New insights into the role of Bartonella effector proteins in pathogenesis

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<ul><li><p>New insights into the role of Bartonella effector proteinsin pathogenesisSabrina Siamer and Christoph Dehio</p><p>Available online at www.sciencedirect.com</p><p>ScienceDirectThe facultative intracellular bacteria Bartonella spp. share a</p><p>common infection strategy to invade and colonize mammals in</p><p>a host-specific manner. Following transmission by blood-</p><p>sucking arthropods, Bartonella are inoculated in the derma and</p><p>then spread, via two sequential enigmatic niches, to the blood</p><p>stream where they cause a long-lasting intra-erythrocytic</p><p>bacteraemia. The VirB/VirD4 type IV secretion system (VirB/D4</p><p>T4SS) is essential for the pathogenicity of most Bartonella</p><p>species by injecting an arsenal of effector proteins into host</p><p>cells. These bacterial effector proteins share a modular</p><p>architecture, comprising domains and/or motifs that confer an</p><p>array of functions. Here, we review recent advances in</p><p>understanding the function and evolutionary origin of this</p><p>fascinating repertoire of host-targeted bacterial effectors.</p><p>Addresses</p><p>Focal Area Infection Biology, Biozentrum, University of Basel, Basel,</p><p>Switzerland</p><p>Corresponding author: Dehio, Christoph (christoph.dehio@unibas.ch)</p><p>Current Opinion in Microbiology 2015, 23:8085</p><p>This review comes from a themed issue on Host-microbe interac-</p><p>tions: bacteria</p><p>Edited by David Holden and Dana Philpott</p><p>http://dx.doi.org/10.1016/j.mib.2014.11.007</p><p>1369-5274/# 2014 Published by Elsevier Ltd.</p><p>IntroductionBartonella spp. are facultative intracellular bacteria thatare responsible for long-lasting intra-erythrocytic bacter-aemia in diverse mammalian reservoir hosts and aretransmitted by blood-sucking arthropods most likelythrough inoculation of skin lesions by contaminatedinsect feces, or contact with infected animals via scratch-ing or bites [1]. Following dermal inoculation, Bartonellacolonize two sequential niches, respectively called der-mal niche (describing the dermal stage of infection) andblood-seeding niche (formerly known as primaryniche), considered to include dendritic and endothelialcells [2]. Subsequently, Bartonella reach the bloodstream where, restricted to the specific reservoir host,they invade erythrocytes and persist intracellularly for theremaining lifetime of the red blood cell [3]. SpecificCurrent Opinion in Microbiology 2015, 23:8085 adaptation to the reservoir host causes no or only milddisease symptoms, while infection of incidental hosts canbe associated with broad spectrum of diseases. Theexception is B. bacilliformis, a human-specific species thatcauses life-threatening disease. Most other human infec-tions are caused by the human-specific species B. quintanaand the zoonotic cat-specific species B. henselae. Theclinical manifestations and treatments of Bartonellainfection have been extensively reviewed elsewhereand therefore will not be covered in this review [4,5].</p><p>Different genomic and phylogenetic studies revealed thatthe genus Bartonella is divided into four lineages plusB. tamiae and B. australis, which occupy ancestral posi-tions [6,7,8]: lineage 1 is solely composed of the deadlypathogen B. bacilliformis. Lineage 2 is composed of rumi-nant-specific species (e.g. deer-specific B. schoenbuchensis)that have acquired the Vbh (VirB-homologous) T4SS thatis closely related to the VirB/D4 T4SS. Lineage 3 iscomposed of species infecting diverse mammals (e.g.fox/dog-specific B. rochalimae and cat-specific B. clarrid-geiae) that have acquired the VirB/D4 T4SS and the mostspecies-rich lineage 4 contains species (e.g. cat-specificB. henselae, human-specific B. quintana, or rat-specificB. tribocorum) that harbor the VirB/D4 and the TrwT4SS [6,7,8]. Both T4SS present in the lineage 4 specieswere shown to be essential for host interaction at differentstages of the infection cycle [9,10]. The Trw T4SS isinvolved in the erythrocyte invasion process by mediatinghost-specific adhesion to these cells [10]. The VirB/D4T4SS contributes to pathogenicity by translocating acocktail of evolutionary-related effector proteins, calledBeps (Bartonella effector proteins) into nucleated cells ofboth the dermal and the blood-seeding niches [2,6,9].Once inside host cells, these Beps target host componentsand modulate cellular processes to the benefit of thebacteria. Therefore, defining the role of the various Bepsis key to gain a better understanding of the pathogenesisof Bartonella. This review summarizes advances made indeciphering the evolution and functional role of the Bepsin Bartonellahost cell interaction.</p><p>Acquisition of the VirB/D4 T4SSThe current model indicates that the radiating lineage3 and 4 have independently acquired the virB/D4 T4SSlocus with at least one effector gene by horizontal genetransfer [6,7]. The similarity of the VirB/D4 T4SS withbacterial conjugation systems suggests that the virB/D4locus was probably acquired via horizontal gene transferfrom a conjugative plasmid. Whole genome shotgunwww.sciencedirect.com</p><p>http://crossmark.crossref.org/dialog/?doi=10.1016/j.mib.2014.11.007&amp;domain=pdfmailto:christoph.dehio@unibas.chhttp://dx.doi.org/10.1016/j.mib.2014.11.007http://www.sciencedirect.com/science/journal/13695274</p></li><li><p>Function of Bartonella effector proteins Siamer and Dehio 81analysis of representative species of lineages 3 and4 demonstrated lineage-specific integration of the virB/D4 locus and the presence of lineage-specific repertoiresof fast evolving bep genes that display a high degree ofinter-species variation. Comparative whole genomeanalysis further indicated that the virB/D4 and bep geneclusters represent a major driver of the adaptive radiationobserved in the lineages 3 and 4 which represent aremarkable example of parallel evolution [6].</p><p>Evolution of the BepsBartonellahost interaction exposes the arsenal of Beps toa strong selective pressure resulting in their rapid evolu-tion, which could explain their modular architecture. Thismodular architecture of the Beps, which is derived from asingle ancestor composed of an N-terminal FIC and C-terminal BID domain, evolved by extensive rounds ofduplication, diversification and reshuffling of domains(Figure 1) [6,11]. The BID domain together with aFigure 1</p><p>Independeduplicatio</p><p>Ancestral effect</p><p>Representative Beps from the lineage 3</p><p>Recombination andadaptive muta</p><p>FIC motif</p><p>FIC B</p><p>FIC BID</p><p>FIC BID</p><p>FIC</p><p>FIC</p><p>FIC</p><p>BID</p><p>BIDBID</p><p>BID</p><p>BID</p><p>BID</p><p>FIC motif</p><p>FIC motif</p><p>FIC motif</p><p>FIC motif</p><p>HPFxxxNG</p><p>xPFxxGNx</p><p>Parallel evolution of the Beps from the lineages 3 and 4. The Beps from the</p><p>the same FIC-BID domain architecture. Subsequently, after lineage-specific</p><p>adaptive mutations, the Beps with a derived domain structure emerged inde</p><p>www.sciencedirect.com positively charged C-tail constitute a secretion signalfor the VirB/D4 T4SS, while the FIC, also present inother bacterial effectors and many more endogenousproteins found in all domains of life, was shown tomediate post-translational modifications such as AMPyla-tion, phosphocholination or phosphorylation [1215].Interestingly, previous studies suggest that the BIDdomain together with a positively charged C-tail, whichevolved as bi-partitie T4SS signal from relaxases ofalphaproteobacterial conjugation systems, was acquiredby the ancestral Bartonella effector protein to facilitateinjection into target cells [11]. This is supported by thefact that in B. grahamii the Vbh T4SS, which is closelyrelated to the VirB/D4 T4SS, is encoded on a plasmid inaddition to a chromosomally encoded copy [16]. The vbhlocus on the plasmid also encodes a toxin called VbhT inB. schoenbuchensis, which harbors the classical FIC-BIDdomain architecture as found conserved in many Beps[13]. It is thus tempting to speculate that the VbhT toxinntn</p><p>or VbhT</p><p> fixation oftions</p><p>Representative Beps from the lineage 4</p><p>ID</p><p>FIC</p><p>FIC FIC</p><p>FIC</p><p>BID BID</p><p>BID</p><p>BID BID</p><p>BID</p><p>BID</p><p>BID</p><p>BID BID</p><p>BID</p><p>BID</p><p>BID</p><p>FIC BID</p><p>FIC BID</p><p>FIC motif</p><p>FIC motif</p><p>FIC motifHPFxxGNx</p><p>xPFxxGNx</p><p>FIC motif</p><p>FIC motif</p><p>Current Opinion in Microbiology</p><p> lineages 3 and 4 evolved possibly from the VbhT toxin, which harbors</p><p> amplifications followed by recombination events and fixation of</p><p>pendently in both lineages 3 and 4.</p><p>Current Opinion in Microbiology 2015, 23:8085</p></li><li><p>82 Host-microbe interactions: bacteriais closely related to the ancestral Bep from which thearsenal of effectors of both lineages 3 and 4 evolvedindependently by successive lineage-specific duplicationsand was further shaped by positive selection of diversifiedgene copies (Figure 1). The arsenal of Beps can be sortedinto ten phylogenetic clades for the lineage 3, and sevenphylogenetic clades for the lineage 4. The FIC-BIDdomain structure, followed by a positively charged C-tail,displays the most abundant effector protein type. How-ever, a subset of effectors both in lineages 3 and 4 harborsother functional modules instead of the FIC domain, liketandem-repeated tyrosine-phosphorylation motifs (EPIYAand EPIYA-related motifs) or additional BID domains [11].These different domain architectures suggest that theseBeps arose from the ancestral domain structure by inde-pendent recombination events (Figure 1). Thus, the evol-utionary diversification of the Beps created a wide array ofadaptive functions dedicated to the cellular interactionwithin the mammalian hosts. This remodeling of pre-existing effector proteins as an evolutionary mechanismto rapidly adapt to the host was described by Stavrinideset al. as terminal reassortment in which effector genesrecombine among themselves or with other geneticelements to give rise to new chimeric effectors [17].</p><p>Interestingly, comparative whole genome analysis of 14Bartonella genomes revealed the presence of a Bartonellagene transfer agent (BaGTA) that represents the mostconserved key innovation driving the explosive radiationsof both lineages 3 and 4 [7]. Gene transfer agents (GTAs)are phage-like elements that generally transfer randompieces of the bacterial genome rather than their ownDNA. GTA genes are located on the host chromosome,and GTAs generally transfer DNA from the producingcell to a recipient cell via a mechanism similar to trans-duction. Although the function of GTAs remains unclearregarding their role in evolution, they provide an efficientmechanism to duplicate and recombine genes [18]. Bepgene duplication and recombination by BaGTA may thushave conferred the high degree of adaptability to diversemammalian reservoir hosts that have led to the explosiveradiations of lineages 3 and 4.</p><p>Actin remodeling and invasome formationHistorically, B. henselae was found to elicit VirB/D4-de-pendent phenotypes in vitro using human umbilical veinendothelial cells (HUVECs), like bacterial internalizationvia the invasome structure, inhibition of apoptosis, oractivation of proinflammatory signaling [19]. This inter-action of Bartonella with endothelial cells in vitro and theclinical descriptions of B. henselae in association with mani-festations in the human vasculature, led to the propositionthat vascular endothelial cells represent a part of theblood-seeding niche, that is colonized initially and fromwhere bacteria are seeded to the blood stream to invadeerythrocytes [2]. Studies of the infection of endothelialcells have shown that the internalization process ofCurrent Opinion in Microbiology 2015, 23:8085 Bartonella starts with endocytosis of one or a few bacteriainto a vacuole called Bartonella-containing vacuole (BCV).This process is then arrested by the activities of severalBeps (i.e. BepC, BepF and BepG in B. henselae), which aretranslocated via the VirB/D4 T4SS into the host cellcytoplasm [2022]. It was shown that both BepG aloneor a combined action of BepC and BepF impedes BCVformation via the inhibition of endocytosis. This leads toaccumulation of bacterial aggregates at the cell surface,which invade the endothelial cells via F-actin-dependentinvasome-mediated internalization [21,22]. Invasome for-mation mediated by combined action of BepC and BepFdepends on F-actin modulation by Rac1/Scar1/Wave/Arp2/3, Cdc42/WASP/Arp2/3 and cofilin1. In contrast, BepG-triggered invasome formation requires only F-actin modu-lation by Rac1/Scar1/Wave/Arp2/3 and Cdc42/WASP/Arp2/3, but not cofilin 1 [21,22]. Interestingly, constitu-tive-active Cdc42 or Rac1 can mimic BepF action in theBepC/BepF dependent invasome formation pathway,suggesting a regulatory role of BepF on the small RhoGTPases. BepF harbors a tyrosine-repeat motif near itsN-terminus and contains three BID domains in the C-terminal part. Although the N-terminal tyrosine-repeatphosphorylation motif is phosphorylated upon transloca-tion via the VirB/D4 T4SS, it is not involved in invasomeformation [23]. Instead, the first two BID domains of BepF,BID-F1 and BID-F2, are sufficient to promote the for-mation of these structures [23]. BepG harbors an array offour BID domains. However, sequence comparisons ofB. henselae BID domains revealed that the BID domainsof BepF and BepG are poorly conserved between eachother suggesting that these effectors may promote inva-some formation by targeting different host functions [11].</p><p>Inhibition of apoptosisBepA mediates protection of endothelial cells from apop-tosis and may thus contribute to the formation of vaso-proliferative tumors as found in bacillary angiomatosis, atypical manifestation of immunocompromised patientsinfected with B. henselae or B. quintana [24,25]. Onthe molecular level, the BID domain of BepA was shownto block endothelial cell apoptosis by direct binding tohost cell adenylyl cyclase, thereby potentiating GaS-de-pendent cAMP production. The elevation of cAMP levelsand consecutive upregulation of cAMP-stimulated geneexpression then leads to inhibition of apoptosis [24].While inhibition of apoptosis is exclusively mediated bythe BID domain of BepA, the FIC domain shows auto-AMPylation and AMPylation of a target from total HeLacell extract [26]. The nature of the host target andphysiological consequences of AMPylation remains tobe demonstrated.</p><p>BepE: an essential protein for BartonelladisseminationRecently, it was shown that HUVEC cells infected withthe DbepE mutant of B. henselae are deficient in rear endwww.sciencedirect.com</p></li><li><p>Function of Bartonella effector proteins Siamer and Dehio 83detachment and undergo cell fragmentation. These phe-notypes require the action of BepC, which possiblyaffects rear detachment during cell migration by modu-lating the actin cytoskeleton [2]. Ectopic expression ofBepE from B. henselae or its orthologs in cells is sufficientto suppress the cell fragmentation phenotype [2]. Theprominent activity of BepE in restoring cell migration ledthe authors to dissect the in vivo function of this effectorprotein. They demonstrated using a rat model and differ-ent routes of infection that BepE is essential to invadesome nucleated cell types in the derma and spread theinfection to the blood stream. As observed for BepA,BepF and BepG, this BepE-dependent phenotype alsorelies on the effector BID domains. The authors assumedthat the migration of Bartonella from the derma to theblood stream could be achieve...</p></li></ul>