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Review
The bacterial type VI secretion machine: yet another
player for protein transport across membranes
Alain Filloux,
1,
Abderrahman Hachani
1,
and Sophie Bleves
2
Correspondence Alain Filloux a.filloux@imperial.ac.uk
(^1) Imperial College London, Division of Cell and Molecular Biology, Centre for Molecular Microbiology and Infection, South Kensington Campus, Flowers Building, London SW7 2AZ, UK (^2) Laboratoire d’Inge´ nierie des Syste` mes Macromole´ culaires, UPR9027, CNRS-IBSM, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France
Several secretion systems have evolved that are widespread among Gram-negative bacteria. Recently, a new secretion system was recognized, which is named the type VI secretion system (T6SS). The T6SS components are encoded within clusters of genes initially identified as IAHP for IcmF-associated homologous proteins, since they were all found to contain a gene encoding an IcmF-like component. IcmF was previously reported as a component of the type IV secretion system (T4SS). However, with the exception of DotU, other T4SS components are not encoded within T6SS loci. Thus, the T6SS is probably a novel kind of complex multi- component secretion machine, which is often involved in interaction with eukaryotic hosts, be it a pathogenic or a symbiotic relationship. The expression of T6SS genes has been reported to be mostly induced in vivo. Interestingly, expression and assembly of T6SSs are tightly controlled at both the transcriptional and the post-translational level. This may allow a timely control of T6SS assembly and function. Two types of proteins, generically named Hcp and VgrG, are secreted via these systems, but it is not entirely clear whether they are truly secreted effector proteins or are actually components of the T6SS. The precise role and mode of action of the T6SS is still unknown. This review describes current knowledge about the T6SS and summarizes its hallmarks and its differences from other secretion systems.
Introduction
Interaction between bacteria and hosts ranges from a commensal collaboration to a competition that may result in host death (Merrell & Falkow, 2004). Such interaction is guided by a communication/signalling game between the host and the pathogen, which, as in a game of chess, aims to influence the way you would like your opponent to play. Among the tools used by bacteria to influence the host response, secretion machines that deliver proteins and toxins into the environment and within a eukaryotic target cell are crucial for virulence and survival within hosts (Caron et al., 2006; Cossart & Sansonetti, 2004; Mota et al., 2005). These proteins are transported across the mem- branes of the bacteria, and eventually of the host, by means of specific devices called secretion systems (SSs). During the last 20 years, it has been found that Gram-negative bacteria have evolved several SSs, which have been identified by types (Fig. 1) (Filloux, 2004; Saier, 2006). Five types have been defined, i.e. type I to type V (T1SS to T5SS). The SSs vary in complexity but all use a single polypeptide or a supra-macromolecular complex to build a path through the bacterial cell envelope. SSs can be recognized by a set of core components used to build up the secretion device. Recent studies have led to the
identification of a new type of SS, named T6SS. The T6SS components are encoded within gene clusters that vary in organization. These clusters were initially named IAHP, for IcmF-associated homologous proteins, because they contain a gene encoding an IcmF-like component (Das & Chaudhuri, 2003). It thus all started with the finding that a known T4SS component, IcmF, was encoded within a conserved gene cluster among Gram-negative bacteria but whose other genes had no homology with T4SS components. These novel genes were likely to encode the components of a novel secretion machine or T6SS.
The type IVB secretion system Legionella pneumophila is a facultative intracellular patho- gen, and when it grows inside human cells or amoebae it is able to inhibit phagosome–lysosome fusion. L. pneumo- phila pathogenesis requires 26 dot (defect in organelle trafficking) and icm (intracellular multiplication) genes (Segal et al., 2005). These genes are essential for altering the endocytic pathway and for replication of L. pneumophila inside the host cells (Sexton & Vogel, 2002). The L. pneumophila dot/icm genes encode components of the type IVB SS, and share extensive similarities with the trb/tra genes found on the IncI plasmids and involved in bacterial
Microbiology (2008), 154, 1570–1583 DOI 10.1099/mic.0.2008/016840-
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conjugation. The type IVA archetypes are the T-DNA transferring system called VirB in Agrobacterium tumefa- ciens and the Tra/Trb conjugative system from IncP plasmids (Christie et al., 2005). The relationship between the B and the A subgroups of the T4SS is limited to a few components.
Whereas the Tra/Trb systems of the IncI plasmids are conjugation machines that deliver nucleoprotein com- plexes (Wilkins & Thomas, 2000), the L. pneumophila type IVB SS (Dot/Icm) is known to deliver proteins into target cells. In L. pneumophila Dot/Icm-dependent effectors have been characterized. Among them, RalF was shown to be required for the localization of ARF (ADP-ribosylation factor) on phagosomes containing L. pneumophila (Nagai et al., 2002).
The DotU/IcmF paradigm In contrast to most dot/icm genes, icmF is partially required for L. pneumophila replication in macrophages (Purcell & Shuman, 1998). The icmF gene is located at one end of the dot/icm cluster, downstream of a gene designated dotU or icmH. DotU and IcmF localize to the L. pneumophila inner membrane (Sexton et al., 2004). IcmF contains several transmembrane domains and a putative Walker A nucleotide-binding motif whereas DotU contains a trans- membrane segment in its C-terminal region. dotU and icmF mutants have similar intracellular growth phenotypes, i.e. partially defective in replication within macrophages (Sexton et al., 2004; Zusman et al., 2004), which suggested that the DotU and IcmF proteins worked together. Furthermore, the lack of IcmF resulted in a reduced level
Fig. 1. Type I–V secretion systems in Gram-negative bacteria. Type I, type III and type IV SSs (left) are believed to transport proteins in one step from the bacterial cytosol to the bacterial cell surface and external medium. In the case of type III and type IV SSs, the proteins are transported from the bacterial cytoplasm to the target cell cytosol. One exception for type IV is the pertussis toxin, which is secreted in two steps and released into the extracellular medium. This exception is represented by the dotted arrow, which connects Sec and the type IV SS. Type II and type V SSs transport proteins in two steps. In that case, proteins are first transported to the periplasm via the Sec or Tat system before reaching the cell surface. Type Va is a putative autotransporter, indicating that the C-terminus of the protein forms the outer-membrane channel (cylinder) whereas the N- terminus (pink line) is exposed to the surface or released by proteolytic cleavage (scissors). C, bacterial cytoplasm; IM, bacterial inner membrane; P, bacterial periplasm; OM, bacterial outer membrane; ECM, extracellular milieu. PM (brown zone), host cell plasma membrane. When appropriate, coupling of ATP hydrolysis to transport is highlighted. Arrows indicate the route followed by transported proteins.
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lacking signal peptides, like the T4SS and T3SS but in contrast to the T2SS (Filloux, 2004).
The T6SS in Salmonella enterica
Early in the discovery of the T6SS, a complete gene cluster, named sci (Salmonella enterica centisome 7 genomic island), was described in S. enterica (Folkesson et al., 2002). The island is 47 kb long and harbours 37 genes. From sciA to sciY, many genes could be identified as core genes for the T6SS, including 9 of the 15 vas genes found in V. cholerae. The Sci island includes genes encoding an Hcp-
like protein (sciK) and a VgrG-like protein (vgrS) (Table 1). In addition, genes involved in fimbrial assembly (saf) or invasin production (pagN) were found. A complete deletion of the Sci genomic island resulted in decreased ability of S. enterica to enter eukaryotic cells. This result is different from data obtained with a sciS (icmF-like) transposon mutant (Parsons & Heffron, 2005). In that case, it was observed that SciS limits intracellular growth in macrophages at late stages of infection and attenuates the lethality of S. enterica in a murine host. sciS was maximally expressed at a late stage of infection, and was shown to be negatively regulated by SsrB, part of the SsrA/SsrB two-
Fig. 2. T6SS gene clusters. R. leguminosarum T6SS genes are labelled impA to impN. V. cholerae genes are indicated by the number of the annotated gene (e.g. VC0107) and when applicable with the given gene name, i.e. vas, hcp or vgrG. The three P. aeruginosa T6SS clusters are presented (HSI-I, HSI-II and HSI-III). The genes are indicated by the number of their annotation (e.g. PA0074) and when applicable with the given gene name. In addition to the gene nomenclature presented by Mougous et al. (2006), genes with unknown function or homologues have been named hsi. The gene letter given corresponds to the R. leguminosarum homologue. Thus hsiA is an impA homologue. We have indicated the gene encoding a putative lipoprotein as lip, the gene encoding the sigma factor activator as sfa and the gene encoding the putative Ser/Thr phosphatase from HSI-I as pppB. In all cases homologous genes are represented with the same colour or motif. In the case of P. aeruginosa HSI-II and V. cholerae distal hcp and vgrG genes have also been indicated. The P. aeruginosa HSI-II orfX gene is bordered with dashed lines indicating that it might be a misannotated gene. The HSI-I cluster is represented from PA0074 to PA0091. It should be noted that PA0071–PA0073 are likely to be part of HSI-I since they are upregulated in a P. aeruginosa retS mutant (Mougous et al., 2006). They have not been indicated in this figure simply because no homologues have yet been reported in other T6SS clusters.
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Table 1.
Some features of T6SSs in various bacteria
Bacterium
T6SS genes
Secreted proteins
Putative secreted
proteins
Regulatory aspects*
Notable features
D
Burkholderia mallei
tss
Hcp1, VgrG
TssB, TssM
VirAG two-component system
Attenuated virulence in hamsters
BMAA1517 (AraC-type regulator)
Edwardsiella ictaluri
eip
Anti-Eip produced during catfish infection
Edwardsiella tarda
evp
EvpC (Hcp)
EsrB (response regulator, SsrB-like)
Attenuated virulence in blue gourami fish
EvpP (Hcp-like)
Temperature-dependent
Escherichia coli
(EAEC)
sci-I
Hcp-like
sci-II
( aai)
AaiC
AaiG (VgrG-like)
AggR (AraC-type regulator)
Francisella tularensis
igl
,^ pdp
,^ pig
PigG (VgrG-like)
MglA master regulatorInduced
in vivo
(macrophages)
Required for intramacrophage growth; noClpV-homologue
Pseudomonas aeruginosa
hsi-I
Hcp
VgrG
Two-component system sensor RetSTwo-component system sensor LadSPpkA/PppA S/T kinase-phosphatase
Attenuated virulence in rat lung infection;anti-Hcp1 produced in CF patients
hsi-II
VgrG2, Hcp
Putative
s
54 activator (PA1663)
Stk1/Stp1 S/T kinase-phosphatase
hsi-III
Hcp
Putative
s
54 activator (PA2359)
No S/T kinase-phosphatase or Fha
Pectobacterium atrosepticum
Hcp1, -2, -3, -4, VgrG
Induced by host-plant extract
Rhizobium leguminosarum
imp
RbsB homologue
Temperature-dependent
No ClpV homologue
ImpM/ImpN S/T kinase-phosphatase
No lipoprotein-encoding gene; signal pep-tide in RbsB; C-terminal extension inDotU
Salmonella enterica^ subspecies I
sci
SciK (Hcp), VgrS(VgrG)
Induced
in vivo
(macrophage and
rabbit ileal loop)
Increased bacterial number in macrophages
Response regulator SsrB
Hypervirulent in mice
Vibrio cholerae
vas
Hcp1, -2, VgrG1, -2, -
Induced
in vivo
(rabbit model for cholera)
RpoN (
(^54) s )
Putative
s
54 activator (VasH)
Fha but no S/T kinase-phosphatase
Attenuated virulence on
Dictyostelium
and
macrophagesVgrG1 covalently cross-links actin
*Regulatory components that control T6SS expression and function have been indicated as well as conditions in which T6SS genes are specifically controlled. The role of the putative
s
54 activator in
expression of T6SS genes has not been demonstrated. However, in
V. cholerae
, a putative
s
54 activator is required for the T6SS-dependent phenotype as well as RpoN (
(^54) s ).
DThe attenuation or hypervirulence is indicated when it has been observed for at least some of the described T6SS mutants.
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avirulent, whereas individual hcp1 or hcp2 single mutants retain virulence. This indicated that T6SS-dependent secretion of at least one Hcp protein is required and sufficient for virulence.
The secretion of Hcp protein was confirmed in P. aeruginosa (Mougous et al., 2006). Each of the P. aeruginosa T6SS clusters contains or is associated with hcp genes, hcp1 (PA0085/HSI-I), hcp2 (PA1512/HSI-II) or hcp3 (PA2367/HSI-III) (Fig. 2). The function of the HSI-I/ T6SS system was studied in a retS background, which allowed overproduction of HSI-I genes and abundant secretion of Hcp1 (Mougous et al., 2006). The secretion of Hcp1 was abolished after introducing a mutation in the icmF1 or clpV1 genes, indicating that, as with V. cholerae, Hcp secretion in P. aeruginosa is T6SS-dependent. In conditions where Hcp secretion is defective the protein was found accumulated in the periplasm (Mougous et al., 2007). This is a puzzling observation, which needs to be confirmed, since Hcp1 does not contain a signal peptide to allow inner membrane translocation. Interestingly, Hcp was found in the sputum of cystic fibrosis (CF) patients colonized with P. aeruginosa, and CF sera contained Hcp antibodies, which suggested that Hcp1 secretion is relevant not only in vitro but also in vivo.
The X-ray crystal structure of P. aeruginosa Hcp1 was obtained at a resolution of 1.95 A˚ (0.195 nm), and showed that the protein could assemble into hexameric rings with an inner diameter of 40 A˚ (4 nm) (Fig. 3) (Mougous et al., 2006). The Hcp function is still unknown, and despite the fact that Hcp is considered as a secreted protein one may consider that it may also be used to assemble a conduit at the bacterial cell surface through which other effector molecules might be transported to the host cell.
The VgrG family
The T6SS-dependent secretion of VgrG proteins was demonstrated in V. cholerae. VgrG2 is encoded by a gene linked to hcp2, and in the vicinity of the T6SS gene clusters
(vas), whereas vgrG3 is located within the vas gene cluster (Pukatzki et al., 2006) (Fig. 2). Mutations in vgrG1 and vgrG2 resulted in lack of Hcp secretion (Pukatzki et al., 2007), suggesting that these proteins may also serve a T6SS function despite the fact that they appeared to be secreted substrates. Several vgrG genes located outside T6SS clusters are found in association with hcp genes. The Vgr proteins were initially considered as accessory components of the Rhs (recombination hot spot) family (Wang et al., 1998b). Rhs components are complex genetic composites, and in E. coli, eight components, RhsA–H, are known. Rhs compo- nents include a core ORF (G/C rich) and an adjacent core extension (A/T-rich) with linked downstream ORFs (A/T- rich). Two ORFs immediately upstream of the rhsG core were identified and named vgrG and hcp (Wang et al., 1998b). Although they are not homologous, vgrG and the RhsG core ORFs are similar in a number of respects. They are both large, their predicted products are hydrophilic, and both are characterized by a regularly repeated peptide motif. These motifs are YDxxGRL(I/T) for the RhsG core protein and a Val-Gly dipeptide motif (Valine glycine repeats) in the case of VgrG. In the case of the RhsE element, a vgr gene, vgrE, was also found upstream of the rhsE core ORF (Wang et al., 1998b). Interestingly, within the S. enterica T6SS gene cluster (sci), in addition to the vgrS gene, two genes were annotated rhs1 and rhs2, rhs encoding a protein with similarity to the E. coli RhsE protein (Folkesson et al., 2002). Characterization of VgrG proteins revealed further remark- able features. In V. cholerae, the VgrG1 C terminus shares a domain (ACD) with the RtxA toxin, which mediates actin cross-linking (Sheahan et al., 2004). The functionality of the VgrG1 ACD domain was tested in COS-7 cells. As with RtxA ACD, ectopic expression of VgrG1 ACD produces cell rounding and actin cross-linking within the transfected cells. The activity of the VgrG1 ACD domain was confirmed in vitro using a purified VgrG1 protein, monomeric G-actin and cytoplasmic extracts of amoebae or macrophages (Pukatzki et al., 2007). The VgrG proteins
Fig. 3. P. aeruginosa Hcp1 structure, reproduced from Mougous et al. (2006) (Science 312 , 1526–1530) with permission from AAAS. Hcp1 forms rings as seen from electron microscopy and single-particle analysis of purified material (inset scale bar, 10 nm). The rings are hexameric as shown by the top view of a ribbon representation. The individual subunits are coloured differently to highlight their organization.
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containing a C-terminal extension were named ‘evolved VgrGs’. In V. cholerae, VgrG3 has a peptidoglycan-binding domain, whereas no extension was found in VgrG2.
Independently of C-terminal extensions, VgrGs share a conserved region, which contains two domains that showed similarities with the gp5 and gp27 proteins that constitute the bacteriophage T4 tail spike. Dimer of trimers of gp5 and gp27 constitutes the tail spike, which is used for puncturing the bacterial envelope and allows DNA injection into the bacterial cytoplasm (Kanamaru et al., 2002; Rossmann et al., 2004). Pull-down experiments using V. cholerae VgrG-directed antibodies revealed that these proteins form homotrimeric or heterotrimeric complexes (Pukatzki et al., 2007). The assembly of the VgrG proteins into a putative tail-spike-like structure suggests that the resulting structure may be used as a puncturing device, to allow perforation of the bacterial cell envelope or of the host cell membrane, or both. In any case, VgrGs appear to be not simply secreted proteins but structural components of the T6SS machine. This is reminiscent of the other family of secreted proteins, Hcp, which form hexameric rings with a central channel of 40 A˚ (4 nm) (Mougous et al., 2006). The tube formed by the bacteriophage T4 gp5/ gp27 complex is about 30 A˚ (3 nm) in diameter (Kanamaru et al., 2002). Pukatzki and colleagues speculated that the VgrG complex may adopt a similar structure and could be surrounded by the Hcp rings, both forming the tube for T6SS substrates. If that were the case, Hcps and VgrGs may be appended at the bacterial surface and possibly released into the culture supernatant by shearing.
The presence of catalytic domains in some VgrG proteins does not support the idea of VgrGs being solely T6SS components. Considering the example of the VgrG1 ACD domain, one has to think that it should be translocated into the host cytosol to be able to promote actin cross-linking. This transport is unlikely to be performed after release of VgrG1 into the extracellular medium, since V. cholerae culture supernatants containing VgrGs were not able to provoke macrophage rounding. This observation anyhow provides evidence that the T6SS is intimately linked with the process of bacteria–host interaction, since remodelling of the cytoskeleton suggests injection of effector proteins/ domains into host cells.
Finally, other proteins have been proposed to be secreted in a T6SS-dependent manner, but whether they are injected into the host cell, attached to the bacterial cell surface or simply released into the milieu needs further investigation. One striking example is the R. leguminosarum RbsB-like protein (ribose-binding protein) reported as a T6SS substrate (Bladergroen et al., 2003). In Gram-negative bacteria, RbsB homologues contain a signal peptide and are localized to the periplasm, where they are involved in binding substrates, such as ribose or the AI-2 signalling molecule (Shao et al., 2007). Bladergroen et al. (2003) confirmed that the R. leguminosarum RbsB does contain a signal peptide, which is not in agreement with T6SS
transporting uniquely substrates lacking signal peptides. This might still be possible since both signal-peptide- containing (pertussis toxin) and signal-peptide-lacking substrates (Helicobacter pylori CagA) are transported in a T4SS-dependent manner (Backert & Meyer, 2006). Whether RbsB secretion in R. leguminosarum is significant remains unclear, but strikingly, a gene encoding an RbsB homologue is found downstream of the vgrG3 gene in the V. cholerae T6SS cluster (Bladergroen et al., 2003).
The key role of threonine phosphorylation in type VI secretion Another remarkable feature of T6SS gene clusters is that they generally encode Ser/Thr kinases and phosphatases (Fig. 2, Table 1). Mukhopadhyay et al. (1999) reported Hank’s-type Ser/Thr kinase and Ser/Thr phosphatase in P. aeruginosa. The corresponding genes, stk1 and stp1, are located downstream of a gene encoding an IcmF-like protein, which is now recognized as part of the HSI-II/ T6SS system of P. aeruginosa. In Yersinia, the Ser/Thr kinase YpkA is involved in T3SS virulence (Cornelis, 1998), and it is considered that some bacterial Ser/Thr kinases have host targets due to their similarity with mammalian proteins. Mukhopadhyay et al. (1999) suggested that Stk and Stp1 could be translocated in host cells by a mechanism involving IcmF and thus the T6SS. It is now clear that it is not the case. In P. aeruginosa, in addition to stk1 and stp1, genes encoding Ser/Thr kinases and phosphatases, ppkA and pppA, respectively, are found in the T6SS/HSI-I cluster. The ppkA gene was previously found specifically induced within the host and required for virulence in neutropenic mice (Wang et al., 1998a; Motley & Lory, 1999). Since the T6SS/HSI-I gene cluster is upregulated in the retS background, Mougous and collaborators could monitor T6SS function when introducing mutations in either ppkA or pppA (Mougous et al., 2007). They showed that PpkA and PppA have antagonistic activities since a ppkA mutation resulted in defective Hcp1 secretion, whereas mutation in pppA resulted in increased Hcp1 secretion. The antagonistic effect of PpkA and PppA on T6SS function was confirmed by investigating T6SS localization. Using a ClpV1-GFP chimera, it was shown that the P. aeruginosa ClpV1 protein is localized in foci in the bacterial cell (Mougous et al., 2006). However, ClpV1 was found evenly distributed throughout the cell in hcp1 or icmF mutants, indicating that the T6SS machine assembles as a macromolecular complex. Interestingly, the localization of ClpV1 into foci was observed in a pppA mutant, but not in a ppkA mutant. This suggested that both T6SS assembly and Hcp1 secretion require phosphorylation by PpkA and are prevented by dephosphorylation through PppA. Mougous and colleagues recognized a gene from the T6SS/HSI-I cluster encoding a protein with an FHA (forkhead-associated) domain. Proteins with FHA domains have affinity for phosphothreonine and have been
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Burkholderia mallei, Burkholderia pseudomallei and Burkholderia thailandensis possess multiple T6SS clusters, i.e. four, six and five, respectively. In B. mallei, one T6SS gene cluster (tssA–tssN) is upregulated upon overproduc- tion of the VirAG two-component system (Schell et al., 2007), which resulted in an increased T6SS-dependent secretion of the Hcp1 protein. A B. mallei hcp mutant is attenuated in the hamster infection model (Schell et al., 2007). As with P. aeruginosa Hcp1, antibodies against B. mallei Hcp1 were found in sera of infected animals. Although VgrG proteins are encoded within Burkholderia T6SS clusters, their secretion has not been demonstrated. Interestingly, a non-Hcp and non-VgrG putative T6SS substrate was proposed to be the B. mallei TssB protein. The tssB gene is part of the T6SS cluster, and whereas a tssB mutant was not impaired in Hcp1 secretion it was attenuated for virulence in hamsters, similarly to a hcp mutant (Table 1). This observation suggested that if TssB is not part of the T6SS machine, it is involved in a T6SS- dependent phenotype and might be a T6SS effector (Schell et al., 2007). Finally, tssM encodes a protein containing an ubiquitin-specific proteinase domain (Schell et al., 2007). Since ubiquitination of proteins does not occur in bacteria, one can speculate that TssM is injected into the host cell.
In the case of Edwardsiella tarda, the T6SS cluster was named evp and contains 16 genes (Zheng et al., 2005; Zheng & Leung, 2007). Mutations in evp genes resulted in attenuated virulence in blue gourami fish (Table 1). A previous report on Edwardsiella ictaluri (Moore et al.,
- identified eip genes similar to the imp genes from Rhizobium, and eip gene products were recognized by the catfish immune system during infection, indicating that they are produced in vivo. In Ed. tarda, it was shown that 13 evp genes were required for secretion of EvpC, an Hcp homologue (Rao et al., 2004), and EvpP, a secreted protein with no homology with either VgrG or Hcp proteins (Zheng & Leung, 2007) (Table 1). Sequence variation between Hcps is high, and EvpP shares several features with this family. It has no canonical signal peptide and runs aberrantly on polyacrylamide gels. Such aberrant migration was observed with V. cholerae Hcps. The formation of a T6SS complex involving the IcmF-like component EvpO was tested using a two-hybrid approach. Relevant interac- tions with EvpA, EvpL (lipoprotein) and EvpN (DotU) were found. No interaction between EvpH (ClpV) and any other Evp components was found, contradictory to results in P. aeruginosa, which suggests an interaction between ClpV1, Hcp1 and IcmF1 (Mougous et al., 2006).
In the case of enteroaggregative E. coli (EAEC), expression of chromosomal genes that are part of a T6SS cluster (aaiA– aaiY; AggR-activated island) is under the control of the virulence-plasmid-encoded AggR (aggregative adherence) transcriptional regulator (Dudley et al., 2006), a member of the AraC family (Sheikh et al., 2002). This plasmid encodes additional virulence factors such as the Pet enterotoxins, the aggregative adherence fimbriae (aafDA) (Nataro et al., 1994) and the dispersin (aap) (Sheikh et al., 2002). The aai cluster is
located in an EAEC pathogenicity island (117 kb) and is not found on the E. coli K-12 genome. It was shown that the aai cluster is required for secretion of AaiC. AaiC has no similarity with Hcp proteins, but like EvpP from Ed. tarda, has a size of about 18–19 kDa and does not contain a signal peptide. The aai cluster contains a gene encoding a VgrG-like protein but its secretion was not observed (Table 1). A second AggR-independent T6SS was found on the EAEC genome. The cluster consists of 21 genes and was named EAECSci-I, whereas the aai cluster was named EAECSci-II. In the EAECSci-I/T6SS cluster an hcp-like gene encodes a product that is secreted in a T6SS-dependent manner (Table 1). In Pectobacterium atrosepticum, which causes diseases in potatoes and other plants, the production of four Hcps was observed (Mattinen et al., 2007). Mutation in one hcp gene did not impair virulence but overproduction of Hcp increased virulence. In vivo induction of T6SS gene expression is likely to occur in P. atrosepticum, since secretion of VgrG and Hcp proteins was induced by addition of potato tuber and stem extracts to the bacterial cultures (Mattinen et al., 2007). In Francisella tularensis, a gene cluster encompassing 16– genes and located on a pathogenicity island (FPI) is needed for intramacrophage growth and virulence in chicken embryos (de Bruin et al., 2007; Nano et al., 2004; Nano & Schmerk, 2007). The organization of these genes is conserved in Francisella species, but is distinct from the well-characterized T6SS organization in other species. Two genes, iglA and iglB, have similarities to impB and impC, respectively. Expression of the iglAB genes is induced during growth in macrophages and is under the positive control of MglA (de Bruin et al., 2007) (Table 1), previously described as a master regulator for Francisella virulence (Lauriano et al., 2004). MglA is similar to the E. coli stringent starvation protein A (SspA), an RNAP- associated protein (Hansen et al., 2005). The mglA gene is linked with mglB, which is also required for intracellular growth of Francisella, and which encodes a protein similar to SspB from E. coli (Baron & Nano, 1998). The pdpB (pathogenicity determinant protein) gene encodes an IcmF-like protein, whereas the pigF (pathogenicity island gene) gene has similarities to dotU. The pigB gene encodes a protein with similarity to the VgrG-family of proteins. The iglD gene encodes a protein with some similarities to ImpJ, particularly in the N-terminal domain. The iglD gene is essential for intracellular replication in primary human monocyte-derived macrophages (Santic et al., 2007). Other genes, iglC, pigC and pigG, seem to be unique to Francisella. Finally, no gene encoding a homologue to ClpV protein is found, which overall makes this system at the limits of variation to classify it as a T6SS.
Evolutionary aspects of T6SSs The screening of databases shows that there are almost 100 different bacterial species with a T6SS but it has been
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experimentally studied in only a few. Whereas it is mainly found in pathogenic or symbiotic bacteria of the phylum Proteobacteria, the T6SS is also found in non-pathogenic soil bacteria such as Pseudomonas putida and Myxococcus xanthus (Bingle et al., 2008). Recently, a phylogenetic analysis distinguished four branches (A–D) within the T6SS tree (Bingle et al., 2008). Interestingly, multiple T6SS clusters from a single bacterium are found in different groups. For example, P. aeruginosa HSI-I is in group A (together with B. pseudomallei and S. typhimurium), HSI-II is in group B (together with R. leguminosarum) and HSI-III is in group D (together with V. cholerae). The three T6SS clusters are found in all P. aeruginosa genome sequences that are available, i.e. PAO1, PA14 and PA7 (http:// www.pseudomonas.com). Yersinia pestis has five T6SS loci, which are spread between the A, C and D groups. This observation indicates that multiple T6SS loci may not have arisen through duplication but rather been acquired by horizontal gene transfer. The T6SS from F. tularensis is phylogenetically distant from the other T6SSs, and constitutes an outward fifth branch. This confirms the weak sequence similarities with other T6SS components that we have described above.
Conclusion
The hallmark of the T6SS is a set of approximately 15 genes found within clusters, which may vary in terms of organization. It is thought that T6SS genes encode components of a secretion machine and specific effectors. In several species, multiple T6SS loci can be found, which in some cases are incomplete. It is not known whether those loci that are most incomplete cooperate with other T6SSs. Consequently, it is difficult to unambiguously define the core components for a functional system. T6SS components that have been discussed are presented in a speculative model in Fig. 4.
Most T6SS clusters contain a gene encoding an AAA+ ATPase, ClpV, which may be the T6SS motor. ClpV should be considered as a core component; however, in R. leguminosarum, which is a T6SS archetype, no clpV-like gene is found in the imp cluster. One possibility is that some T6SSs use a protein encoded by a clpV gene located elsewhere on the chromosome. In order to cross the bacterial cell envelope, a membrane channel is necessary. Apart from DotU and IcmF none of the T6SS components possess transmembrane domains. There is a putative outer- membrane lipoprotein but this is unlikely to form a pore in the membrane if it is only anchored via the lipid modification.
The VgrG proteins from V. cholerae may form a puncturing device similar to the bacteriophage tail spike. This device may be used to perforate the bacterial envelope, and further the host cell membrane, to transport effectors all the way. Another class of proteins, the Hcps, have been shown to form hexameric rings, which may form an extracellular conduit that extends or surrounds the tube
formed by the VgrG proteins. These observations are difficult to reconcile with the idea that Hcps and VgrGs are the T6SS substrates. One may compare this with the T3SS, which transports the translocator proteins to be inserted into the host cell membrane and subsequently the effectors to be translocated into the host cell cytosol (Edqvist et al., 2007). T6SSs could contribute to assembling VgrGs and Hcps outside the bacterial cell, into a putative conduit that
Fig. 4. Schematic representation of a T6SS. Some T6SS components discussed in the text are displayed in the bacterial cell envelope. The ClpV ATPase (orange) may help to transport Hcp (yellow) and VgrG (dark green) across the cell envelope. Although Hcps could be secreted independently, they are represented as a channel through and outside the cell envelope as discussed in the text. The ‘VgrG’ puncturing device (dark green arrows) could be involved either in injecting the C-terminus of evolved VgrG (dark green circle) into the eukaryotic cell or in releasing VgrG into the milieu or at the bacterial cell surface. Lip (pink) is a putative outer-membrane lipoprotein. IcmF (blue) and DotU (red) are inner-membrane proteins. In some cases ClpV has been shown to interact with IcmF. The level of phosphorylation of the Fha protein (brown) regulates T6SS activity. STK (dotted light green) is the Ser/Thr kinase, whereas STP (light green) is the Ser/ Thr phosphatase. The colour coding corresponds to that used for the corresponding genes in Fig. 2. C, bacterial cytoplasm; IM, bacterial inner membrane; P, bacterial periplasm; OM, bacterial outer membrane; ECM, extracellular milieu. PM (brown zone), host cell plasma membrane.
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