Virulence-associated type IV secretion systems of Bartonella

Virulence-associated type IV secretionsystems of BartonellaGunnar Schroder and Christoph Dehio

Division of Molecular Microbiology, Biozentrum of the University of Basel, CH-4056 Basel, Switzerland

Type IV secretion systems (T4SSs) are transport

machineries of Gram-negative bacteria that mediate

interbacterial DNA-transfer, and secretion of virulence

factors into eukaryotic target cells. A growing number of

human pathogenic bacteria use T4SSs for intercellular

delivery of effector molecules that modify host cellular

functions in favour of the pathogen. Recent advances in

studying the molecular mechanisms of Bartonella

pathogenesis have provided evidence for the central

roles of two distinct T4SSs, VirB/VirD4 and Trw, in the

ability of the bacteria to colonize, invade and persist

within either vascular endothelial cells or erythrocytes,

respectively. The identification of VirB/VirD4-trans-

ported substrates and the delineation of their secretion

signal have paved the way towards understanding the

molecular mechanisms underlying Bartonella–host cell

interaction and modulation, as well as the exploitation

of this system for engineered substrate delivery into

mammalian target cells.

Introduction

Type IV secretion systems (T4SSs) are molecular transportmachineries that are ancestrally related to bacterialconjugation systems. Conjugation accounts for the efficientspread of genetic traits among bacteria and, therefore, is amajor cause of the rapid dissemination of antibioticresistance genes. Bacterial pathogens targeting eukaryotichost cells have adopted theseDNA secretion systems for thedelivery of virulence factors [1,2]. Prominent examples arethe Cag system of the gastric pathogen Helicobacter pyloriand the Dot/Icm system of the pneumonic pathogenLegionella pneumophila. The Cag system directs thetransfer of the CagA protein into gastric epithelial cells.After transfer, CagA induces morphological changes in thehost cell by interacting with multiple signaling molecules[3]. In the Dot/Icm system of L. pneumophila, severalsecreted substrates have been identified [4,5]. Some ofthese effectors probably enable intracellular survival ofL. pneumophila in a specialized organelle resembling theendoplasmatic reticulum (ER). Following the initial uptakeof thebacteria intoaphagosomal compartment, theeffectorsprevent endocytosis and recruit ER-derived vesicles to thephagosomal membrane to mimic an ER compartment inwhich they can replicate [6].

Bartonellae are pathogens of humans and mammals, inwhich they can cause a variety of diseases that result from

Corresponding author: Dehio, C. ([email protected]).Available online 2 June 2005

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bacterial colonization of erythrocytes and vascular endo-thelial cells (ECs) [7]. Each Bartonella species possessesone or several primary (reservoir) hosts, in which it causesa long-lasting intraerythrocytic bacteremia. In addition,the bacterium is often capable of infecting secondary(incidental) hosts, in which it is unable to persist withinerythrocytes. However, ECs represent a major target celltype for bacterial colonization in both reservoir andincidental hosts. Transmission among reservoir hosts orfrom reservoir to incidental hosts occurs via blood-suckingarthropods or via direct contact. The damage inflictedupon the host ranges from subclinical (almost no damage)to highly virulent (strong damage) and often depends onthe immunological status of the host. Two Bartonellaspecies that infect humans as primary hosts are known:Bartonella bacilliformis and Bartonella quintana [7].B. bacilliformis is the first species described in the genusBartonella. This pathogen is the causative agent ofCarrion’s disease, a biphasic disease that is endemic inthe Andes due to the limited spread of its arthropodtransmission vector, the sandfly Lutzomyia verrucarum.The acute phase of Carrion’s disease is characterized byan intraerythrocytic bacteremia, which results in an oftenfatal hemolytic anemia (Oroya fever). In cases of survivalof the patient, the subsequent chronic phase manifests byvasculoproliferative lesions of the skin (Verruga peruana)due to the proliferation of colonized ECs [8]. By contrast,B. quintana is a wide-spread but less aggressive humanpathogen transmitted by the human body louse, Pediculushumanus. It causes a cycling fever (Trench fever) as themanifestation of intraerythrocytic bacteremia in acuteinfection, endocarditis and, in cases of chronic infectionof immunocompromized patients, vasculoproliferativeskin lesions (bacillary angiomatosis) [8]. Within thegenus, Bartonella henselae is responsible for most casesof human disease and represents the prototype of thezoonotic bartonellae. B. henselae causes intraerythrocyticbacteremia in cats and is transmitted by the cat flea(Ctenocephalides felis) within the feline reservoir. Trans-mission to the incidental human host typically occurs bycat scratch or bite. Cat scratch disease (CSD), character-ized by an inflammation of lymph nodes, is the typicalmanifestation of B. henselae infection in humans. Endo-carditis, bacteremia with fever and neuroretinitis repre-sent other clinical manifestations. Immunosuppressioncan lead to chronic infection of the vasculature, manifest-ing either as bacillary angiomatosis or as bacillary peliosis(resulting from vasculoproliferation in inner organs) [7,9].

Review TRENDS in Microbiology Vol.13 No.7 July 2005

d. doi:10.1016/j.tim.2005.05.008

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TRENDS in Microbiology

CY

EX

IM

PP

OM

S

S

PG

10 10 88

1 19 9

7

3 3

66

4

4 444

4

5

5 5

5

7

11D4

2

ATP ADP+Pi

Figure 1. Model of the VirB/VirD4 type IV secretion system (T4SS) machinery. The

T4SS machinery is a multi-component protein complex spanning the inner and

outer membranes of Gram-negative bacteria. This transporter enables secretion of

substrates from the bacterial cytoplasm directly into the cytoplasm of infected host

cells or into the extracellular milieu. In VirB/VirD4-like T4SS, 11 components,

homologues of Agrobacterium tumefaciens VirB2–VirB11 and VirD4, are typically

required for secretion (reviewed in [25]). VirB2–VirB11 form a trans-envelope

channel and an extracellular pilus structure. The presence of T4SS pili in

bartonellae remains to be shown; their general role in T4SS is thought to be in

mediating initial contact with a target cell via adherence to cellular receptors. For

secretion of dedicated T4SS substrates, VirD4, the coupling protein (T4CP) is

additionally required. Its function is to deliver the substrates to the VirB channel

and, along with VirB4 and VirB11, to energize their transport across the channel.

Colour code: yellow, pilus-associated extracellular components of the T4SS

machinery; blue, all other components of the T4SS machinery (pore complex and

energizers); pink, T4CP; green, T4SS substrates. Abbreviations: CY, cytoplasm; EX,

extracellular milieu; IM, inner membrane; OM, outer membrane; PG, peptidoglycan

layer; PP, periplasm.

Review TRENDS in Microbiology Vol.13 No.7 July 2005 337

In recent years, animal and in vitro models haveenabled the study of the molecular and cellular basis ofBartonella pathogenesis [7,10]. Two distinct T4SSs,VirB/VirD4 and Trw, as well as a set of Bartonella-trans-located effector proteins (Beps), have now been identifiedas bona fide pathogenicity factors of bartonellae [11–13].Here, we outline the characteristics of these secretionsystems and highlight their importance for Bartonella–host cell interactions, as well as for general aspects oftype IV secretion.

The VirB/VirD4 T4SS of Bartonella

The first indication for the presence of a T4SS inBartonella was obtained when exploring the geneticlocus of a 17-kDa protein, previously identified as animmunodominant antigen of B. henselae [14,15]. The17-kDa protein was revealed to be a VirB5-like represen-tative of a T4SS comprizing the full set of homologuestypically found in VirB-like T4SSs, including the func-tionally associated T4SS coupling protein (T4CP) VirD4(Figure 1). VirB/VirD4-like T4SSs are probably present inall Bartonella species, although this has not been provenfor B. bacilliformis [16]. The Bartonella VirB/VirD4system is highly conserved between the species in whichthe encoding loci have been sequence-analyzed (B. henselae,B. quintana and Bartonella tribocorum) [17–19]. Thegenes encoding the Bartonella VirB/VirD4 system arecollinear and sequence-related to the T4SSs of conjugativeplasmids, with closest similarity to the ones of rhizobac-terial origin (including the Agrobacterium tumefacienscryptic plasmid pAT; Figure 2). The A. tumefaciensVirB/VirD4 T-DNA transfer system and other virulence-related T4SSs of mammalian pathogens (e.g. the VirBsystem of Brucella spp.) are more distantly related [18].The VirB/VirD4 system of bartonellae is part of aBartonella-specific pathogenicity island (PAI), which addi-tionally encodesa set of proteins thathavebeen identifiedasVirB/VirD4-translocated substrates (Beps) [12].

Role of the VirB/VirD4 system in pathogenesis

The requirement of the VirB/VirD4 secretion system forinfectivity of bartonellae was demonstrated in an animalmodel, showing that virB4 or virD4 deletion mutants ofB. tribocorum lost their ability to cause intraerythrocyticbacteremia in infected rats [18]. Segregation analysis ofthe complementation plasmids further indicated thatVirB4 and VirD4 are required at an early infectionphase before the onset of bacteremia occurs on days 4 or5. This observation suggested that the VirB/VirD4 systemis required for colonizing a primary infection niche, whichis thought to comprize ECs as major target cell type [20].An in vitro infection model of cultured human ECs hasdemonstrated that the corresponding VirB/VirD4 systemof B. henselae is indeed mediating most of the cellularphenotypes associated with B. henselae infection ofhuman ECs [12,13]. These phenotypes consist of: (i) amassive cytoskeleton rearrangement, resulting in theengulfment of bacterial aggregates (a phenotype called‘invasome’ formation); (ii) nuclear factor kB-dependentproinflammatory activation, characterized by chemokinesecretion and expression of cell adhesion proteins; and

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(iii) enhanced cell survival as a result of anti-apoptosis.The subversion of multiple EC functions by the VirB/VirD4system is considered to be crucial for both the establish-ment of infection in the primary niche of the reservoir hostand the formation of vasculoproliferative lesions inimmunocompromized patients [7]. Invasome-mediateduptake is considered to represent a specific mechanismfor EC colonization [21] and might be reflected by thecharacteristic bacterial aggregates found in close associ-ation with proliferating ECs within bacillary angiomatosislesions [22,23]. The VirB/VirD4-dependent proinflamma-tory activation of ECs is thought to mediate therecruitment of phagocytes, which, upon activation byB. henselae infection, are triggered to release pro-angiogenic factors (such as vascular endothelial growthfactor, VEGF) that promote EC proliferation in a



49 %23 4 5 6 7 8 9 10 11

A1

virD4

C E F1 F2

23 4 5 6 7 8 9 10 11 virD4

52%

61% 91% 73% 50%∆ ∆ 32% 45% ∆

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Ø 43%

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oriT

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C D E F G

B. henselae

B. quintana

A. tumefaciens

A B

virB bepvirD4

avhB tra

Ø 86%

Figure 2. The virB/virD4/bep PAI of Bartonella. Genetic organization of the Bartonella-specific virB/virD4/bep pathogenicity island (PAI) and its phylogenetic relationship to the

AvhB/Tra conjugation system (avh/tra loci) of the Agrobacterium tumefaciens cryptic plasmid pAT. The degree of conservation for individual genes or groups of genes (in

average, Ø) is indicated in percentages of amino acid identity of the translated gene products. The virB and virD4 genes are highly conserved between bartonellae (also

including Bartonella tribocorum) and show a considerable degree of identity towards the avhB/tra genes of A. tumefaciens plasmid pAT. By contrast, the bep genes are less

conserved between different Bartonella species and have no counterparts in other T4SSs. However, the bep genes encode the novel BID domain (boxes coloured in red),

which, after its identification in bartonellae, was also found to be present in conjugative relaxases like the one encoded by traA of the AvhB/Tra conjugation system. Colour

code: yellow, pilus-associated extracellular components of the T4SS machinery; blue, all other components of the T4SS machinery (pore complex and energizers); pink,

T4CP; green, T4SS substrates.


paracrine manner [21]. The VirB/VirD4-mediated anti-apoptosis of ECs supposedly protects the cellular habitatof Bartonella in the primary niche and contributesindirectly to vasculoproliferative lesion formation due toincreased cell survival [13,21]. Interestingly, the directmitogenic stimulation of ECs described as a prominentphenotype ofB. henselae infection proved to be VirB/VirD4-independent and – at high bacterial titers and longinfection times – was even found to be counteracted byVirB/VirD4-mediated cytostatic or cytotoxic effects [12,13].There is an obvious need for an animal model of vasculo-proliferative lesion formation by B. henselae that enablesassessment of the in vivo relevance of the VirB/VirD4-dependent cellular phenotypes ofECs studied so far in vitro.

Molecular basis of VirB/VirD4-mediated virulence

A yeast two-hybrid interaction study of the VirB systemof B. henselae has largely confirmed the protein inter-actions earlier identified in other VirB-like T4SSs [24].The VirB/VirD4 system of A. tumefaciens, as well as thehomologous mating pair formation (Mpf)/T4CP system ofconjugative plasmids, which have been characterizedextensively, serve here as a reference [25]. In additionto the known interactions, a novel interaction pair,VirB3–VirB5, was identified and characterized biochemi-cally in B. henselae [24]. Together, these data fit well intothe model view of the T4SS machinery (Figure 1).

The discovery of the virB/virD4/bep PAI in B. henselaehas led to the identification of seven VirB/VirD4-translocated substrates (BepA–BepG; Figure 2). Similar


to VirB/VirD4, BepA–BepG are required for invasion,proinflammatory activation and antiapoptotic protectionof ECs [12]. Thus, BepA–BepG are likely to represent themolecular effectors mediating VirB/VirD4-dependentcellular responses in infected ECs. The Bep substratesdisplay a modular structure. Each of the substratescontains one or several copies of a domain that isconserved among the Beps but has not been describedpreviously. This domain was later identified as beingpart of a translocation signal and was thus termedBep-intracellular delivery (BID) domain [12] (see subse-quent section). BepA, BepB and BepC additionally harboran N-terminal domain, which is homologous to the widelydistributed bacterial protein family of Fic (filamentationinduced by cAMP) proteins [26]. The function of Ficproteins is not well understood. In Escherichia coli, Fic1mediates a cAMP-inducible filamentation phenotype andis proposed to be involved in the regulation of bacterial celldivision [26]. The presence of a Fic domain in the secretedsubstrates BepA–BepC rather suggests a role in theeukaryotic host cell. Indeed, a search for Fic-likeproteins in mammals retrieves the huntingtin-interactingprotein E (HypE, [27]), suggesting that the Fic-domain inBepA–BepC might have a function related to HypE.However, this function remains to be determined becausenothing is known about HypE except for its interactionwith a pathogenic variant of huntingtin [27]. BepD, BepEand BepF each contain several peptide motifs at theirN-termini that resemble eukaryotic tyrosine-phosphoryl-ation motifs. An assay monitoring the phosphorylation



state of BepD has indeed shown that, following transloca-tion, BepD becomes tyrosine-phosphorylated by hostcellular kinases [12]. Importantly, tyrosine-phosphoryl-ation is also a feature of the H. pylori T4SS substrateCagA, which, after phosphorylation, induces changes inthe tyrosine phosphorylation state of distinct cellularproteins [28]. Whether BepD–BepF induce a similarcascade, and whether this directly or indirectly mediatesany of the VirB/VirD4/Bep-dependent phenotypes,remains to be elucidated. BepG is unique among theBeps because it appears to be entirely composed of BIDdomains. The assessment of the contribution of individualBep substrates to the observed phenotypes of EC infectionis a major topic for future research.

Identification of a bipartite T4SS signal

The repeated occurrence of a conserved domain (BIDdomain) in each of the Bep substrates has suggested thatthis domain might be responsible for mediating substratetransport via the VirB/VirD4 system. Using a reporterassay (Cre Recombinase Assay for Translocation, CRAfT),the translocation signal of the Bep substrates wasdelineated [12]. The BID domain plus the C-terminal,positively charged tail sequence of the Bep substrateswere thus shown to constitute a bipartite translocationsignal [12]. A search for related translocation signals inother T4SSs has identified BID-like domains in therelaxases of rhizobial conjugation systems, such as TraAof the A. tumefaciens AvhB/Tra system (Figure 2) [12].Conjugative relaxases are known to cleave, covalentlybind to and deliver plasmid-derived DNA into recipientbacteria by virtue of the conjugative T4SS machinery(Mpf/T4CP system). However, the domain mediatingT4SS-specific translocation is so far unknown. The phylo-genetic relationship between the BID domains of the Bepsand the relaxases suggests that the pathogen Bartonellahas adopted the putative secretion signal of relaxases fortranslocation of effector proteins into host cells.

In conjugation systems, relaxases are coupled to theMpf secretion channel via specific interaction with theT4CP [29]. Analogously, the protein substrates ofA. tumefaciens interact and co-localize with the T4CPVirD4 [30]. The T4CP probably forms a pore-like entry tothe secretion channel and through nucleotide-binding/hydrolysis might drive translocation of the substrates.Remarkably, the T4CPs of conjugation systems encodingBID-containing relaxases (e.g. the T4CP TraG of theA. tumefaciens AvhB/TraG system) form a separate phylo-genetic cluster together with the T4CPs (i.e. VirD4s) ofbartonellae [12]. This implies that the T4CPs and theinteraction domains of their cognate substrates have co-evolved, constituting a pairwise key-lock system. Experi-mental evidence for the notion of a BID-related T4SSsignal in relaxases has been provided: the BID-likedomain and tail sequence of TraA was shown to directVirB/VirD4-dependent translocation from B. henselae intohuman ECs [12]. Similar to the Bep substrates, theC-terminal tail sequence of TraA is positively charged.The translocation efficiency of the secretion signal of TraAlies in the same range as the one measured for individual


Bep substrates, providing further evidence for a ratherrecent acquisition of this signal by B. henselae [12].

The identification of a bipartite, C-terminal T4SSsignal in bartonellae is the first example of a clearlydefined translocation signal in T4SS substrates. TheC-terminal domains of the A. tumefaciens VirB/VirD4substrates VirD2, VirD5, VirE2, VirE3 and VirF have alsobeen shown to direct VirB/VirD4-dependent secretion.However, the only consensus between these secretiondomains is a positively charged C-terminus, whichappears to be crucial for functionality as a secretion signal[31]. A positive charge at the C-terminus could represent acommon feature of the substrates of VirB/VirD4-likeT4SSs. This seems not to be the case for the more distantlyrelated type IVb secretion systems (T4bSSs), as exempli-fied by the Dot/Icm system of L. pneumophila [32]. Thetransfer of the Dot/Icm substrate RalF was shown recentlyto be equally dependent on a C-terminal translocationsignal, yet a positive charge does not appear to be a featureof this signal [32]. For the B. henselae VirB/VirD4 system,the clear delineation of a secretion signal and thespecificity of its targeting into human ECs have openedthe possibility to use this system as a tool for EC study, aswell as for therapeutic purposes. The exploitation of thesystem for protein and DNA vaccine delivery is currentlybeing investigated.

The Trw T4SS of Bartonella

Apart from the VirB/VirD4 system, bartonellae encode asecond T4SS, Trw, which is essential for pathogenicity. Asshown in the B. tribocorum-rat model, the Trw system isrequired for intraerythrocytic parasitism of Bartonella[11]. Deletion of the essential virB10-like trwE gene inB. tribocorum results in a non-bacteremic infection course.However, B. tribocorum trwE mutants probably stillretain their ability to colonize the primary infectionniche (i.e. the vascular endothelium), as indicated by theshort-termed appearance and subsequent rapid clearanceof bacteria in the blood at the time point at which intra-erythrocytic bacteremia normally initiates [7]. Thus, theTrw system might not be required for infection of ECs butrather for colonization of erythrocytes and/or for intra-erythrocytic replication. The finding that the Trw systemis upregulated during in vitro infection of ECs [11] furthersuggests that the bacteria might gain competence forsubsequent erythrocyte infection during their transientinhabitation of ECs.

The Trw system of Bartonella represents a paradigmfor a T4S system that has recently been acquired byhorizontal transfer from a distantly related species,followed by functional diversification by adaptive evolu-tion [17]. It displays the characteristic features of a PAIand shares an extensive homology with the Trw system ofthe IncW broad-host range plasmid R388, which wasoriginally isolated from E. coli. The trw genes ofBartonella are collinear with the respective genes ofplasmid R388, except for the presence of multiple tandemgene duplications of trwL and trwJ–trwH in Bartonella(Figure 3). The homology towards the Trw system of R388goes up to 80% of amino acid sequence identity.Complementation experiments with mutants of R388


53–55%

63%80% 55% 79%72% 25%36–46%

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B. tribocorumtrw

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J5 I5

J4 I4

J2 I2


80%

KorB KorA eex oriT

KorB KorA

K J

Figure 3. The Trw type IV secretion system (T4SS) of Bartonella. The loci encoding the Trw systems of Bartonella triborum and of plasmid R388 are shown for comparison,

highlighting their similarity on the level of amino acid identity (percentages are indicated), as well as on the level of transcriptional regulation (promoters containing

KorA/KorB binding boxes are indicated by red arrows). Colour code: yellow, pilus-associated extracellular components of the T4SS machinery; blue, all other components of

the T4SS machinery (pore complex and energizers); pink, T4CP; green, T4SS substrates. The two regions containing gene duplications (trwL and trwJIH) are superimposed

on the alignment for clarity. In Bartonella quintana and Bartonella henselae, the trw locus is similar but varies in the numbers of trwL (eight copies versus seven in Bartonella

tribocorum) and trwJIH duplications (two copies versus five in B. tribocorum).


have shown that this homology even extends to the level offunctional interchangeability. Also the transcription regu-latory circuit (KorA/KorB repressor, binding to kor boxsequences) is identical and interchangeable between bothsystems [11]. However, a remarkable difference betweenboth systems is the lack of the T4CP-encoding trwB genein Bartonella. In plasmid R388, TrwB is essential forsubstrate (i.e. TrwC) delivery to the secretion channel,therefore, the lack of TrwB in Bartonella suggests thatthis system might not be able to transfer any substrates.At present, it is unknown whether there is cross-talkbetween the VirB/VirD4 and the Trw T4SSs of Bartonella.It is conceivable that the T4CP VirD4 is functional forsubstrate delivery to both T4SSs, in analogy to theversatile interaction of T4CPs with Mpf systems observedin conjugation [33]. The multiple copies of trwL and trwJin the Bartonella trw locus supposedly encode variants ofsurface-exposed pilus components (homologues of VirB2and VirB5, respectively). The other duplicated genes, trwIand trwH, encode homologues of VirB6 and VirB7, whichare required for pilus elongation and for pilus anchorageto the outer membrane, respectively. Thus, the presence ofmultiple copies of these components in the Trw system ofBartonella indicates that a primary function of the systemcould be to produce multiple pilus variants. Identical korbox containing promoter sequences are found in front ofeach copy of trwJ, therefore, the expression of the multiplevariants of TrwJ–TrwH is probably co-regulated. Inaddition, the variants of trwL seem to be co-expressedbecause the trwL genes all localize immediately down-stream of a putative KorA/KorB-regulated promoter. It is


therefore unlikely that the different variants of TrwL andTrwJ/TrwI/TrwH are expressed in a phase-variablemanner. If the assumption that T4SS pili serve forattachment to target cells holds true, the production ofmultiple Trw pilus variants might mediate binding tovarious surface structures of erythrocytes, which couldvary among different blood groups or between differentspecies.

Adaptive evolution of T4SSs

The two T4SSs of Bartonella represent paradigms for theadaptive evolution of T4SSs from interbacterial conju-gation machineries to trans-kingdom delivery systems forvirulence proteins or to other molecular functions relatedto pathogen–host cell interaction (Figure 4). In case of theVirB/VirD4 system, the ancestral conjugation machinerywas adopted for the transfer of effector protein substrates(Beps) into target host cells. To this aim, the secretionsignal for VirB/VirD4-mediated translocation was appar-ently adopted from conjugative relaxases [12]. The con-servation of such a secretion signal in the Bep substratesis the first example for the adaptive evolution of T4SSsubstrates from relaxase ancestors. Most probably, theT4SS signal of the substrates mediates a specific inter-action with the cytoplasmic ‘gatekeeper’ of the secretionchannel, the T4CP. In case of the Trw T4SS, the parentalconjugation system has evolved rather differently. Tan-dem duplications of pilus-related genes have occurredwith the deletion of the T4CP, suggesting an evolution intoa virulence device specified for mediating cellular attach-ment. The occurrence of such tandem duplications is a


Trw

?

Erythrocyte

Bartonella spp.

Endothelial cell

Subversion ofcellular functions:• cytoskeleton (invasion)• proinflammatory activation• antiapoptosis

Bartonella spp.

VirB/VirD4

Beps

Inter-bacterial nucleoprotein transfer(conjugative plasmid transfer)

Horizontal transfer andfunctional diversification

by adaptive evolution

Trans-kingdomprotein transfer

Trans-kingdomsubstrate transferor adhesion tohost cells?

TraA

A. tumefaciens(pAT)

E. coli(R388)

AvhB/TraG Trw


Figure 4. A model of the evolution of type IV secretion systems (T4SSs) in Bartonella and their role in bacterial pathogenesis. The two T4SSs of Bartonella have probably

evolved recently from two distinct bacterial conjugation systems (i.e. AvhB/Tra and Trw) of conjugative plasmids that are present in related a2-proteobacteria (i.e. pAT of

Agrobacterium tumefaciens) or that have a broad-host-range (i.e. the IncW plasmid R388), respectively. The VirB/VirD4 system is required for colonization of ECs by

mediating the intracellular delivery of effector proteins (BepA–BepG). Translocation of the Beps provokes distinct cellular phenotypes. These effectors carry a translocation

signal (indicated by black circles) that has been adopted from the relaxase substrate (TraA) of the ancestral conjugation system. The Trw system is required for colonization of

erythrocytes. It is uncertain whether the Trw system serves for secretion of substrates or whether its function is primarily to produce variable bacterial surface structures (pili)

for cell adhesion.


unique feature of the Trw system of Bartonella and couldrepresent a novel kind of functional divergence of a T4SS.

Conclusions

Since the initial discovery of T4SSs in the genusBartonella, the importance of these secretion systems formediating virulence has rapidly become apparent. TheVirB/VirD4 T4SS was found to translocate effectorproteins (Beps) into ECs, triggering a series of hostresponses that might be crucial for enabling bacterialcolonization of the endothelium. Furthermore, the TrwT4SS proved to be essential for erythrocyte colonization,possibly through the production of adhesive surfacestructures. Together, the two T4SSs of bartonellaerepresent essential virulence factors required for hostcell interactions at different stages or different routes ofBartonella infection. An apparent shortcoming is thatstudies on the role of these T4SSs have been conducted indifferent infection models for in vitro and in vivo studies,respectively. Given the pace at which research onBartonella pathogenesis has advanced recently, a consist-ent model enabling direct comparison between in vitro andin vivo data might soon become available. Nevertheless,the study of the T4SSs of Bartonellae has advanced ourconception of the type IV secretion process. It has becomeapparent that bacterial conjugation systems can evolverapidly into functionally divergent secretion machineriesserving for pathogenesis. The identification of thesecretion signal for Bartonella VirB/VirD4-mediatedtransport could be regarded as the most importanthallmark because it represents the first incidence of aT4SS where such a signal is clearly defined. Tracing the


cellular targets of the translocated effectors will, in future,help to shed light on the molecular mechanisms under-lying Bartonella–EC interactions. Furthermore, theVirB/VirD4 secretion system of Bartonella can now beexploited for delivering engineered substrates into ECs,paving the way for a wide range of applications.

AcknowledgementsFinancial support was given to G.S. by the Deutsche Forschungsge-meinschaft, grant SCHR 988/1–1, and to C.D. by the Swiss NationalScience Foundation, grant 3100–061777.

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Virulence-associated type IV secretion systems of Bartonella

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