CCC GAA TTC GCA CCC ACC ACG ATC GGA TTG 3’
EcoR1
5’ CCC GGA TCC AAG GGG TGG CCA CTT TCG CCC 3’
BamH1
entF2 forward
entF2 reverse
entF2
(540 bp)
5’ CCC GGA TCC ACC CCG GCC TCG TCA GTG CAA 3’
BamH1
5’ CCC GCA TGC GCG CAC TTC CGG TCG CCA ATG 3’
Sph1
entF forward
entF reverse
entF gene
(1410 bp)
5’ GGG GGA TCC TTG GTC CCA ATT TGT CAA CCG GGT 3’
BamH1
5’ GGG TCT AGA TCA TGG CAA ACG GCG GCG AAG ATC 3’
Xba1
entF/P forward
entF/P reverse
entF gene with its
promoter
(1710 bp)
5’ CCCC GTC GAC CCA CGA TCG GAT TGC GCT GCC CAG CGC 3’
Sal1
5’ CCC TCT AGA TCA ATG ATG ATG ATG ATG ATG TGG CAA
Xba1
ACG GCG GCG AAG ATC GCT CAC 3’
Page 78
68
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