Sistema Secretor tipo VI

Sistema Secretor tipo VI

(Parte 1 de 4)

Review The bacterial type VI secretion machine: yet another player for protein transport across membranes

Alain Filloux,1,2 Abderrahman Hachani1,2 and Sophie Bleves2

Correspondence Alain Filloux a.filloux@imperial.ac.uk

1Imperial College London, Division of Cell and Molecular Biology, Centre for Molecular Microbiology and Infection, South Kensington Campus, Flowers Building, London SW7 2AZ, UK

2Laboratoire d’Ingenierie des Systemes Macromoleculaires, 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 multicomponent 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 membranes 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 S, named T6S. 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 pathogen, and when it grows inside human cells or amoebae it is able to inhibit phagosome–lysosome fusion. L. pneumophila 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 S, 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.0.2008/016840-0

1570 2008/016840 G 2008 SGM Printed in Great Britain conjugation. The type IVA archetypes are the T-DNA transferring system called VirB in Agrobacterium tumefaciens 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 complexes (Wilkins & Thomas, 2000), the L. pneumophila type IVB S (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 transmembrane 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 I 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 I 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 S. Type I and type V Ss 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.

The bacterial type VI secretion machine http://mic.sgmjournals.org 1571 of DotU protein, suggesting that the two proteins interact. It was also shown that the lack of DotU and/or IcmF affected the stability of three other Dot proteins. This suggests that DotU/IcmF assist in assembly and stability of a functional Dot–Icm complex and that in their absence the complex is not maintained in a fully active form (Sexton et al., 2004; VanRheenen et al., 2004).

IcmF and DotU orthologues have been found in a wide range of Gram-negative bacterial species. In many cases, these genes are linked, but no other T4SS genes have been found in their vicinity. Instead, another set of conserved genes are systematically found, which were originally known as IAHP (Das & Chaudhuri, 2003). Each gene cluster encodes at least a dozen proteins with various degrees of conservation.

The Rhizobium leguminosarum Imp system

The first report describing genes belonging to IAHP clusters was published in 1997 (Roest et al., 1997). Rhizobium leguminosarum is a plant symbiont, which induces formation of nitrogen-fixing nodules. The host specificity is determined by the Sym plasmid (Van Brussel et al., 1986). The authors described a R. leguminosarum strain capable of nodulation on Vicia sativa but not on Pisum sativum and Vicia hirsuta. In these last species small nodules were formed on the plant roots, but they were not able to fix nitrogen. This strain was designated RBL5523. Tn5 mutagenesis on RBL5523 allowed the identification of a mutant, RBL5787, restored in its ability to nodulate P. sativum and V. hirsuta. Because the locus is important for nodulation or subsequent stages of symbiosis, it was named imp (impaired in nodulation). In 2003, another study reported the sequence of a 3 kb region around the Tn5 inserted in RBL5787 (Bladergroen et al., 2003), revealing a putative operon of 14 genes (Fig. 2) named impA–impN. These genes mostly encode proteins with unknown function, but ImpK and ImpL have similarities with DotU and IcmF, respectively. ImpK is similar to DotU but has a C-terminal extension with similarity to OmpA, an Escherichia coli outer-membrane protein, and to the flagellar torque-generating protein MotB (De Mot & Vanderleyden, 1994). This conserved C-terminal domain is described as a peptidoglycan-anchoring domain. DotU and IcmF are known T4SS-like components, which encouraged the authors to test whether imp mutants were defective in protein secretion. They showed that four proteins were lacking in the culture supernatant of the imp mutant (RLB5787), as compared to RLB5523, including a homologue of the signal-peptide-containing protein RbsB (ribose-binding protein). Furthermore, they showed that when spent growth medium of RBL5523 was used to inoculate the imp mutant RBL5787, a reduction in nodules and nitrogen fixation could be observed. In conclusion, they suggested that the Imp system encoded components of a secretion machine and that Imp-dependently secreted proteins could block the colonization/infection process in pea plants. Obviously, a new S was born, even though not yet named T6SS.

The T6SS in Vibrio cholerae

The work defining the imp locus in R. leguminosarum has been concomitant with work on Vibrio cholerae, which started with the characterization of an IcmF homologue. The V. cholerae icmF gene was identified as being induced in vivo in a rabbit model of infection (Das et al., 2000, 2002). However, the most significant work that revealed the role of the V. cholerae IAHP cluster, and which identified it as the new T6SS, was published in 2006 (Pukatzki et al., 2006). The authors used the O37 serogroup V. cholerae strain V52, which, in contrast to the O1 strain, is capable of evading amoeboid killing when plated with Dictyostelium discoideum. Transposon mutagenesis on strain V52 identified a series of mutants that were attenuated for their virulence on Dictyostelium. Transposon mapping located most of the insertions in a cluster of genes called vas for ‘virulence associate secretion’. Interestingly, an insertion was found in vasK (VAC0120), which corresponds to the previously identified icmF-like gene (Das et al., 2002). The other vas genes, namely VCA0107 to VCA0120, had homologies with most of the imp genes previously described (Fig. 2) (Bladergroen et al., 2003). This indicated that the imp genes from R. leguminosarum and the vas genes from V. cholerae encoded a related system, whose role is important for pea infection and Dictyostelium killing, respectively. Like the imp gene cluster, the vas cluster encodes IcmF and DotU homologues. It should be noted that the DotU homologue of V. cholerae (VCA0115/VasF) does not contain the OmpA/ MotB C-terminal extension found in ImpK, which indicated that this is not a hallmark for DotU orthologues encoded within IAHP/T6SS gene clusters. All the hallmark features found within characterized T6SSs are described in Table 1. Pukatzki et al (2006) investigated whether the Vas system may be involved in protein secretion. Analysis of vas mutants’ culture supernatant, and comparison with the parental strain, revealed the absence of a 28 kDa protein, which appeared to be Hcp, a ‘Haemolysin A co-regulated protein’ (Williams et al., 1996), and of proteins called VgrG. Analysis of an O1 serogroup strain sensitive to Dictyostelium, N16961, showed that it failed to secrete Hcp and VgrG proteins, similarly to the V52 vas mutants. Dictyostelium resistance thus appeared to be dependent on the secretion of Hcp and VgrG proteins. It should be noted that in the study by Pukatzki et al. (2006), secretion of VgrG1 and VgrG2 was shown to be Hcp-dependent, even though it was unclear how Hcp could serve this role. The VgrG1, VgrG2, VgrG3 and Hcp proteins do not contain an N-terminal signal peptide. Interestingly, secreted V. cholerae proteins such as chitinase, neuraminidase, PrtV and HlyA contain a signal peptide and are still secreted in the vas mutants. These observations suggested that the Vas/ IAHP/T6SS is responsible for the secretion of proteins

A. Filloux, A. Hachani and S. Bleves 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)o r 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-I and HSI-II). 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-I and V. cholerae distal hcp and vgrG genes have also been indicated. The P. aeruginosa HSI-I 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.

The bacterial type VI secretion machine http://mic.sgmjournals.org 1573

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, VgrG1 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-I (aai) AaiC AaiG (VgrG-like) AggR (AraC-type regulator)

Francisella tularensis igl , pdp , pig PigG (VgrG-like) MglA master regulator

Induced in vivo (macrophages)

Required for intramacrophage growth; no ClpV-homologue

Pseudomonas aeruginosa hsi-I Hcp1 VgrG1 Two-component system sensor RetS

Two-component system sensor LadS PpkA/PppA S/T kinase-phosphatase

Attenuated virulence in rat lung infection; anti-Hcp1 produced in CF patients hsi-I VgrG2, Hcp2 Putative s54 activator (PA1663)

Stk1/Stp1 S/T kinase-phosphatase hsi-I Hcp3 Putative s54 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 peptide in RbsB; C-terminal extension in DotU

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, -3 Induced in vivo (rabbit model for cholera)

Putative s54 activator (VasH)

Fha but no S/T kinase-phosphatase

Attenuated virulence on Dictyostelium and macrophages VgrG1 covalently cross-links actin

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