Bartonella and its survival in different microenvironments 

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Free Fe(II) and its transport

Under acidic and anaerobic conditions, ferrous iron Fe(II) predominates. This soluble iron can diffuse freely through the outer membrane porins and is transported through the cytoplasmic membrane via a high-affinity ferrous ion uptake (feo) system in many Gram-negative bacteria (Kammler et al., 1993). In Escherichia coli, feoA and B encode for the Fe(II) transporter (Kammler et al., 1993) (Fig.1). These feo genes are conserved in many bacteria, but feoB is often present in the absence of feoA (Andrews et al., 2003). The feoB gene is present in ~50% of the bacterial genomes sequenced to date (Hantke, 2003). FeoB was identified as an ATP-dependent transporter (Kammler et al., 1993) and has been shown to function as a G protein (Hantke, 2003). Feo mutants of Salmonella enterica and E. coli colonize mouse intestine less efficiently than the wild type (Stojiljkovic et al., 1993; Tsolis et al., 1996). In Legionella pneumophila, disruption of feoB decreases Fe(II) uptake, leading to reduced intracellular growth (Robey and Cianciotto, 2002). FeoB is the most important iron uptake system for Helicobacter pylori, which colonizes the stomach, an acidic environment (Velayudhan et al., 2000).

Transferrin and lactoferrin iron transport

Many bacterial species such as Neisseria and Haemophilus influenzae have transferrin and/or lactoferrin receptors (Cornelissen and Sparling, 1994; Fuller et al., 1998). Iron is stripped by these receptors from transferrin and lactoferrin and iron-free proteins are released. The functional transferrin receptor in Neisseria consists of TbpA and TbpB subunits. TbpA is analogous to TonB-dependent outer membrane receptors and TbpB is a lipoprotein anchored in the outer leaflet of the outer membrane (Legrain et al., 1993). TbpA is strictly required, whereas the requirement for TbpB is not as stringent. Uptake of iron by TbpA is TonB-ExbB-ExbD and pmf-dependent (Cornelissen et al., 1997). The bipartite lactoferrin LbpAB receptors of Neisseria (Schryvers et al., 1998) and H. influenzae (Ekins et al., 2004) function similarly, with only Fe(III) crossing the outer membrane. In the periplasm, Fe(III) binds FbpA in association with the bipartite receptor TbpAB (Gomez et al., 1998). FbpA shuttles
Fe(III) across the periplasm to an ABC permease in the cytoplasmic membrane (Ekins et al., 2004). This ABC permease translocates ferric iron across the cytoplasmic membrane into the cytosol.

Siderophores and hemophores

Synthesis and excretion of siderophores

To overcome the problem of the low solubility of Fe (III), many bacteria synthesize and secrete strong iron chelators: siderophores. Siderophores chelate iron from the extracellular medium; the iron-loaded siderophore is then taken up by the cell through a specific uptake system (Fig.2). Siderophores are low-molecular-weight compounds (400 to 1,000 Da) produced by microorganisms. They are synthesized by non-ribosomal cytoplasmic peptide synthetases resembling machinery used for the biosynthesis of peptide antibiotics. Following their synthesis, siderophores are excreted into the extracellular medium. These molecules are thought to be too large to diffuse through the envelope. In E. coli, entS, which is located in the enterobactin biosynthesis and transport gene cluster, encodes a membrane protein belonging to the major facilitator superfamily. The proteins of this family are involved in proton motive-force-dependent membrane efflux pumps. The protein EntS was shown to be directly involved in enterobactin export (Furrer et al., 2002).

Hemophores

Hemophores have been found in Gram-negative and Gram-positive bacteria. Hemophores are either anchored to the cell surface or released into the extracellar medium to capture free heme or to extract heme from hemoproteins such as hemoglobin in the external medium and present it to specific outer membrane receptors (Wandersman and Delepelaire, 2004, 2012).

The HasA hemophore

HasA from Serratia marcescens was the first hemophore to be discovered (Letoffe et al., 1994a). Later, hemophores were identified in P. aeruginosa (Letoffe et al., 1998), P. fluorescens (Idei et al., 1999), Y. pestis ((Rossi et al., 2001), Bacillus anthracis (Fabian et al., 2009) and Porphyromonas gingivalis (Gao et al., 2010). HasA hemophores are conserved in several Gram-negative species (Wandersman and Delepelaire, 2012).
In S. marcescens, HasA, a 19 kDa monomer, exhibits very high affinity for heme (Kd~10-11M) at 1:1 stoichiometry (Izadi-Pruneyre et al., 2006). The crystal structure of holo HasA shows a globular protein with a two-faced fold (4 α-helices on one face and 7 β-strands on the other) and a heme pocket with potential iron ligands (Arnoux et al., 1999). The heme iron atom is ligated by tyrosine 75 and histidine 32 (Letoffe et al., 2001). Histidine 83 also plays an important role in heme binding, since it stabilizes Tyr-75 and strengthens the Tyr-75 iron coordination bond (Wolff et al., 2002) (Fig. 3).
HasA is secreted by ABC transporters made up of three envelope proteins associated in a protein complex (Letoffe et al., 1994b) (Fig 4). This complex includes an inner membrane ATPase, HasD, a membrane fusion protein, HasE, located in the inner membrane and HasF, an outer membrane protein of the TolC family (Wandersman and Delepelaire, 1990) (Fig 4).
HasA is able to capture heme from hemoglobin, holo-hemopexin and myoglobin (Benevides-Matos and Biville, 2010; Wandersman and Delepelaire, 2004). In S.
marcescens, heme is directly taken up at concentrations higher than 10-6M in the presence of iron depletion. Hemophore HasA is required to mediate heme uptake only under strong iron depletion conditions and low heme concentrations (lower than 10-7M) (Benevides-Matos and Biville, 2010).

The HxuCBA system of H. influenzae

Another hemophore system has been described in H. influenzae that lacks the heme biosynthetic pathway and requires exogenous heme for aerobic growth (White and Granick, 1963). In H. influenzae, the gene cluster hxuC, hxuB, and hxuA is required for heme-hemopexin utilization (Cope et al., 1995). HuxA is a 100 kDa protein that can be found on the H. influenzae cell surface and in culture supernatants (Cope et al., 1994). In the reconstituted E. coli system, most HuxA remains associated with the cell and only a small fraction of HxuA is released into the extracellular medium (Fournier et al., 2011). HxuA is secreted by a signal-peptide-dependent pathway requiring one helper protein, HxuB (Cope et al., 1995). HxuB was hypothesized as being involved in release of soluble HxuA from the cell surface (Cope et al., 1995). Unlike HasA, there is no heme capture by HxuA, but heme is released into the medium when HxuA interacts with heme-hemopexin (Wandersman and Delepelaire, 2012). In reconstituted E. coli strain C600△hemA, expression of HxuC alone enabled use of free heme or hemoglobin as a heme source (Fournier et al., 2011). However, co-expression of HxuA, HxuB and HxuC in C600ΔhemA is required for heme acquisition from heme-loaded hemopexin (Fournier et al., 2011). It was shown that when HxuA interacts with holo-hemopexin, heme is released from hemopexin and immediately binds its cognate receptor HxuC, which transports heme through the outer membrane (Fournier et al., 2011).

Receptors for siderophores and heme/hemophores

Receptors for siderophores

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Loaded siderophore complexes are too large to freely pass through the porins. The first step in ferri-siderophore internalization requires a specific outer membrane receptor having high affinity (Kd 0.1-100nM) for the ferri-siderophore complex (Stintzi and Raymond, 2000). Outer membrane receptors involved in recognition and transport of iron-loaded siderophores are specific, but have structural and functional characteristics in common (Koster, 2001). These receptors all display a plug and barrel organization (Biville, 2008; Krewulak and Vogel, 2008). The N-terminus part of the protein, which constitutes the plug, is folded inside a β-barrel anchored in the outer membrane. The plug delimits two interacting surfaces, one on the outside of the membrane and the other on the periplasmic side. The substrate sits on the top of the plug, facing the extracellular medium. This brings it into contact with residues of the extracellular loops and the plug apices. There is no channel large enough to accommodate the substrate. Determination of the crystal structure of FecA complexed with ferric citrate and of FhuA complexed with iron-containning ferrichrome has provided information on the transport process (Locher et al., 1998; Yue et al., 2003). Substrate binding triggers small changes in the conformation of the plus apices, with larger movements observed on the periplasmic side where the TonB box is found (Ferguson and Deisenhofer, 2004). The TonB box consists of five conserved residues towards the N-terminus of outer membrane siderophore receptors. These residues have been implicated in the functioning of the TonB complex (Postle, 1993). It is thought that substrate binding leads to closure of the outside loops around the substrate and movement on the periplasmic side, allowing the TonB complex to function. Closing of the outside loops prevents the substrate from being released into the medium (Eisenhauer et al., 2005).

Receptors for heme

Heme receptors were subdivided into three categories: the first group recognizes free heme, the second recognizes host hemoproteins (Fig 5) and the third interacts with hemophores (Fig 4) (Tong and Guo, 2009). The receptors that recognize hemoproteins and hemophores can also take up free heme. All these receptors share overall common structural attributes within the family and also share significant homology with siderophore receptors (Wilks and Burkhard, 2007).
The outer membrane receptor binds heme free or bound to hemoproteins, and transports it into the periplasm via a TonB-dependent process (Fig 5). Inside the periplasm, heme is bound by the heme periplasmic binding protein, which addresses it to an inner membrane ATP binding cassette (ABC) transporter (Fig 5). Inside the cytoplasm, heme is rapidly transferred to a heme oxygenase (HO)/heme-degrading protein which are able to release iron from heme (Fig. 5).

The TonB-ExbB-ExbD complex

Transportation of ferri-siderophores or heme through the outer membrane is energy-dependent. This transport against a concentration gradient depends on a complex of three proteins in the cytoplasmic membrane:TonB, ExbB and ExbD (Moeck and Coulton, 1998). This TonB-ExbB-ExbD complex transduces the energy generated by the electrochemical gradient across the cytoplasmic membrane (Bradbeer, 1993). TonB-ExbB-ExbD proteins are found in many Gram-negative bacteria. The structure and function of this system has been extensively studied in E. coli.
The TonB protein is associated with the inner membrane, with a large part of the protein occupying the periplasmic space (Fig. 6). TonB spans the periplasm and directly contacts outer membrane active transport proteins. ExbB and ExbD are integral cytoplasmic membrane proteins. ExbB has three transmembrane segments and its N-terminus projects into the periplasm (Fig. 6). ExbD has only one transmembrane domain and most of the protein is in the periplasm (Braun and Braun, 2002) (Fig. 6). The three proteins, ExbB, ExbD and TonB, seem to act as a complex, since ExbB and ExbD interact with each other in vitro (Braun et al., 1996).

Transport across the periplasm and cytoplasmic membrane

Transport of ferri-siderophore complexes and heme across the periplasmic space and the cytoplasmic membrane is mediated by periplasmic binding proteins associated with inner membrane transporters. The periplasmic binding proteins shuttle ferri-siderophore or heme from the outer membrane receptor or the periplasm and deliver it to a cognate permease in the inner membrane (Koster, 2001). The periplasmic binding proteins are less specific than the outer membrane receptors. ABC permeases are responsible for transport across the inner membrane. ABC permeases consist of a periplasmic domain and an inner membrane complex energized by an ABC ATPase (Koster, 2001).

Fate of the ferri-siderophore and heme in the cytoplasm

After internalization, iron must be released from the siderophore. Two mechanisms have been proposed for release of iron from the ferri-siderophore into the cytoplasm. Spontaneous dissociation of the ferri-siderophore may be related to the relatively low affinity of siderophores for Fe (II). A specific ferric reductase is probably required for this pathway (Matzanke et al., 2004). The second mechanism involves intracellular breakdown of the ferri-siderophore, implying that siderophores are used only once. In E. coli, the use of ferri-enterobactin requires esterase activity encoded by the fes gene, located in a cluster of genes involved in enterobactin biosynthesis and uptake. Fes is a cytoplasmic esterase that hydrolyzes ferri-enterobactin, producing 2,3-dihydroxybenzoyl serine (Brickman and McIntosh, 1992). Iron is released from heme as soon as it is transported to the cytoplasm (see paper one, Introduction).

Table of contents :

Chapter one Bartonella and its survival in different microenvironments 
1. Analysis of Bartonellae genomes
2. Diseases caused in humans; diagnosis and treatment of Bartonellosis
3. The cycle of Bartonella in the mammalian reservoir host
4. Bartonella and erythrocyte parasitism
5. Bartonella interaction with endothelial cells
6. Bartonellae and their vectors
Chapter two Iron/Heme uptake in Bartonella 
1. Free Fe(II) and its transport
2. Transferrin and lactoferrin iron transport
3. Siderophore and Hemophore
4. Receptors for siderophores and heme/hemophore
5. The TonB-ExbB-ExbD complex
6. Transport across the periplasm and cytoplasmic membrane
7. Fate of the ferri-siderophore and heme in the cytoplasm
8. Regulation of iron and heme uptake in Gram-negative bacteria
9. The heme uptake system in Bartonella
Chapter three Research significance and Purpose of the work 
Results
Paper one Heme degrading protein HemS is involved in oxidative stress response of Bartonella henselae
State of the art concerning the fate of heme in the cytoplasm
Paper two Heme binding proteins of Bartonella henselae are required when undergoing oxidative stress during cell and flea invasion
State of the art concerning outer membrane heme binding proteins of Bartonella
1. Outer membrane heme binding proteins in Gram-negative bacteria
2. The finding of heme binding proteins in Bartonella and their structure
3. Heme binding proteins cannot transport heme
4. Heme binding proteins and interactions with the infected host
5. Regulation of expression of heme binding proteins in Bartonella
Discussion Discussion and perspectives
Supplement Paper Identification of a novel nanoRNase in Bartonella
References References

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