Exploring cargo transport mechanics in the type IV secretion systems

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TRENDS in Microbiology

Vol.13 No.7 July 2005

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Exploring cargo transport mechanics in the type IV secretion systems Jianxiong Li1, Sharon G. Wolf 2, Michael Elbaum3 and Tzvi Tzfira1 1

Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA Electron Microscopy Unit, Weizmann Institute of Science, Rehovot 76100, Israel 3 Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel 2

Type IV secretion systems (T4SSs) are used by various bacteria to deliver protein and DNA molecules to a wide range of target cells. These include systems that are directly involved in pathogenesis, such as the secretion of pertussis toxin by Bordetella pertussis into human cells and the delivery of single-stranded DNA (ssDNA) into plants by Agrobacterium. These complex systems are composed of proteins that span the bacterial cytoplasm. The Agrobacterium T4SS is composed of 12 virulence proteins and delivers its transferred ssDNA and several virulence protein substrates to a variety of eukaryotic cells. Recent studies on the Agrobacterium T4SS have revealed new information on the localization and structure of its proteins in the bacteria, the biochemical properties of its transport signal, the route of a DNA substrate through the secretion system, and the initial point of contact of the system with its host. These findings have expanded our knowledge and understanding of the still mostly obscure structure and function of the T4SSs. Introduction Type IV secretion systems (T4SS), which are present in a large number of bacterial species, are used to transfer DNA and proteins across the bacterial cell envelope. For example, these systems serve during conjugation of plasmid DNA between different bacterial cells and as translocation systems for transferring proteins into eukaryotic cells during infection (reviewed in [1]). The Agrobacterium VirB/D4 system is a T4SS used to deliver ssDNA molecules (T-strand or T-DNA) and a set of virulence (Vir) proteins into a wide range of host cells (reviewed in [2]). A network of interactions between the 12 virB- and virD-encoded components of the Agrobacterium VirB/D4 system has been revealed using various biochemical and genetic assays [3–5]. A schematic model representing current knowledge of the VirB/D4 secretion apparatus suggests a supermolecular structure that spans several bacterial compartments (Figure 1). Despite a range of biochemical, functional and genetic assays (reviewed in [2]), the exact cellular localization of many VirB proteins is still unknown and the physical structure of the secretion system assembly has never been solved. Moreover, the mechanism of T4SS-mediated substrate translocation through the bacterial and host-cell membrane Corresponding author: Tzfira, T. ([email protected]). Available online 26 May 2005 www.sciencedirect.com

barriers is still obscure. Recently, using a wide range of experimental approaches, which include cellular localization, bioinformatics, and genetic and functional, as well as novel assays, some fundamental insights into the structure and function of the Agrobacterium VirB/D4 secretion system have been gained. Functional assays help characterize the biochemical properties of the T4SS substrate-export signal Using a genetic system, the export of several Vir proteins has been shown to be T4SS-dependent [6]. VirE2, which

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Figure 1. The VirB/D4 Type IV secretion systems (T4SS) of the Agrobacterium as a supermolecular structure. The VirB4 and VirB11 ATPases and the VirD4 coupling protein are located predominantly at the cytoplasmic face of the bacterial inner membrane, whereas VirB6, a highly hydrophobic protein, most likely spans the inner membrane several times. VirD4 recognizes and recruits the export substrates by their positively charged export signal and directs them to the export apparatus. The VirB8 and VirB10 proteins have an N-terminal cytoplasmic domain, a transmembrane domain and a C-terminal periplasmic domain, suggesting that they span the inner membrane and partially localize to the bacterial cytoplasm and periplasm. VirB2, VirB3 and VirB5 are present at the periplasm, and the small lipoproteins VirB7 and VirB9 produce a disulfide-linked complex, which is localized to the bacterial outer membrane. At least three VirB proteins are required for assembly of the Agrobacterium T-pilus, with VirB2 being the major, and VirB5 and VirB7 the minor, components (reviewed in [2]). The T-pilus is most likely required for the initial contact with the host cell but its function in the actual substrate transfer still needs to be determined. This figure is reproduced, with permission, from Ref. [15].

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coats the T-strand, has been reported to interact and localize with VirD4 at the bacterial cell pole [7], suggesting VirD4-mediated recognition of Vir proteins as an export substrate. Vergunst et al. [8] used a ‘Cre-recombinase reporter assay for translocation’ (CRAfT) to elucidate the VirF translocation signal and to study how the exported proteins are recognized by the T4SS. In their assay, GFP was used as a sensitive reporter for CreTVir transport from Agrobacterium to plant cells (Figure 2b). Using a series of deletions and point mutations, the authors defined and mapped the C-terminal export signal of the VirF protein and by aligning their results with export signals of other Vir proteins, as well as Mesorhizobium loti proteins Msi059 and Msi061 and RSF1010 MobA [8], they identified the hydrophilic and positively charged export signal consensus R-X(7)-R-X-R-X-R-X-X(n) sequence (Figure 2b). The authors also reported that the presence of T-strand is not required for the export of the Cre–VirD2 chimeric protein, thus suggesting that VirD2 (which is covalently bound to the T-strand) provides the export signal for transfer of the T-strand–VirD2 complex [6]. Additional studies, aimed toward revealing the exportsignal interactions with other components of the Agrobacterium VirB/D4 secretion system, are required to provide a comprehensive understanding of the protein export mechanism. (a)

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Immunofluorescence studies reveal the subcellular localization of VirB6 to the cell pole VirB6 is an integral inner-membrane protein [9], which interacts with the two outer-membrane proteins VirB7 and VirB9 [3] and affects the formation of VirB7–VirB7 and VirB7–VirB9 complexes [3,10]. Thus, VirB6 is an important structural component, which might function as a link between the inner and outer membrane components of the VirB/D4 T4SS. Nevertheless, the possibility that VirB6 is just a peripheral component of the transport apparatus has not been ruled out [3,10]. VirD4, a coupling factor delivering the protein and DNA substrates to the transport machinery, was observed to localize to the bacteria cell pole [11], which suggests that other integral proteins of the transport apparatus might localize to the same sub-cellular location. Indeed, using immunofluorescence microscopy, Judd et al. [12] co-localized VirB6 to VirD4 at the bacterial cell pole (Figure 2c). Using Agrobacterium strains that express a defined set of VirB proteins, the authors discovered that deletion of any of the VirB7 to VirB11 proteins abolishes the specific subcellular localization of VirB6. Supported by reports on the network of interactions between various VirB proteins, the authors suggested that VirB6 is a core component of the Agrobacterium T4SS and that it is most likely involved in the actual assembly of the transport apparatus. In their

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VirB4 predicted structure Bacteria VirF RPIARSIKTAHDDARAELMSADRPRSTRGL VirE3 LPIPSPKPKSARSMIFEGSRPRERSTSRGF VirE2 FVRPEPASRPISDSRRIYE-SRPRSQSVNSF VirD5 EAETEKAIDELDDRRVYDPRDRAQDKAFKR VirD2 PKRPRDRHDGELGGRKRARGNRRDDGRGGT CONSENSUS r R r r

Vir proteins export signals Figure 2. Structural and functional analysis of the VirB/D4 Type IV secretion systems (T4SS) of the Agrobacterium. (a) The Agrobacterium T-pilus protein VirB2 interacts with the Arabidopsis host protein BTI1 (VirB2-interacting protein 1). BTI is preferentially localized to the periphery of Arabidopsis root cells (upper panel, GFP-BTI) but not to the cell wall. How BTI interacts with the Agrobacterium T-pilus still need to be studied but it is a plant receptor that is essential for the efficient Agrobacterium-mediated transformation of Arabidopsis cells. (b) A functional Cre-recombinase reporter assay for translocation (CRAfT) enables analysis of Vir protein export from Agrobacterium to host cells. Using this assay, expression of a Cre fusion to the target protein (e.g. the VirF export signal, Cre::VirF37C) in Agrobacterium cells activates the GFP reporter gene in transgenic Arabidopsis plants (upper panel). The assay was used to determine that a positive charge is an essential characteristic of the C-terminal consensus transport signal (lower panel). (c) Immunofluorescence microscopy studies revealed that VirB6 co-localizes with VirD4 to the Agrobacterium cell pole (upper panel), and a comparative analysis (lower panel, left hand side) of the VirB4 C-terminus (turquoise) with TrwB (gold) leads to a predicted hexameric structure for VirB4 (lower panel, right hand side). VirB6-VirD4 co-localization to the cell pole indicates that VirB6 is a component of the transport apparatus, whereas the VirB4 conserved hexameric structure suggests its structural and functional role in the T4SS. Various images in the figure are reproduced, with permission, from Refs. [8,12,14,15,17]. www.sciencedirect.com

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proposed model, complexes of VirB7–VirB9 dock on the outer membrane, whereas complexes of VirB8–VirB10 form on the inner membrane. These complexes then interact with each other to form a four-protein complex of VirB7–VirB10 to which VirB6 is then finally recruited by VirB11 [12]. Bioinformatics analysis suggests a docking function for VirB4 Three of the Agrobacterium proteins, VirB4, VirB11 and VirD4, are the most conserved components of the T4SS in a variety of pathogens. They all have NTP-binding domains, which suggests an energy-consuming process for their assembly or function [13]. Previous studies predicted hexameric ring structures for VirB11 and VirD4, based on their sequence homology to the Helicobacter pylori HP20525 and Escherichia coli TrwB proteins, respectively. Middelton et al. [14] used a wide range of bioinformatics tools to predict the structure of VirB4, the largest component of the T4SS. Specifically, the authors predicted that the C-terminus of VirB4 and the cytoplasmic domain of TrwB are evolutionarily related and share similar structure folding and function. A comparative model between the crystalline structures of TrwB and VirB4 enabled the authors to predict the 3D structure of a single VirB4 molecule and to suggest that, like TrwB, VirB4 forms a hexamer (Figure 2c). The structural similarity between VirB4 and VirD4, and the interaction of VirD4 with VirB11 and VirB4 [4,13], could suggest that VirB4 and VirD4 compete for binding with VirB11. Under this scenario, VirB4 would only be essential for the initial structuring of the channel but not for its function, and most likely would be excluded from some types of T4SS channels. Nevertheless, the high conservation and presence of both VirB4- and VirD4-like proteins in various T4SSs led the authors to propose that VirB4 is required both functionally and structurally during the transport of protein and DNA substrates through the Agrobacterium T4SS [14]. In their model, the authors suggest that VirB4 is anchored to the inner membrane by its N-terminus and undergoes conformational changes at its C-terminus. These changes occur in the presence of VirD4, which temporarily replaces the VirB4 C-terminus, docks to VirB11 and releases its substrate to the channel. T-strand immunoprecipitation defines its route through the secretion channel VirD2 could provide the export signal for the transfer of the T-strand–VirD2 complex [8] and the mechanism underlying T-strand recognition by additional components of the Agrobacterium T4SS channel was only recently studied [15]. By designing a sensitive assay to immunoprecipitate T-strand molecules with components of the secretion system, Cascales and Christie [15] identified the T-strand points of interaction with various components of the VirB/D4 T4SS of the Agrobacterium. The authors cross-linked T-strand molecules to VirB proteins and studied the precipitated DNA–protein complexes by PCR. Precipitation of VirD4–T-strand complexes from wild-type, but not from virB- and virD2-mutant Agrobacterium cells, provided the first direct evidence that VirD4 www.sciencedirect.com

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recruits VirD2–T-strand complexes to the T4SS. Furthermore, because VirD4–T-strand precipitation was independent of VirB proteins, the authors suggested that VirD4 is the first component of the T4SS to participate in the first step of the cascade of events that leads to T-strand translocation into the host cell. Interestingly, the authors found that T-strand molecules precipitate at abundant levels with five of the eleven proteins, – VirB2, VirB6, VirB8, VirB9 and VirB11 (designated class I proteins) – and at minor, albeit detectable levels with VirB4, VirB5, VirB7 and VirB10 (designated class II proteins). Although co-precipitation of T-strand molecules with class I proteins represents a direct interaction between the T-strand and a specific VirB protein, interaction of T-strand molecules with class II proteins is thought to be indirect and to be mediated by interactions between VirB proteins from both classes. Using a set of Agrobacterium mutants defective for the production of each of the class I proteins, the authors could study at which stage of export the T-strand interacts with each VirB protein, enabling them to define the route of the T-strand through the VirB/D4 secretion apparatus. It begins with VirD2-T-strand recruitment by VirD4 to the VirB11 ATPases, followed by its transfer to VirB6, the integral trans-membrane channel that spans the inner cell membranes of the bacteria. From there the T-strand reaches VirB8, which might be involved in translocating it into the periplasm, where it finally meets VirB2 and VirB9, components of the outer membrane and the T-pilus, on its way to the host cell. Baiting the VirB2 plant receptor The T-pilus is composed of at least three proteins, with VirB2 being the major component and VirB5 and VirB7 the minor ones [16]. The exact function of the Agrobacterium T-pilus is still debated but it mostly likely undergoes direct contact with the host cell, at least during the initial stages of the transformation process. Using the yeast two-hybrid system, Hwang and Gelvin [17] identified several plant proteins that interact with VirB2 specifically. Pre-incubation of Agrobacterium cells with VirB2-interacting protein 1 (BTI1), one of three related proteins, resulted in decreased transformation efficiency of the host plant cells. Downregulation of BTI1 in transgenic plants resulted in lower transformation susceptibility, whereas BTI1 overexpression rendered the plants more highly susceptible. Subcellular localization of BTI–GFP in plant cells showed preferential localization to the cell periphery but not to the cell wall (Figure 2a). The presence of two putative transmembrane domains in BTI1 led the authors to suggest its association with the plasma membrane, the Golgi and/or the ER; however, how the T-pilus interacts with BTI or other proteins at the plantcell surface still need to be studied. Nevertheless, these results demonstrate clearly the crucial role of host proteins in the initial interaction of Agrobacterium with its host. Concluding remarks The importance of the T4SS reaches far beyond its function in T-DNA and protein transfer from Agrobacterium to plant cells because T4SSs are used by various pathogens

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to provoke diseases in humans and animals [18]. In this review, we give only a small sampling of the significant progress achieved in recent years in revealing new structural and functional aspects of T4SSs. Although we have concentrated on several unique reports from various groups demonstrating their contribution to understanding the Agrobacterium VirB/D4 transport apparatus, it is safe to assume that only a combined effort, bridging between bioinformatics, structural biology and functional studies in the bacteria and the host cells, will eventually lead to a complete understanding of T4SS structure and function. Acknowledgements We thank the authors cited in this work for their contribution to the figures and we apologize to those who were not cited due to space constraints. The work in our laboratories is supported by grants from the Human Frontiers Science Program (HFSP) to T.T. and M.E. and the US-Israel Bi-national Agricultural Research and Development Fund (BARD) to T.T., S.G.W. and M.E.

References 1 Ding, Z. et al. (2003) The outs and ins of bacterial type IV secretion substrates. Trends Microbiol. 11, 527–535 2 Christie, P.J. (2004) Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems. Biochim. Biophys. Acta 1694, 219–234 3 Jakubowski, S.J. et al. (2003) Agrobacterium tumefaciens VirB6 protein participates in formation of VirB7 and VirB9 complexes required for type IV secretion. J. Bacteriol. 185, 2867–2878 4 Ward, D.V. et al. (2002) Peptide linkage mapping of the Agrobacterium tumefaciens vir-encoded type IV secretion system reveals protein subassemblies. Proc. Natl. Acad. Sci. U. S. A. 99, 11493–11500 5 Das, A. and Xie, Y.H. (2000) The Agrobacterium T-DNA transport pore proteins VirB8, VirB9, and VirB10 interact with one another. J. Bacteriol. 182, 758–763 6 Vergunst, A.C. et al. (2000) VirB/D4-dependent protein translocation from Agrobacterium into plant cells. Science 290, 979–982

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7 Atmakuri, K. et al. (2003) VirE2, a type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens. Mol. Microbiol. 49, 1699–1713 8 Vergunst, A.C. et al. (2005) Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium. Proc. Natl. Acad. Sci. U. S. A. 102, 832–837 9 Das, A. and Xie, Y.H. (1998) Construction of transposon Tn3phoA: its application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins. Mol. Microbiol. 27, 405–414 10 Hapfelmeier, S. et al. (2000) VirB6 is required for stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens. J. Bacteriol. 182, 4505–4511 11 Kumar, R.B. and Das, A. (2002) Polar location and functional domains of the Agrobacterium tumefaciens DNA transfer protein VirD4. Mol. Microbiol. 43, 1523–1532 12 Judd, P.K. et al. (2005) The type IV secretion apparatus protein VirB6 of Agrobacterium tumefaciens localizes to a cell pole. Mol. Microbiol. 55, 115–124 13 Atmakuri, K. et al. (2004) Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion. Mol. Microbiol. 54, 1199–1211 14 Middleton, R. et al. (2005) Predicted hexameric structure of the Agrobacterium VirB4 C terminus suggests VirB4 acts as a docking site during type IV secretion. Proc. Natl. Acad. Sci. U. S. A. 102, 1685–1690 15 Cascales, E. and Christie, P.J. (2004) Definition of a bacterial type IV secretion pathway for a DNA substrate. Science 304, 1170–1173 16 Lai, E.M. and Kado, C.I. (2000) The T-pilus of Agrobacterium tumefaciens. Trends Microbiol. 8, 361–369 17 Hwang, H.H. and Gelvin, S.B. (2004) Plant proteins that interact with VirB2, the Agrobacterium tumefaciens pilin protein, mediate plant transformation. Plant Cell 16, 3148–3167 18 Llosa, M. and O’Callaghan, D. (2004) Euroconference on the Biology of Type IV Secretion Processes: bacterial gates into the outer world. Mol. Microbiol. 53, 1–8

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Letter

TLR2: for or against Candida albicans? M. Luisa Gil1, Didier Fradelizi2 and Daniel Gozalbo1 1 2

Department of Microbiology and Ecology, University of Valencia, Burjassot, Spain Institut Cochin, INSERM CNRS University Paris 5, Paris, France

In a recent issue of Trends in Microbiology, Netea and coworkers presented their opinion that toll-like receptors (TLRs) are involved in escape from the defense mechanisms of the host [1]. In their article, the authors clearly identified three major TLR-mediated escape mechanisms that are used by microbial pathogens, such as Yersinia, Mycobacterium and Candida. Here, we wish to comment on the roll of TLR2 in Candida albicans infections. Netea’s interesting hypothesis, that TLR2 expression might confer to mice an increased susceptibility to C. albicans infection through TLR2-mediated immunosuppression, either by premature or biased anti-inflammatory effects, Corresponding author: Gil, M.L. ([email protected]). Available online 31 May 2005 www.sciencedirect.com

should be regarded with caution because it is based on controversial arguments. The case against Netea’s group has presented experimental results previously [2], which indicated that the TLR2-mediated response to C. albicans was biased towards an antiinflammatory response with induction of interleukin (IL)-10 (Th2 cytokine), but with no induction of significant pro-inflammatory cytokine, tumor necrosis factor a (TNF-a). Such results contradict those from at least three groups. Jouault et al. [3] and Roeder et al. [4] have demonstrated unambiguously that C. albicans cell-wallassociated phospholipomannan is the ligand of TLR2, the engagement of which induces macrophage activation with