Available online at www.sciencedirect.com
ScienceDirect New insights into the role of Bartonella effector proteins in pathogenesis Sabrina Siamer and Christoph Dehio The facultative intracellular bacteria Bartonella spp. share a common infection strategy to invade and colonize mammals in a host-specific manner. Following transmission by bloodsucking arthropods, Bartonella are inoculated in the derma and then spread, via two sequential enigmatic niches, to the blood stream where they cause a long-lasting intra-erythrocytic bacteraemia. The VirB/VirD4 type IV secretion system (VirB/D4 T4SS) is essential for the pathogenicity of most Bartonella species by injecting an arsenal of effector proteins into host cells. These bacterial effector proteins share a modular architecture, comprising domains and/or motifs that confer an array of functions. Here, we review recent advances in understanding the function and evolutionary origin of this fascinating repertoire of host-targeted bacterial effectors. Addresses Focal Area Infection Biology, Biozentrum, University of Basel, Basel, Switzerland Corresponding author: Dehio, Christoph ([email protected])
Current Opinion in Microbiology 2015, 23:80–85 This review comes from a themed issue on Host-microbe interactions: bacteria Edited by David Holden and Dana Philpott
http://dx.doi.org/10.1016/j.mib.2014.11.007 1369-5274/# 2014 Published by Elsevier Ltd.
Introduction Bartonella spp. are facultative intracellular bacteria that are responsible for long-lasting intra-erythrocytic bacteraemia in diverse mammalian reservoir hosts and are transmitted by blood-sucking arthropods most likely through inoculation of skin lesions by contaminated insect feces, or contact with infected animals via scratching or bites [1]. Following dermal inoculation, Bartonella colonize two sequential niches, respectively called ‘dermal niche’ (describing the dermal stage of infection) and ‘blood-seeding niche’ (formerly known as ‘primary niche’), considered to include dendritic and endothelial cells [2]. Subsequently, Bartonella reach the blood stream where, restricted to the specific reservoir host, they invade erythrocytes and persist intracellularly for the remaining lifetime of the red blood cell [3]. Specific Current Opinion in Microbiology 2015, 23:80–85
adaptation to the reservoir host causes no or only mild disease symptoms, while infection of incidental hosts can be associated with broad spectrum of diseases. The exception is B. bacilliformis, a human-specific species that causes life-threatening disease. Most other human infections are caused by the human-specific species B. quintana and the zoonotic cat-specific species B. henselae. The clinical manifestations and treatments of Bartonella infection have been extensively reviewed elsewhere and therefore will not be covered in this review [4,5]. Different genomic and phylogenetic studies revealed that the genus Bartonella is divided into four lineages plus B. tamiae and B. australis, which occupy ancestral positions [6,7,8]: lineage 1 is solely composed of the deadly pathogen B. bacilliformis. Lineage 2 is composed of ruminant-specific species (e.g. deer-specific B. schoenbuchensis) that have acquired the Vbh (VirB-homologous) T4SS that is closely related to the VirB/D4 T4SS. Lineage 3 is composed of species infecting diverse mammals (e.g. fox/dog-specific B. rochalimae and cat-specific B. clarridgeiae) that have acquired the VirB/D4 T4SS and the most species-rich lineage 4 contains species (e.g. cat-specific B. henselae, human-specific B. quintana, or rat-specific B. tribocorum) that harbor the VirB/D4 and the Trw T4SS [6,7,8]. Both T4SS present in the lineage 4 species were shown to be essential for host interaction at different stages of the infection cycle [9,10]. The Trw T4SS is involved in the erythrocyte invasion process by mediating host-specific adhesion to these cells [10]. The VirB/D4 T4SS contributes to pathogenicity by translocating a cocktail of evolutionary-related effector proteins, called Beps (Bartonella effector proteins) into nucleated cells of both the ‘dermal’ and the ‘blood-seeding’ niches [2,6,9]. Once inside host cells, these Beps target host components and modulate cellular processes to the benefit of the bacteria. Therefore, defining the role of the various Beps is key to gain a better understanding of the pathogenesis of Bartonella. This review summarizes advances made in deciphering the evolution and functional role of the Beps in Bartonella–host cell interaction.
Acquisition of the VirB/D4 T4SS The current model indicates that the radiating lineage 3 and 4 have independently acquired the virB/D4 T4SS locus with at least one effector gene by horizontal gene transfer [6,7]. The similarity of the VirB/D4 T4SS with bacterial conjugation systems suggests that the virB/D4 locus was probably acquired via horizontal gene transfer from a conjugative plasmid. Whole genome shotgun www.sciencedirect.com
Function of Bartonella effector proteins Siamer and Dehio 81
analysis of representative species of lineages 3 and 4 demonstrated lineage-specific integration of the virB/ D4 locus and the presence of lineage-specific repertoires of fast evolving bep genes that display a high degree of inter-species variation. Comparative whole genome analysis further indicated that the virB/D4 and bep gene clusters represent a major driver of the adaptive radiation observed in the lineages 3 and 4 which represent a remarkable example of parallel evolution [6].
Evolution of the Beps Bartonella–host interaction exposes the arsenal of Beps to a strong selective pressure resulting in their rapid evolution, which could explain their modular architecture. This modular architecture of the Beps, which is derived from a single ancestor composed of an N-terminal FIC and Cterminal BID domain, evolved by extensive rounds of duplication, diversification and reshuffling of domains (Figure 1) [6,11]. The BID domain together with a
positively charged C-tail constitute a secretion signal for the VirB/D4 T4SS, while the FIC, also present in other bacterial effectors and many more endogenous proteins found in all domains of life, was shown to mediate post-translational modifications such as AMPylation, phosphocholination or phosphorylation [12–15]. Interestingly, previous studies suggest that the BID domain together with a positively charged C-tail, which evolved as bi-partitie T4SS signal from relaxases of alphaproteobacterial conjugation systems, was acquired by the ancestral Bartonella effector protein to facilitate injection into target cells [11]. This is supported by the fact that in B. grahamii the Vbh T4SS, which is closely related to the VirB/D4 T4SS, is encoded on a plasmid in addition to a chromosomally encoded copy [16]. The vbh locus on the plasmid also encodes a toxin called VbhT in B. schoenbuchensis, which harbors the classical FIC-BID domain architecture as found conserved in many Beps [13]. It is thus tempting to speculate that the VbhT toxin
Figure 1
Ancestral effector VbhT FIC motif
FIC
BID
Independent duplication
FIC motif
FIC motif
BID
FIC
BID
FIC
FIC motif
FIC motif
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BID
FIC
Recombination and fixation of adaptive mutations
BID
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HPFxxxNG
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xPFxxGNx
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xPFxxGNx
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Representative Beps from the lineage 3
BID BID
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Representative Beps from the lineage 4 Current Opinion in Microbiology
Parallel evolution of the Beps from the lineages 3 and 4. The Beps from the lineages 3 and 4 evolved possibly from the VbhT toxin, which harbors the same FIC-BID domain architecture. Subsequently, after lineage-specific amplifications followed by recombination events and fixation of adaptive mutations, the Beps with a derived domain structure emerged independently in both lineages 3 and 4. www.sciencedirect.com
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82 Host-microbe interactions: bacteria
is closely related to the ancestral Bep from which the arsenal of effectors of both lineages 3 and 4 evolved independently by successive lineage-specific duplications and was further shaped by positive selection of diversified gene copies (Figure 1). The arsenal of Beps can be sorted into ten phylogenetic clades for the lineage 3, and seven phylogenetic clades for the lineage 4. The FIC-BID domain structure, followed by a positively charged C-tail, displays the most abundant effector protein type. However, a subset of effectors both in lineages 3 and 4 harbors other functional modules instead of the FIC domain, like tandem-repeated tyrosine-phosphorylation motifs (EPIYA and EPIYA-related motifs) or additional BID domains [11]. These different domain architectures suggest that these Beps arose from the ancestral domain structure by independent recombination events (Figure 1). Thus, the evolutionary diversification of the Beps created a wide array of adaptive functions dedicated to the cellular interaction within the mammalian hosts. This remodeling of preexisting effector proteins as an evolutionary mechanism to rapidly adapt to the host was described by Stavrinides et al. as ‘terminal reassortment’ in which effector genes recombine among themselves or with other genetic elements to give rise to new chimeric effectors [17]. Interestingly, comparative whole genome analysis of 14 Bartonella genomes revealed the presence of a Bartonella gene transfer agent (BaGTA) that represents the most conserved key innovation driving the explosive radiations of both lineages 3 and 4 [7]. Gene transfer agents (GTAs) are phage-like elements that generally transfer random pieces of the bacterial genome rather than their own DNA. GTA genes are located on the host chromosome, and GTAs generally transfer DNA from the producing cell to a recipient cell via a mechanism similar to transduction. Although the function of GTAs remains unclear regarding their role in evolution, they provide an efficient mechanism to duplicate and recombine genes [18]. Bep gene duplication and recombination by BaGTA may thus have conferred the high degree of adaptability to diverse mammalian reservoir hosts that have led to the explosive radiations of lineages 3 and 4.
Actin remodeling and invasome formation Historically, B. henselae was found to elicit VirB/D4-dependent phenotypes in vitro using human umbilical vein endothelial cells (HUVECs), like bacterial internalization via the invasome structure, inhibition of apoptosis, or activation of proinflammatory signaling [19]. This interaction of Bartonella with endothelial cells in vitro and the clinical descriptions of B. henselae in association with manifestations in the human vasculature, led to the proposition that vascular endothelial cells represent a part of the ‘blood-seeding niche’, that is colonized initially and from where bacteria are seeded to the blood stream to invade erythrocytes [2]. Studies of the infection of endothelial cells have shown that the internalization process of Current Opinion in Microbiology 2015, 23:80–85
Bartonella starts with endocytosis of one or a few bacteria into a vacuole called Bartonella-containing vacuole (BCV). This process is then arrested by the activities of several Beps (i.e. BepC, BepF and BepG in B. henselae), which are translocated via the VirB/D4 T4SS into the host cell cytoplasm [20–22]. It was shown that both BepG alone or a combined action of BepC and BepF impedes BCV formation via the inhibition of endocytosis. This leads to accumulation of bacterial aggregates at the cell surface, which invade the endothelial cells via F-actin-dependent invasome-mediated internalization [21,22]. Invasome formation mediated by combined action of BepC and BepF depends on F-actin modulation by Rac1/Scar1/Wave/Arp2/ 3, Cdc42/WASP/Arp2/3 and cofilin1. In contrast, BepGtriggered invasome formation requires only F-actin modulation by Rac1/Scar1/Wave/Arp2/3 and Cdc42/WASP/ Arp2/3, but not cofilin 1 [21,22]. Interestingly, constitutive-active Cdc42 or Rac1 can mimic BepF action in the BepC/BepF dependent invasome formation pathway, suggesting a regulatory role of BepF on the small Rho GTPases. BepF harbors a tyrosine-repeat motif near its N-terminus and contains three BID domains in the Cterminal part. Although the N-terminal tyrosine-repeat phosphorylation motif is phosphorylated upon translocation via the VirB/D4 T4SS, it is not involved in invasome formation [23]. Instead, the first two BID domains of BepF, BID-F1 and BID-F2, are sufficient to promote the formation of these structures [23]. BepG harbors an array of four BID domains. However, sequence comparisons of B. henselae BID domains revealed that the BID domains of BepF and BepG are poorly conserved between each other suggesting that these effectors may promote invasome formation by targeting different host functions [11].
Inhibition of apoptosis BepA mediates protection of endothelial cells from apoptosis and may thus contribute to the formation of vasoproliferative tumors as found in bacillary angiomatosis, a typical manifestation of immunocompromised patients infected with B. henselae or B. quintana [24,25]. On the molecular level, the BID domain of BepA was shown to block endothelial cell apoptosis by direct binding to host cell adenylyl cyclase, thereby potentiating GaS-dependent cAMP production. The elevation of cAMP levels and consecutive upregulation of cAMP-stimulated gene expression then leads to inhibition of apoptosis [24]. While inhibition of apoptosis is exclusively mediated by the BID domain of BepA, the FIC domain shows autoAMPylation and AMPylation of a target from total HeLa cell extract [26]. The nature of the host target and physiological consequences of AMPylation remains to be demonstrated.
BepE: an essential protein for Bartonella dissemination Recently, it was shown that HUVEC cells infected with the DbepE mutant of B. henselae are deficient in rear end www.sciencedirect.com
Function of Bartonella effector proteins Siamer and Dehio 83
detachment and undergo cell fragmentation. These phenotypes require the action of BepC, which possibly affects rear detachment during cell migration by modulating the actin cytoskeleton [2]. Ectopic expression of BepE from B. henselae or its orthologs in cells is sufficient to suppress the cell fragmentation phenotype [2]. The prominent activity of BepE in restoring cell migration led the authors to dissect the in vivo function of this effector protein. They demonstrated using a rat model and different routes of infection that BepE is essential to invade some nucleated cell types in the derma and spread the infection to the blood stream. As observed for BepA, BepF and BepG, this BepE-dependent phenotype also relies on the effector BID domains. The authors assumed that the migration of Bartonella from the derma to the blood stream could be achieved intracellularly in dendritic cells. This is supported by the fact that a trans-well migration assay showed an inhibition of dendritic cell migration upon infection with the B. henselae DbepDEF
mutant, while complementation with BepE restores it [2]. These new results on the function of BepE allowed the authors to extend the concept of the ‘blood-seeding niche’ (formerly known as ‘primary niche’) to ‘dermal niche’, which describes the dermal stage of infection [2]. Moreover, the authors demonstrated that BepE acts via its second BID domain on the RhoA signaling pathway to most likely preserve cells from fragmentation [2]. Indeed, they observed that BepE interferes with the inhibitory effect of the C3-based Rho inhibitor 1, which is known to ADP-ribosylate RhoA [27]. Altogether, the experimental studies showed that the Beps are essential for the primary stage of infection (Figure 2). It is surprising to see that most Bep-dependent phenotypes of B. henselae that have been studied so far rely on the BID domain, which evolved as a secretion signal and acquired, over the course of evolution, diverse functions, with distinct phenotypic properties. Until now,
Figure 2
1 Blood-sucking arthropod
9
2
Dermis Dendritic cells 3 BepE Bartonella 8
Dermal niche
4 Migration 5
7
Blood seeding niche Invasome formation
Vasculature
BepF
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6 Capillary lumen
Inhibition of apoptosis BepA
Current Opinion in Microbiology
Model of the Bartonella infection process in the reservoir hosts. (1) Bartonella replicate first in the midgut of the arthropod vector and are excreted in the feces. (2) When the arthropods suck blood on mammals, it provides a local source of irritation followed by scratching and inoculation of Bartonella-containing insect feces into the derma. (3) Subsequently, Bartonella colonize the ‘dermal niche’, a term proposed by Okujava et al. (see Ref. [2]). This ‘dermal niche’ includes most likely dendritic cells, that are considered to disseminate Bartonella towards the ‘blood seeding niche’ (formerly known as ‘primary niche’) a process that strictly depends on the effector BepE (4). In the ‘blood-seeding niche’, bacteria are considered to colonize endothelial cells, which likely require the action of BepC, BepF, BepG and BepA (5). From this ‘blood seeding niche’, the bacteria are periodically seeded into the bloodstream where they invade erythrocytes and reinfect the cells of the ‘blood seeding niche’ (6). After limited replication inside the red blood cell (7), they persist in the ‘intraerythrocytic niche’ for the remaining life-span of the red blood cell (8) and are thus competent for transmission by a bloodsucking arthropod (9). www.sciencedirect.com
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Table 1 Beps studied and their function in Bartonella pathogenicity Beps
Bartonella species
BepA BepC
B. henselae B. henselae
BepE
B. henselae B. tribocorum
BepE
B. henselae
BepF
B. henselae
BepG
B. henselae
Bep2
B. rochalimae
Phenotype Inhibition of apoptosis F-actin modulation Trigger invasome formation Trigger cell fragmentation Preserves ECs from fragmentation and blocks defects of DCs migration caused by other Beps Unknown F-actin modulation Trigger invasome formation F-actin modulation Trigger invasome formation Unknown
Domain implicated BID domain Unknown
BID domains 1 and 2
Tyrosine-phosphorylation motif BID domains 1 and 2 Multiple BID domains FIC domain
Target
References
Adenylyl cyclase Related to Rac1 and Cdc42 signaling, Cofilin1-dependent RhoA signaling pathway
[25,26] [2,21,22]
Csk SHP2 Related Rac1 and Cdc42 signaling, Cofilin1-dependent Related to Rac1 and Cdc42 signaling, Cofilin1-independent AMPylation of Vimentin
[28]
[2]
[21–23] [21,22] [29]
Abbreviations: ECs, endothelial cells; DCs, dendritic cells.
no phenotype in vivo or in vitro has been shown to depend on the tyrosine phosphorylation motifs or the FIC domain of any Bep from lineage 4. BepD, BepE and BepF from the lineage 4 and Bep9 from the lineage 3 were shown to be tyrosine-phosphorylated in human cells [6,11,28]. More particularly, using a proteomic screen to identify potential targets of all known tyrosine-phosphorylated bacterial effectors, BepE was shown to interact with Csk upon phosphorylation on Tyr37 and with SHP2 upon phosphorylation with Tyr64. Although this result was confirmed by a co-immunopurification assay, further work is required to elucidate the biological relevance of these tyrosine-phosphorylation and subsequent protein interactions [28]. All the Beps discussed and their implications in Bartonella pathogenicity are listed in Table 1.
Beps from the lineage 3: opening the black box While the Bep effectors of lineage 4 and particularly of B. henselae have been extensively studied, the functions of the Beps of lineage 3 are still poorly understood. As described above, these Beps are classified in 10 phylogenetic clades and most of them, except the clade 9, harbor the classical FIC-BID domain organization [6]. Recently, Pieles et al. showed by an unbiased mass spectrometrybased approach that Bep2 from B. rochalimae AMPylates via its FIC domain the intermediate filament protein vimentin [29]. Although the contribution to virulence by this AMPylation remains to be elucidated, this method paves the way for identifying AMPylation targets for other FIC domains of Beps from lineages 3 and 4. This approach can further be adapted to study other posttranslational modifications for which isotopically labeled substrates are available. As it was shown that FIC domains can mediate diverse post-translational modifications [12–15], this approach offers a potentially powerful means to identify both new targets of Beps and their FIC domain-mediated post-translational modifications. Current Opinion in Microbiology 2015, 23:80–85
Concluding remarks Recent advances have enhanced our understanding of how the repertoire of Beps finely manipulates host cell processing steps enabling Bartonella to interact, colonize, proliferate and persist in host cells. Here we have reviewed the evolutionary diversification of these effector proteins, their function and their phenotypic properties. Although they share a modular architecture, they acquired different functions within host cells. Some effectors, like BepC, BepF and BepG, appear to act in concert whereas others, like BepC and BepE, appear to be antagonistic. However, despite the progress made to elucidate the functions of several Beps from lineage 4, the roles of the ones from the lineage 3 remain to be elucidated. One of the big challenges in the study of bacterial effectors is that they exert their function(s) in concert with other effectors and therefore are regulated both temporally and spatially. We hope that new approaches developed will enhance our understanding of the contribution of these proteins to the Bartonella–host interaction, making the next few years very exciting in this regard.
Acknowledgements We would like to thank Alexander Harms, Dr. Rusudan Okujava and Dr. Maxime Que´batte for critical reading of the manuscript. This work was supported by Grant 310030B_149886 from the Swiss National Science Foundation (SNSF, www.snf.ch), advanced Grant 340330 (FicModFun) from the European Research Council (ERC), and Grant 51RTP0_151029 for the research and Technology Development (RTD) project TargetInfectX in the frame of systemsX.ch (www.systemX.ch), the Swiss Initiative for System Biology.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1. Chomel BB, Boulouis HJ, Breitschwerdt EB, Kasten RW, VayssierTaussat M, Birtles RJ, Koehler JE, Dehio C: Ecological fitness and strategies of adaptation of Bartonella species to their hosts and vectors. Vet Res 2009, 40:29. www.sciencedirect.com
Function of Bartonella effector proteins Siamer and Dehio 85
2.
Okujava R, Guye P, Lu YY, Mistl C, Polus F, Vayssier-Taussat M, Halin C, Rolink AG, Dehio C: A translocated effector required for Bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors. PLoS Pathog 2014, 10:e1004187. Identified BepE as an essential protein for Bartonella dissemination from the dermal site of inoculation to the blood stream.
3.
Pulliainen AT, Dehio C: Persistence of Bartonella spp. stealth pathogens: from subclinical infections to vasoproliferative tumor formation. FEMS Microbiol Rev 2012, 36:563-599.
4. Angelakis E, Raoult D: Pathogenicity and treatment of Bartonella infections. Int J Antimicrob Agents 2014, 44:16-25. A comprehensive review covering the clinical aspect of Bartonella infections. 5.
Harms A, Dehio C: Intruders below the radar: molecular pathogenesis of Bartonella spp.. Clin Microbiol Rev 2012, 25:42-78. A comprehensive review covering the molecular and cellular basis of pathogenesis by Bartonella spp.
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Engel P, Salzburger W, Liesch M, Chang CC, Maruyama S, Lanz C, Calteau A, Lajus A, Medigue C, Schuster SC, Dehio C: Parallel evolution of a type IV secretion system in radiating lineages of the host-restricted bacterial pathogen Bartonella. PLoS Genet 2011, 7:e1001296.
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Guy L, Nystedt B, Toft C, Zaremba-Niedzwiedzka K, Berglund EC, Granberg F, Naslund K, Eriksson AS, Andersson SG: A gene transfer agent and a dynamic repertoire of secretion systems hold the keys to the explosive radiation of the emerging pathogen Bartonella. PLoS Genet 2013, 9:e1003393. A study which proposed an updated Bartonella phylogeny based on the sequencing of six new genomes and description of the highly conserved GTA in Bartonella genomes.
8.
Zhu Q, Kosoy M, Olival KJ, Dittmar K: Horizontal transfers and gene losses in the phospholipid pathway of Bartonella reveal clues about early ecological niches. Genome Biol Evol 2014, 6:2156-2169. A new phylogenetic study highlighting the impact of horizontal gene transfer on Bartonellae lineage evolution.
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Saenz HL, Engel P, Stoeckli MC, Lanz C, Raddatz G, VayssierTaussat M, Birtles R, Schuster SC, Dehio C: Genomic analysis of Bartonella identifies type IV secretion systems as host adaptability factors. Nat Genet 2007, 39:1469-1476.
10. Vayssier-Taussat M, Le Rhun D, Deng HK, Biville F, Cescau S, Danchin A, Marignac G, Lenaour E, Boulouis HJ, Mavris M et al.: The Trw type IV secretion system of Bartonella mediates host-specific adhesion to erythrocytes. PLoS Pathog 2010, 6:e1000946. 11. Schulein R, Guye P, Rhomberg TA, Schmid MC, Schroder G, Vergunst AC, Carena I, Dehio C: A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells. Proc Natl Acad Sci USA 2005, 102:856-861. 12. Cruz JW, Rothenbacher FP, Maehigashi T, Lane WS, Dunham CM, Woychik NA: Doc toxin is a kinase that inactivates elongation factor tu. J Biol Chem 2014, 289:7788-7798. This study demonstrates the phosphorylation activity of the Doc toxin which represents a new catalytic activity for the FIC protein family. 13. Engel P, Goepfert A, Stanger FV, Harms A, Schmidt A, Schirmer T, Dehio C: Adenylylation control by intra- or intermolecular active-site obstruction in FIC proteins. Nature 2012, 482:107-110. A structure–function study reporting that the Bep ancestral protein VbhT of B. schoenbuchensis forms a toxin–antitoxin module together with the small VbhA protein. 14. Mukherjee S, Liu X, Arasaki K, McDonough J, Galan JE, Roy CR: Modulation of Rab GTPase function by a protein phosphocholine transferase. Nature 2011, 477:103-106.
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15. Worby CA, Mattoo S, Kruger RP, Corbeil LB, Koller A, Mendez JC, Zekarias B, Lazar C, Dixon JE: The FIC domain: regulation of cell signaling by adenylylation. Mol Cell 2009, 34:93-103. 16. Berglund EC, Frank AC, Calteau A, Vinnere Pettersson O, Granberg F, Eriksson AS, Naslund K, Holmberg M, Lindroos H, Andersson SG: Run-off replication of host-adaptability genes is associated with gene transfer agents in the genome of mouse-infecting Bartonella grahamii. PLoS Genet 2009, 5:e1000546. 17. Stavrinides J, Ma W, Guttman DS: Terminal reassortment drives the quantum evolution of type III effectors in bacterial pathogens. PLoS Pathog 2006, 2:e104. 18. Lang AS, Zhaxybayeva O, Beatty JT: Gene transfer agents: phage-like elements of genetic exchange. Nat Rev Microbiol 2012, 10:472-482. A comprehensive review covering the characteristics of GTAs. 19. Dehio C: Infection-associated type IV secretion systems of Bartonella and their diverse roles in host cell interaction. Cell Microbiol 2008, 10:1591-1598. 20. Eicher SC, Dehio C: Bartonella entry mechanisms into mammalian host cells. Cell Microbiol 2012, 14:1166-1173. 21. Rhomberg TA, Truttmann MC, Guye P, Ellner Y, Dehio C: A translocated protein of Bartonella henselae interferes with endocytic uptake of individual bacteria and triggers uptake of large bacterial aggregates via the invasome. Cell Microbiol 2009, 11:927-945. 22. Truttmann MC, Rhomberg TA, Dehio C: Combined action of the type IV secretion effector proteins BepC and BepF promotes invasome formation of Bartonella henselae on endothelial and epithelial cells. Cell Microbiol 2011, 13:284-299. 23. Truttmann MC, Guye P, Dehio C: BID-F1 and BID-F2 domains of Bartonella henselae effector protein BepF trigger together with BepC the formation of invasome structures. PLoS One 2011, 6:e25106. 24. Pulliainen AT, Pieles K, Brand CS, Hauert B, Bohm A, Quebatte M, Wepf A, Gstaiger M, Aebersold R, Dessauer CW, Dehio C: Bacterial effector binds host cell adenylyl cyclase to potentiate GaS-dependent cAMP production. Proc Natl Acad Sci USA 2012, 109:9581-9586. This functional study reveals host adenylate cyclase as a target for the anti-apoptotic activity of BepA. 25. Schmid MC, Scheidegger F, Dehio M, Balmelle-Devaux N, Schulein R, Guye P, Chennakesava CS, Biedermann B, Dehio C: A translocated bacterial protein protects vascular endothelial cells from apoptosis. PLoS Pathog 2006, 2:e115. 26. Palanivelu DV, Goepfert A, Meury M, Guye P, Dehio C, Schirmer T: FIC domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA. Protein Sci 2011, 20:492-499. 27. Worthylake RA, Lemoine S, Watson JM, Burridge K: RhoA is required for monocyte tail retraction during transendothelial migration. J Cell Biol 2001, 154:147-160. 28. Selbach M, Paul FE, Brandt S, Guye P, Daumke O, Backert S, Dehio C, Mann M: Host cell interactome of tyrosinephosphorylated bacterial proteins. Cell Host Microbe 2009, 5:397-403. 29. Pieles K, Glatter T, Harms A, Schmidt A, Dehio C: An experimental strategy for the identification of AMPylation targets from complex protein samples. Proteomics 2014, 14:1048-1052. New proteomic approach to study post-translational modifications of host proteins by bacterial effectors.
Current Opinion in Microbiology 2015, 23:80–85
ScienceDirect New insights into the role of Bartonella effector proteins in pathogenesis Sabrina Siamer and Christoph Dehio The facultative intracellular bacteria Bartonella spp. share a common infection strategy to invade and colonize mammals in a host-specific manner. Following transmission by bloodsucking arthropods, Bartonella are inoculated in the derma and then spread, via two sequential enigmatic niches, to the blood stream where they cause a long-lasting intra-erythrocytic bacteraemia. The VirB/VirD4 type IV secretion system (VirB/D4 T4SS) is essential for the pathogenicity of most Bartonella species by injecting an arsenal of effector proteins into host cells. These bacterial effector proteins share a modular architecture, comprising domains and/or motifs that confer an array of functions. Here, we review recent advances in understanding the function and evolutionary origin of this fascinating repertoire of host-targeted bacterial effectors. Addresses Focal Area Infection Biology, Biozentrum, University of Basel, Basel, Switzerland Corresponding author: Dehio, Christoph ([email protected])
Current Opinion in Microbiology 2015, 23:80–85 This review comes from a themed issue on Host-microbe interactions: bacteria Edited by David Holden and Dana Philpott
http://dx.doi.org/10.1016/j.mib.2014.11.007 1369-5274/# 2014 Published by Elsevier Ltd.
Introduction Bartonella spp. are facultative intracellular bacteria that are responsible for long-lasting intra-erythrocytic bacteraemia in diverse mammalian reservoir hosts and are transmitted by blood-sucking arthropods most likely through inoculation of skin lesions by contaminated insect feces, or contact with infected animals via scratching or bites [1]. Following dermal inoculation, Bartonella colonize two sequential niches, respectively called ‘dermal niche’ (describing the dermal stage of infection) and ‘blood-seeding niche’ (formerly known as ‘primary niche’), considered to include dendritic and endothelial cells [2]. Subsequently, Bartonella reach the blood stream where, restricted to the specific reservoir host, they invade erythrocytes and persist intracellularly for the remaining lifetime of the red blood cell [3]. Specific Current Opinion in Microbiology 2015, 23:80–85
adaptation to the reservoir host causes no or only mild disease symptoms, while infection of incidental hosts can be associated with broad spectrum of diseases. The exception is B. bacilliformis, a human-specific species that causes life-threatening disease. Most other human infections are caused by the human-specific species B. quintana and the zoonotic cat-specific species B. henselae. The clinical manifestations and treatments of Bartonella infection have been extensively reviewed elsewhere and therefore will not be covered in this review [4,5]. Different genomic and phylogenetic studies revealed that the genus Bartonella is divided into four lineages plus B. tamiae and B. australis, which occupy ancestral positions [6,7,8]: lineage 1 is solely composed of the deadly pathogen B. bacilliformis. Lineage 2 is composed of ruminant-specific species (e.g. deer-specific B. schoenbuchensis) that have acquired the Vbh (VirB-homologous) T4SS that is closely related to the VirB/D4 T4SS. Lineage 3 is composed of species infecting diverse mammals (e.g. fox/dog-specific B. rochalimae and cat-specific B. clarridgeiae) that have acquired the VirB/D4 T4SS and the most species-rich lineage 4 contains species (e.g. cat-specific B. henselae, human-specific B. quintana, or rat-specific B. tribocorum) that harbor the VirB/D4 and the Trw T4SS [6,7,8]. Both T4SS present in the lineage 4 species were shown to be essential for host interaction at different stages of the infection cycle [9,10]. The Trw T4SS is involved in the erythrocyte invasion process by mediating host-specific adhesion to these cells [10]. The VirB/D4 T4SS contributes to pathogenicity by translocating a cocktail of evolutionary-related effector proteins, called Beps (Bartonella effector proteins) into nucleated cells of both the ‘dermal’ and the ‘blood-seeding’ niches [2,6,9]. Once inside host cells, these Beps target host components and modulate cellular processes to the benefit of the bacteria. Therefore, defining the role of the various Beps is key to gain a better understanding of the pathogenesis of Bartonella. This review summarizes advances made in deciphering the evolution and functional role of the Beps in Bartonella–host cell interaction.
Acquisition of the VirB/D4 T4SS The current model indicates that the radiating lineage 3 and 4 have independently acquired the virB/D4 T4SS locus with at least one effector gene by horizontal gene transfer [6,7]. The similarity of the VirB/D4 T4SS with bacterial conjugation systems suggests that the virB/D4 locus was probably acquired via horizontal gene transfer from a conjugative plasmid. Whole genome shotgun www.sciencedirect.com
Function of Bartonella effector proteins Siamer and Dehio 81
analysis of representative species of lineages 3 and 4 demonstrated lineage-specific integration of the virB/ D4 locus and the presence of lineage-specific repertoires of fast evolving bep genes that display a high degree of inter-species variation. Comparative whole genome analysis further indicated that the virB/D4 and bep gene clusters represent a major driver of the adaptive radiation observed in the lineages 3 and 4 which represent a remarkable example of parallel evolution [6].
Evolution of the Beps Bartonella–host interaction exposes the arsenal of Beps to a strong selective pressure resulting in their rapid evolution, which could explain their modular architecture. This modular architecture of the Beps, which is derived from a single ancestor composed of an N-terminal FIC and Cterminal BID domain, evolved by extensive rounds of duplication, diversification and reshuffling of domains (Figure 1) [6,11]. The BID domain together with a
positively charged C-tail constitute a secretion signal for the VirB/D4 T4SS, while the FIC, also present in other bacterial effectors and many more endogenous proteins found in all domains of life, was shown to mediate post-translational modifications such as AMPylation, phosphocholination or phosphorylation [12–15]. Interestingly, previous studies suggest that the BID domain together with a positively charged C-tail, which evolved as bi-partitie T4SS signal from relaxases of alphaproteobacterial conjugation systems, was acquired by the ancestral Bartonella effector protein to facilitate injection into target cells [11]. This is supported by the fact that in B. grahamii the Vbh T4SS, which is closely related to the VirB/D4 T4SS, is encoded on a plasmid in addition to a chromosomally encoded copy [16]. The vbh locus on the plasmid also encodes a toxin called VbhT in B. schoenbuchensis, which harbors the classical FIC-BID domain architecture as found conserved in many Beps [13]. It is thus tempting to speculate that the VbhT toxin
Figure 1
Ancestral effector VbhT FIC motif
FIC
BID
Independent duplication
FIC motif
FIC motif
BID
FIC
BID
FIC
FIC motif
FIC motif
FIC
BID
BID
FIC
Recombination and fixation of adaptive mutations
BID
FIC motif
BID
BID
BID
FIC motif
FIC
HPFxxxNG
FIC
BID
BID
BID
FIC
BID
BID
BID
xPFxxGNx
BID
BID
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BID FIC motif
xPFxxGNx
FIC
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FIC motif HPFxxGNx
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Representative Beps from the lineage 3
BID BID
FIC
FIC motif
FIC
BID
Representative Beps from the lineage 4 Current Opinion in Microbiology
Parallel evolution of the Beps from the lineages 3 and 4. The Beps from the lineages 3 and 4 evolved possibly from the VbhT toxin, which harbors the same FIC-BID domain architecture. Subsequently, after lineage-specific amplifications followed by recombination events and fixation of adaptive mutations, the Beps with a derived domain structure emerged independently in both lineages 3 and 4. www.sciencedirect.com
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82 Host-microbe interactions: bacteria
is closely related to the ancestral Bep from which the arsenal of effectors of both lineages 3 and 4 evolved independently by successive lineage-specific duplications and was further shaped by positive selection of diversified gene copies (Figure 1). The arsenal of Beps can be sorted into ten phylogenetic clades for the lineage 3, and seven phylogenetic clades for the lineage 4. The FIC-BID domain structure, followed by a positively charged C-tail, displays the most abundant effector protein type. However, a subset of effectors both in lineages 3 and 4 harbors other functional modules instead of the FIC domain, like tandem-repeated tyrosine-phosphorylation motifs (EPIYA and EPIYA-related motifs) or additional BID domains [11]. These different domain architectures suggest that these Beps arose from the ancestral domain structure by independent recombination events (Figure 1). Thus, the evolutionary diversification of the Beps created a wide array of adaptive functions dedicated to the cellular interaction within the mammalian hosts. This remodeling of preexisting effector proteins as an evolutionary mechanism to rapidly adapt to the host was described by Stavrinides et al. as ‘terminal reassortment’ in which effector genes recombine among themselves or with other genetic elements to give rise to new chimeric effectors [17]. Interestingly, comparative whole genome analysis of 14 Bartonella genomes revealed the presence of a Bartonella gene transfer agent (BaGTA) that represents the most conserved key innovation driving the explosive radiations of both lineages 3 and 4 [7]. Gene transfer agents (GTAs) are phage-like elements that generally transfer random pieces of the bacterial genome rather than their own DNA. GTA genes are located on the host chromosome, and GTAs generally transfer DNA from the producing cell to a recipient cell via a mechanism similar to transduction. Although the function of GTAs remains unclear regarding their role in evolution, they provide an efficient mechanism to duplicate and recombine genes [18]. Bep gene duplication and recombination by BaGTA may thus have conferred the high degree of adaptability to diverse mammalian reservoir hosts that have led to the explosive radiations of lineages 3 and 4.
Actin remodeling and invasome formation Historically, B. henselae was found to elicit VirB/D4-dependent phenotypes in vitro using human umbilical vein endothelial cells (HUVECs), like bacterial internalization via the invasome structure, inhibition of apoptosis, or activation of proinflammatory signaling [19]. This interaction of Bartonella with endothelial cells in vitro and the clinical descriptions of B. henselae in association with manifestations in the human vasculature, led to the proposition that vascular endothelial cells represent a part of the ‘blood-seeding niche’, that is colonized initially and from where bacteria are seeded to the blood stream to invade erythrocytes [2]. Studies of the infection of endothelial cells have shown that the internalization process of Current Opinion in Microbiology 2015, 23:80–85
Bartonella starts with endocytosis of one or a few bacteria into a vacuole called Bartonella-containing vacuole (BCV). This process is then arrested by the activities of several Beps (i.e. BepC, BepF and BepG in B. henselae), which are translocated via the VirB/D4 T4SS into the host cell cytoplasm [20–22]. It was shown that both BepG alone or a combined action of BepC and BepF impedes BCV formation via the inhibition of endocytosis. This leads to accumulation of bacterial aggregates at the cell surface, which invade the endothelial cells via F-actin-dependent invasome-mediated internalization [21,22]. Invasome formation mediated by combined action of BepC and BepF depends on F-actin modulation by Rac1/Scar1/Wave/Arp2/ 3, Cdc42/WASP/Arp2/3 and cofilin1. In contrast, BepGtriggered invasome formation requires only F-actin modulation by Rac1/Scar1/Wave/Arp2/3 and Cdc42/WASP/ Arp2/3, but not cofilin 1 [21,22]. Interestingly, constitutive-active Cdc42 or Rac1 can mimic BepF action in the BepC/BepF dependent invasome formation pathway, suggesting a regulatory role of BepF on the small Rho GTPases. BepF harbors a tyrosine-repeat motif near its N-terminus and contains three BID domains in the Cterminal part. Although the N-terminal tyrosine-repeat phosphorylation motif is phosphorylated upon translocation via the VirB/D4 T4SS, it is not involved in invasome formation [23]. Instead, the first two BID domains of BepF, BID-F1 and BID-F2, are sufficient to promote the formation of these structures [23]. BepG harbors an array of four BID domains. However, sequence comparisons of B. henselae BID domains revealed that the BID domains of BepF and BepG are poorly conserved between each other suggesting that these effectors may promote invasome formation by targeting different host functions [11].
Inhibition of apoptosis BepA mediates protection of endothelial cells from apoptosis and may thus contribute to the formation of vasoproliferative tumors as found in bacillary angiomatosis, a typical manifestation of immunocompromised patients infected with B. henselae or B. quintana [24,25]. On the molecular level, the BID domain of BepA was shown to block endothelial cell apoptosis by direct binding to host cell adenylyl cyclase, thereby potentiating GaS-dependent cAMP production. The elevation of cAMP levels and consecutive upregulation of cAMP-stimulated gene expression then leads to inhibition of apoptosis [24]. While inhibition of apoptosis is exclusively mediated by the BID domain of BepA, the FIC domain shows autoAMPylation and AMPylation of a target from total HeLa cell extract [26]. The nature of the host target and physiological consequences of AMPylation remains to be demonstrated.
BepE: an essential protein for Bartonella dissemination Recently, it was shown that HUVEC cells infected with the DbepE mutant of B. henselae are deficient in rear end www.sciencedirect.com
Function of Bartonella effector proteins Siamer and Dehio 83
detachment and undergo cell fragmentation. These phenotypes require the action of BepC, which possibly affects rear detachment during cell migration by modulating the actin cytoskeleton [2]. Ectopic expression of BepE from B. henselae or its orthologs in cells is sufficient to suppress the cell fragmentation phenotype [2]. The prominent activity of BepE in restoring cell migration led the authors to dissect the in vivo function of this effector protein. They demonstrated using a rat model and different routes of infection that BepE is essential to invade some nucleated cell types in the derma and spread the infection to the blood stream. As observed for BepA, BepF and BepG, this BepE-dependent phenotype also relies on the effector BID domains. The authors assumed that the migration of Bartonella from the derma to the blood stream could be achieved intracellularly in dendritic cells. This is supported by the fact that a trans-well migration assay showed an inhibition of dendritic cell migration upon infection with the B. henselae DbepDEF
mutant, while complementation with BepE restores it [2]. These new results on the function of BepE allowed the authors to extend the concept of the ‘blood-seeding niche’ (formerly known as ‘primary niche’) to ‘dermal niche’, which describes the dermal stage of infection [2]. Moreover, the authors demonstrated that BepE acts via its second BID domain on the RhoA signaling pathway to most likely preserve cells from fragmentation [2]. Indeed, they observed that BepE interferes with the inhibitory effect of the C3-based Rho inhibitor 1, which is known to ADP-ribosylate RhoA [27]. Altogether, the experimental studies showed that the Beps are essential for the primary stage of infection (Figure 2). It is surprising to see that most Bep-dependent phenotypes of B. henselae that have been studied so far rely on the BID domain, which evolved as a secretion signal and acquired, over the course of evolution, diverse functions, with distinct phenotypic properties. Until now,
Figure 2
1 Blood-sucking arthropod
9
2
Dermis Dendritic cells 3 BepE Bartonella 8
Dermal niche
4 Migration 5
7
Blood seeding niche Invasome formation
Vasculature
BepF
BepG
BepC
6 Capillary lumen
Inhibition of apoptosis BepA
Current Opinion in Microbiology
Model of the Bartonella infection process in the reservoir hosts. (1) Bartonella replicate first in the midgut of the arthropod vector and are excreted in the feces. (2) When the arthropods suck blood on mammals, it provides a local source of irritation followed by scratching and inoculation of Bartonella-containing insect feces into the derma. (3) Subsequently, Bartonella colonize the ‘dermal niche’, a term proposed by Okujava et al. (see Ref. [2]). This ‘dermal niche’ includes most likely dendritic cells, that are considered to disseminate Bartonella towards the ‘blood seeding niche’ (formerly known as ‘primary niche’) a process that strictly depends on the effector BepE (4). In the ‘blood-seeding niche’, bacteria are considered to colonize endothelial cells, which likely require the action of BepC, BepF, BepG and BepA (5). From this ‘blood seeding niche’, the bacteria are periodically seeded into the bloodstream where they invade erythrocytes and reinfect the cells of the ‘blood seeding niche’ (6). After limited replication inside the red blood cell (7), they persist in the ‘intraerythrocytic niche’ for the remaining life-span of the red blood cell (8) and are thus competent for transmission by a bloodsucking arthropod (9). www.sciencedirect.com
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Table 1 Beps studied and their function in Bartonella pathogenicity Beps
Bartonella species
BepA BepC
B. henselae B. henselae
BepE
B. henselae B. tribocorum
BepE
B. henselae
BepF
B. henselae
BepG
B. henselae
Bep2
B. rochalimae
Phenotype Inhibition of apoptosis F-actin modulation Trigger invasome formation Trigger cell fragmentation Preserves ECs from fragmentation and blocks defects of DCs migration caused by other Beps Unknown F-actin modulation Trigger invasome formation F-actin modulation Trigger invasome formation Unknown
Domain implicated BID domain Unknown
BID domains 1 and 2
Tyrosine-phosphorylation motif BID domains 1 and 2 Multiple BID domains FIC domain
Target
References
Adenylyl cyclase Related to Rac1 and Cdc42 signaling, Cofilin1-dependent RhoA signaling pathway
[25,26] [2,21,22]
Csk SHP2 Related Rac1 and Cdc42 signaling, Cofilin1-dependent Related to Rac1 and Cdc42 signaling, Cofilin1-independent AMPylation of Vimentin
[28]
[2]
[21–23] [21,22] [29]
Abbreviations: ECs, endothelial cells; DCs, dendritic cells.
no phenotype in vivo or in vitro has been shown to depend on the tyrosine phosphorylation motifs or the FIC domain of any Bep from lineage 4. BepD, BepE and BepF from the lineage 4 and Bep9 from the lineage 3 were shown to be tyrosine-phosphorylated in human cells [6,11,28]. More particularly, using a proteomic screen to identify potential targets of all known tyrosine-phosphorylated bacterial effectors, BepE was shown to interact with Csk upon phosphorylation on Tyr37 and with SHP2 upon phosphorylation with Tyr64. Although this result was confirmed by a co-immunopurification assay, further work is required to elucidate the biological relevance of these tyrosine-phosphorylation and subsequent protein interactions [28]. All the Beps discussed and their implications in Bartonella pathogenicity are listed in Table 1.
Beps from the lineage 3: opening the black box While the Bep effectors of lineage 4 and particularly of B. henselae have been extensively studied, the functions of the Beps of lineage 3 are still poorly understood. As described above, these Beps are classified in 10 phylogenetic clades and most of them, except the clade 9, harbor the classical FIC-BID domain organization [6]. Recently, Pieles et al. showed by an unbiased mass spectrometrybased approach that Bep2 from B. rochalimae AMPylates via its FIC domain the intermediate filament protein vimentin [29]. Although the contribution to virulence by this AMPylation remains to be elucidated, this method paves the way for identifying AMPylation targets for other FIC domains of Beps from lineages 3 and 4. This approach can further be adapted to study other posttranslational modifications for which isotopically labeled substrates are available. As it was shown that FIC domains can mediate diverse post-translational modifications [12–15], this approach offers a potentially powerful means to identify both new targets of Beps and their FIC domain-mediated post-translational modifications. Current Opinion in Microbiology 2015, 23:80–85
Concluding remarks Recent advances have enhanced our understanding of how the repertoire of Beps finely manipulates host cell processing steps enabling Bartonella to interact, colonize, proliferate and persist in host cells. Here we have reviewed the evolutionary diversification of these effector proteins, their function and their phenotypic properties. Although they share a modular architecture, they acquired different functions within host cells. Some effectors, like BepC, BepF and BepG, appear to act in concert whereas others, like BepC and BepE, appear to be antagonistic. However, despite the progress made to elucidate the functions of several Beps from lineage 4, the roles of the ones from the lineage 3 remain to be elucidated. One of the big challenges in the study of bacterial effectors is that they exert their function(s) in concert with other effectors and therefore are regulated both temporally and spatially. We hope that new approaches developed will enhance our understanding of the contribution of these proteins to the Bartonella–host interaction, making the next few years very exciting in this regard.
Acknowledgements We would like to thank Alexander Harms, Dr. Rusudan Okujava and Dr. Maxime Que´batte for critical reading of the manuscript. This work was supported by Grant 310030B_149886 from the Swiss National Science Foundation (SNSF, www.snf.ch), advanced Grant 340330 (FicModFun) from the European Research Council (ERC), and Grant 51RTP0_151029 for the research and Technology Development (RTD) project TargetInfectX in the frame of systemsX.ch (www.systemX.ch), the Swiss Initiative for System Biology.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1. Chomel BB, Boulouis HJ, Breitschwerdt EB, Kasten RW, VayssierTaussat M, Birtles RJ, Koehler JE, Dehio C: Ecological fitness and strategies of adaptation of Bartonella species to their hosts and vectors. Vet Res 2009, 40:29. www.sciencedirect.com
Function of Bartonella effector proteins Siamer and Dehio 85
2.
Okujava R, Guye P, Lu YY, Mistl C, Polus F, Vayssier-Taussat M, Halin C, Rolink AG, Dehio C: A translocated effector required for Bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors. PLoS Pathog 2014, 10:e1004187. Identified BepE as an essential protein for Bartonella dissemination from the dermal site of inoculation to the blood stream.
3.
Pulliainen AT, Dehio C: Persistence of Bartonella spp. stealth pathogens: from subclinical infections to vasoproliferative tumor formation. FEMS Microbiol Rev 2012, 36:563-599.
4. Angelakis E, Raoult D: Pathogenicity and treatment of Bartonella infections. Int J Antimicrob Agents 2014, 44:16-25. A comprehensive review covering the clinical aspect of Bartonella infections. 5.
Harms A, Dehio C: Intruders below the radar: molecular pathogenesis of Bartonella spp.. Clin Microbiol Rev 2012, 25:42-78. A comprehensive review covering the molecular and cellular basis of pathogenesis by Bartonella spp.
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Engel P, Salzburger W, Liesch M, Chang CC, Maruyama S, Lanz C, Calteau A, Lajus A, Medigue C, Schuster SC, Dehio C: Parallel evolution of a type IV secretion system in radiating lineages of the host-restricted bacterial pathogen Bartonella. PLoS Genet 2011, 7:e1001296.
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Guy L, Nystedt B, Toft C, Zaremba-Niedzwiedzka K, Berglund EC, Granberg F, Naslund K, Eriksson AS, Andersson SG: A gene transfer agent and a dynamic repertoire of secretion systems hold the keys to the explosive radiation of the emerging pathogen Bartonella. PLoS Genet 2013, 9:e1003393. A study which proposed an updated Bartonella phylogeny based on the sequencing of six new genomes and description of the highly conserved GTA in Bartonella genomes.
8.
Zhu Q, Kosoy M, Olival KJ, Dittmar K: Horizontal transfers and gene losses in the phospholipid pathway of Bartonella reveal clues about early ecological niches. Genome Biol Evol 2014, 6:2156-2169. A new phylogenetic study highlighting the impact of horizontal gene transfer on Bartonellae lineage evolution.
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Current Opinion in Microbiology 2015, 23:80–85
