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Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae

2015; Springer Nature; Volume: 34; Issue: 16 Linguagem: Inglês

10.15252/embj.201591163

1460-2075

Daniel Unterweger, Benjamin Kostiuk, Rina Ötjengerdes, A. M. Wilton, Laura Diaz‐Satizabal, Stefan Pukatzki,

Escherichia coli research studies

Article20 July 2015Open Access Source Data Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae Daniel Unterweger Daniel Unterweger Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Benjamin Kostiuk Benjamin Kostiuk Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Rina Ötjengerdes Rina Ötjengerdes Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Ashley Wilton Ashley Wilton Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Laura Diaz-Satizabal Laura Diaz-Satizabal Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Stefan Pukatzki Corresponding Author Stefan Pukatzki Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Daniel Unterweger Daniel Unterweger Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Benjamin Kostiuk Benjamin Kostiuk Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Rina Ötjengerdes Rina Ötjengerdes Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Ashley Wilton Ashley Wilton Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Laura Diaz-Satizabal Laura Diaz-Satizabal Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Stefan Pukatzki Corresponding Author Stefan Pukatzki Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Author Information Daniel Unterweger1, Benjamin Kostiuk1, Rina Ötjengerdes1, Ashley Wilton1, Laura Diaz-Satizabal1 and Stefan Pukatzki 1 1Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada *Corresponding author. Tel: +1 780 492 0904; E-mail: [email protected] The EMBO Journal (2015)34:2198-2210https://doi.org/10.15252/embj.201591163 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines. Synopsis Pandemic V. cholerae share T6SS-dependent effectors with no conserved translocation motifs. Vibrio cholerae encodes an array of Tap-1 adaptors with variable ends, presumably generated through recombination, in order to translocate diverse effectors. Proteins of the DUF4123 superfamily, including Tap-1 from V. cholerae, are encoded in T6SS gene clusters. Vibrio cholerae Tap-1 is required for interaction with and secretion through the T6SS of the effector TseL. Different bacterial strains harbor chimeric forms of Tap-1 specific for their cognate T6SS-secreted effectors. The effectors TseL and VgrG-1 from pandemic strains contain an actin cross-linking domain. Introduction Vibrio cholerae is a Gram-negative bacterium commonly found in the aquatic environment. The species V. cholerae is diverse, with over 200 described different serogroups. Only the O1 serogroup strains cause pandemic cholera, a potentially deadly disease characterized by severe watery diarrhea (Harris et al, 2012). In the environment and in the human host, V. cholerae encounters competitors ranging from eukaryotic cells to prokaryotes of the same or other species. One mechanism of competition employed by V. cholerae requires direct contact and the type VI secretion system (T6SS) (MacIntyre et al, 2010; Unterweger et al, 2012). Genes of the T6SS are encoded in one large cluster and two auxiliary gene clusters (Pukatzki et al, 2006). This secretion system forms a contractile puncturing device with homology to the tail-spike complex of the T4 bacteriophage inside the bacterial cell (Leiman et al, 2009). Contraction of the device's outer sheath ejects an inner tube from one bacterium into a neighboring cell (Basler et al, 2012). In the current model for this device, diverse effector proteins are localized to the conserved tip of the inner tube formed of Hcp and are ejected together with the Hcp tube (Ho et al, 2014; Russell et al, 2014; Zoued et al, 2014). Upon delivery of the effector proteins into the neighboring cell, the effectors have toxic effects unless inhibited by immunity proteins (Brooks et al, 2013; Dong et al, 2013). These immunity proteins protect cells from a T6SS-mediated attack by binding and deactivating T6SS effectors (Brooks et al, 2013; Zhang et al, 2014). Vibrio cholerae uses the T6SS effectors VgrG-1, VgrG-3, TseL, and VasX (Brooks et al, 2013; Dong et al, 2013; Miyata et al, 2013) among others (Shneider et al, 2013; Altindis et al, 2015). These effectors are encoded by pandemic V. cholerae strains and also by the O37 serogroup strain V52, which has an active T6SS under laboratory conditions (Pukatzki et al, 2006; Unterweger et al, 2014). V52 is employed in this study as a representative strain that relies on this effector set. VgrG proteins are important for their structural role as part of a trimeric complex at the distal end of the Hcp tube (Leiman et al, 2009). VgrG-1 and VgrG-3 belong to a group of proteins with unique enzymatic C-terminal extensions attached to a core domain. Based on the covalent linkage of the C-terminal effector domain to the VgrG core domain, they are referred to as class 1 effectors (Shneider et al, 2013). The C-terminal domain of VgrG-1 contains an actin cross-linking domain that is toxic to phagocytic eukaryotic cells, such as murine macrophages (Sheahan et al, 2004; Pukatzki et al, 2007; Ma et al, 2009). The C-terminal domain of VgrG-3 contains a peptidoglycan-binding domain and confers toxicity toward other bacteria (Brooks et al, 2013; Dong et al, 2013). The genes encoding VgrG proteins are located upstream of TseL- and VasX-encoding genes. TseL and VasX are proposed to non-covalently bind surface features of VgrG proteins and are thus referred to as class 2 or cargo effectors (Shneider et al, 2013). VasX is a pore-forming effector that, by interfering with the inner membrane potential, lyses prokaryotic competitors (Miyata et al, 2011, 2013). TseL is a phospholipase (Dong et al, 2013; Russell et al, 2013) that degrades membranes of prokaryotic prey. The anti-prokaryotic effectors VgrG-3, VasX, and TseL are inhibited by their cognate immunity proteins TsiV3, TsiV2, and TsiV1, respectively. Anti-prokaryotic T6SS effector and immunity proteins are encoded in T6SS effector modules that are likely mobile elements exchanged among V. cholerae strains (Unterweger et al, 2014). One effector module is found in each of the T6SS auxiliary gene clusters 1 and 2, and in the large gene cluster. The effector module in each gene cluster can differ from strain to strain. The gene clusters are distributed over both chromosomes of V. cholerae and also contain genes encoding additional T6SS proteins. Different strains encode different sets of effector modules, yet outside these modules, retain conserved genes of the T6SS, including genes encoding structural and regulatory components and an ATPase. The effector module set of any given V. cholerae strain determines the strain's T6SS effector repertoire and controls its interaction with other bacteria. For example, bacteria of two V. cholerae strains with different module sets kill each other in a T6SS-dependent manner (incompatible strains) because they carry different immunity proteins in their effector module sets (Unterweger et al, 2014). In contrast, two strains with the same module set do not die when attacking each other, but coexist (compatible strains) because they carry immunity proteins to the same effectors (Unterweger et al, 2014). We refer to this phenomenon as the "compatibility rule". We observed that pandemic strains of V. cholerae harbor the same T6SS effector module set (Unterweger et al, 2014). Phylogenetic analysis of pandemic and non-pandemic V. cholerae strains and the effector module sets they harbor provide multiple signs of horizontal gene transfer of T6SS effector modules between distantly related strains (Unterweger et al, 2014). The power of these effectors depends on the environment the bacteria compete in. For example, the potent effector VasX creates pores in the membranes of prokaryotic competitors that only kill the cell in an environment of low osmolarity (Miyata et al, 2013). This environment is apparently not provided in the infant rabbit model system, in which T6SS-mediated killing depends on VgrG-3 but not VasX (Fu et al, 2013). One class of diverse T6SS effectors is known to contain a conserved PAAR domain for recognition as T6SS substrates (Shneider et al, 2013). In contrast, effectors encoded in any of the three modules do not share a conserved domain, suggesting an alternative, PAAR-independent translocation mechanism. Here, we demonstrate that translocation of the effector TseL (encoded in auxiliary cluster 1) depends on VgrG-1, and a previously uncharacterized protein that we name the type VI secretion system adaptor protein 1 (Tap-1). The genes vgrG-1 and tap-1 are located immediately upstream of tseL. Tap-1 is a chimeric adaptor protein with conserved core region and varying N- and C-terminal segments. Vibrio cholerae strains encode a diverse set of chimeric Tap-1 proteins to enable secretion of various effectors in strain backgrounds with varying VgrG-1 proteins. Our analysis suggests that the chimeric adaptor proteins result from diversifying selection and recombination at a highly conserved site within tap-1. Results T6SS gene clusters encode proteins of the DUF4123 superfamily The anti-prokaryotic T6SS effector TseL is encoded in the auxiliary cluster 1 of pandemic V. cholerae O1 serogroup strains and other clinical isolates such as the O37 serogroup strain V52 (Dong et al, 2013). We set out to elucidate the mechanism by which the T6SS effector TseL is translocated into neighboring cells. We took a candidate approach to decipher the molecular events that lead to TseL translocation. First, we evaluated the contribution of the uncharacterized ~33-kDa protein Tap-1, encoded immediately upstream of tseL (Fig 1A). BLAST (Altschul et al, 1990) analysis revealed that Tap-1 belongs to a superfamily of proteins with the domain of unknown function, DUF4123 (Fig 1B). This domain is about 120 amino acids in length and contains multiple conserved motifs: YLLLD, SPYxxLVxL, HLRxLLxV, and LFRFYDPxVL (Fig EV1A). DUF4123-containing proteins are found in over 100 bacterial species. Five representative species with DUF4123-encoding genes as part of their T6SS clusters are depicted in Fig 1C. DUF4123-containing proteins often contain additional conserved domains, including Fha, ZapA, and PWWP domains, all of which are involved in protein–protein interactions (Fig EV1B). Genes encoding the DUF4123 superfamily proteins are commonly found downstream of vgrG or putative effector-encoding genes in predicted T6SS gene clusters (Fig 1C). Some species encode multiple proteins of the DUF4123 superfamily in their genomes. For example, in addition to Tap-1, V. cholerae strain N16961 encodes VasW (VCA0019) (Fig 1C). We previously demonstrated that VasW is necessary for the secretion of and killing mediated by VasX, the pore-forming effector encoded directly downstream of vasW (Miyata et al, 2013). Even though Tap-1 and VasW belong to the same superfamily, the two proteins share < 20% identity in their amino acid sequences (Fig EV1C). Click here to expand this figure. Figure EV1. Proteins of the DUF4123 superfamily WebLogo of the four characteristic motifs within DUF4123 from a sequence alignment of the 100 most diverse members of the DUF4123 superfamily. Color of the letter indicates hydrophilic (blue), neutral (green), or hydrophobic (black) character of the amino acid. Value on the y-axis indicates sequence conservation at a particular site on the x-axis. If multiple amino acids are shown at one position, proportional height indicates relative frequency. Cartoon (drawn to scale) of nine members of the DUF4123 superfamily from various bacterial species, showing the modular domain architecture of DUF4123 superfamily proteins that combine various domains with DUF4123. Conserved domains are colored. Alignment of the amino acid sequences of Tap-1 and VasW. Download figure Download PowerPoint Figure 1. Tap-1 belongs to the superfamily of DUF4123 proteins Graphical depiction of the T6SS auxiliary gene cluster 1 of Vibrio cholerae strain N16961. Gene names are indicated above each gene. Tap-1 (yellow) is outlined in bold, and its locus tag is indicated below the gene. Graphical depiction of Tap-1 indicating the domain with homology to DUF4123. Graphical depiction of DUF4123-containing gene clusters drawn to scale. The bacterial species encoding the genes are indicated on the left. All genes encoding proteins of the DUF4123 superfamily (yellow) are outlined in bold and labeled with their locus tag. Data information: A legend for color coding is shown below (C). Legend indicates function predicted by BLAST. Genes of unknown function are colored in gray. Genes outside the operons are colored in white. Download figure Download PowerPoint In summary, tap-1 encodes a protein of the DUF4123 superfamily that is found in T6SS gene clusters of many bacterial species. Its conserved nature and physical proximity to T6SS effectors suggests a conserved role for Tap-1 in T6SS function. Tap-1 is required for TseL translocation Tap-1 is encoded upstream of tseL. Based on their close proximity, we investigated whether Tap-1 is required for TseL secretion and subsequent TseL-mediated killing. To determine the secretion requirements for TseL, V52 and an isogenic tap-1 mutant were maintained in LB broth until they reached the mid-logarithmic phase of growth. TseL was found in the pellet and supernatant from V52, but was absent from the supernatant of the V52 mutant lacking tap-1 (Fig 2A). In trans complementation of the tap-1 null mutation restored detection of TseL in the supernatant. DnaK—a cytoplasmic protein used as a lysis control—was found in the pellet but not supernatant. We conclude that Tap-1 is required for T6SS-mediated translocation of TseL. To determine whether the secretion defect of the tap-1 mutant is specific for TseL or affects T6SS-mediated secretion universally, we analyzed Hcp secretion in V52∆tap-1, as Hcp secretion is the hallmark of a functional secretion system. We observed that Hcp is still secreted in the absence of Tap-1 (Fig 2A), suggesting that Tap-1 is necessary for the secretion of TseL but not for other T6SS proteins. Tap-1 was not detected in culture supernatants by us and others, suggesting that Tap-1 is retained in TseL-secreting cells (Appendix Fig S1 and Altindis et al, 2015). Figure 2. Tap-1 is required for TseL-mediated killing Tap-1 is required for the secretion of TseL. Pellet (P) and supernatant (S) of bacterial cultures were analyzed by SDS–PAGE. The result of immunoblotting with antisera against TseL and Hcp and purified antibodies against DnaK is shown. The asterisk marks an unspecific band, detected by the antiserum against TseL, present in the pre-immunized serum. One representative experiment of three independent experiments is shown. Tap-1 is required for TseL-mediated killing. Wild-type C6706 or C6706 lacking tsiV1 were exposed to the indicated V52 mutants in a killing assay. The y-axis indicates the number of surviving C6706. Tap-1 is required for TseL function. Killing assays in which V52∆tap-1 was mixed with wild-type C6706 or mutants lacking tsiV1, tsiV2, or tsiV3. The y-axis indicates surviving C6706. Data information: In (B, C), the arithmetic mean ± SD of three independent experiments each performed in duplicate is shown. P-values of a two-tailed, unpaired Student's t-test are indicated. Source data are available online for this figure. Source Data for Figure 2A [embj201591163-sup-0003-SDataFig2A.tif] Download figure Download PowerPoint To determine whether the inability of a tap-1 mutant to secrete TseL prevents killing of other prokaryotic cells in a TseL-dependent manner, we performed a killing assay in which V52 or V52∆tap-1 (predator) was mixed with C6706 or a C6706 mutant lacking the cognate immunity gene tsiV1 (prey). Under these conditions, C6706 represses its T6SS while maintaining the expression of immunity genes (Miyata et al, 2013). The C6706∆tsiV1 mutant allowed us to analyze TseL-mediated killing because TsiV1 deactivates TseL in the bacterium under attack (here C6706) (Dong et al, 2013; Unterweger et al, 2014). Predator (V52) and prey (C6706) were mixed and plated on nutrient agar plates. After 4 h at 37°C, surviving V52 and C6706 were enumerated. The lack of tap-1 abolished TseL-mediated killing by V52, comparable to a mutant lacking tseL (Fig 2B). Complementation with episomal tap-1 restored TseL-mediated killing. Expression of tap-1 in trans in V52 had no effect on the survival of wild-type C6706 but killed the mutant lacking tsiV1, indicating that Tap-1 acts on TseL. These results show that TseL-mediated killing depends on Tap-1. To test whether Tap-1 is required for the secretion of effectors in addition to TseL, we determined whether Tap-1 also controls VasX- and VgrG-3-mediated killing. As indicator strains for VasX- and VgrG-3-mediated killing, we used C6706 mutant prey lacking the cognate immunity gene tsiV2 and tsiV3, respectively. The absence of tap-1 did not abolish killing mediated by VasX or VgrG-3 (Fig 2C). These results show that Tap-1 is specific for the effector TseL. A similar observation was made for VasW, the second protein of the DUF4123 superfamily in V. cholerae. VasW is only required for the effector VasX, encoded downstream of vasW, but is dispensable for TseL translocation (Appendix Fig S2). Similarly, a T6SS adaptor with a DUF1795 domain in Serratia marcescens also appeared to be specific for a single effector (Diniz & Coulthurst, 2015). Taken together, these data indicate that Tap-1 is required for the secretion of TseL, allowing V. cholerae to engage in TseL-mediated killing. VgrG-1 is necessary for secretion of and killing by TseL The observation that Tap-1 is dedicated to TseL translocation encouraged us to probe other proteins encoded in auxiliary cluster 1 that act in concert with Tap-1. VgrG-1 is encoded immediately upstream of tap-1 (Fig 1A). VgrG-1 in V52 is a structural component of the secretion system and is the carrier of an effector domain with actin cross-linking activity (Sheahan et al, 2004; Pukatzki et al, 2007; Ma et al, 2009). To determine whether VgrG-1 has a dual function (actin cross-linking and TseL translocation), we performed Western blot analysis on pellet and supernatant fractions of V52 and a vgrG-1 mutant. As reported previously (Pukatzki et al, 2007), deletion of vgrG-1 lowers the amounts of Hcp secreted by cells (Fig 3A). However, TseL secretion was abolished. Providing the vgrG-1 mutant with episomal VgrG-1 restored TseL secretion (Fig 3A). These observations indicate that VgrG-1 is required for the secretion of TseL. Figure 3. VgrG-1 is required for TseL-mediated killing TseL secretion depends on VgrG-1. Western blot analysis of pellet (P) and supernatant (S) samples of the indicated strains. Samples were immunoblotted with antisera against TseL and Hcp and purified antibodies against DnaK. The unspecific band detected by rabbit serum is marked with an asterisk. One representative of three experiments is shown. VgrG-1 is required for TseL-mediated killing. Killing assay with wild-type V52 or indicated mutants and wild-type C6706 or a mutant lacking the immunity gene tsiV1. The y-axis indicates surviving C6706. VasX- and VgrG-3-mediated killing but not TseL-mediated killing occurs in the absence of VgrG-1. V52∆vgrG-1 was mixed in a killing assay with wild-type C6706 or mutants lacking immunity genes tsiV1, tsiV2, or tsiV3 (specific for TseL, VasX, and VgrG-3, respectively). The y-axis indicates surviving C6706. Data information: In (B, C), the mean ± SD of three independent experiments each performed in duplicate is shown. The P-values of a two-tailed, unpaired Student's t-test are indicated. Source data are available online for this figure. Source Data for Figure 3A [embj201591163-sup-0004-SDataFig3A.tif] Download figure Download PowerPoint Next, we used the killing assay to determine whether TseL-mediated killing occurred in the absence of VgrG-1. Parental V52 and mutants lacking tseL or vgrG-1 were mixed with C6706 or C6706∆tsiV1. A V52 mutant lacking vgrG-1 lost its ability for TseL-mediated killing unless provided with episomal VgrG-1 (Fig 3B). These results show that vgrG-1 is necessary for TseL-mediated killing. To determine whether VgrG-1 is required for the translocation of VasX and VgrG-3 in addition to TseL, we performed killing assays using C6706 mutants lacking TsiV2 or TsiV3, the cognate immunity proteins for VasX and VgrG-3, respectively. TseL-mediated killing was abolished in the absence of vgrG-1, while VasX- and VgrG-3-mediated killing still occurred (at reduced rates) in the absence of vgrG-1 (Fig 3C). While VgrG-1 and VgrG-3 differ in their C-termini, they share common core domains that could attract cargo effectors. To test whether VgrG-3 is necessary for TseL function, similar to VgrG-1, we determined the requirement of VgrG-3 for TseL secretion and TseL-mediated killing (Fig EV2). A vgrG-1-deficient mutant was unable to secrete TseL, while secretion of TseL was still observed in a mutant lacking vgrG-3 (Fig EV2A). In the absence of vgrG-3, VgrG-3-mediated killing was abolished, but TseL-mediated killing was still observed (Fig EV2B and C). Even though VgrG-3 might be involved in the secretion of and killing by TseL, our results indicate that VgrG-3, unlike VgrG-1, is dispensable for TseL function. Click here to expand this figure. Figure EV2. VgrG-3 is not necessary for the secretion of and killing by TseL TseL secretion of a vgrG-3 deletion mutant. Pellet and supernatant of indicated bacterial cultures were analyzed by SDS–PAGE. The results of immunoblotting with antisera against TseL and purified antibodies against DnaK are shown. Observation of TseL- and VasX-mediated killing in the absence of vgrG-3. The indicated mutants of C6706 were exposed to V52∆vgrG-3 in a killing assay to analyze TseL-, VasX-, and VgrG-3-mediated killing. The y-axis indicates surviving C6706. Effector-mediated killing by V52. Killing assay similar to (B). Data information: In (B, C), the arithmetic mean ± SD of three independent experiments each performed in duplicate is shown. P-values of a two-tailed, unpaired Student's t-test are indicated. Source data are available online for this figure. Download figure Download PowerPoint To identify the domain of VgrG-1 required for TseL-mediated killing, we created VgrG-1 truncation mutants and tested their ability to restore TseL-mediated killing and Hcp secretion of a tseL-deletion strain (Fig EV3A, Appendix Fig S4). A truncated version of VgrG-1 missing the C-terminal actin cross-linking domain (ACD) (1–679) restored TseL-mediated killing and Hcp secretion similar to complementation with full-length VgrG-1 (Fig EV3B and C). An ACD-mutant of VgrG-1 lacking additionally amino acids 642–679 abolished TseL-mediated killing but maintained Hcp secretion. This 37-amino-acid-long sequence between position 642–679 connects the gp5-like domain with the actin cross-linking domain of VgrG-1 and is absent from VgrG-2 and VgrG-3 (Fig EV3D). We conclude that amino acids 642–679 are necessary for VgrG-1 to mediate killing by TseL. In summary, these results indicate that VgrG-1 is necessary for the secretion of and subsequent killing by TseL. Click here to expand this figure. Figure EV3. Linker domain of VgrG-1 required for TseL-mediated killing Graphical depiction of VgrG-1. Amino acid sequence of the highlighted region 642–679 is shown. Thirty-seven amino acids of VgrG-1 are sufficient to restore TseL-mediated killing. Killing assay in which a tsiV1-deficient mutant of C6706 is exposed to wild-type V52 or V52∆vgrG-1 provided with the indicated constructs of vgrG-1 in trans. The arithmetic mean ± SD of log-transformed data of three independent experiments, each performed in duplicate, is shown. P-values of a two-tailed, unpaired t-test are indicated. V52∆vgrG-1 expressing truncated VgrG-1 versions secretes high levels of Hcp. Pellet and supernatant of V52 strains tested in (B) were analyzed by SDS–PAGE. The results of immunoblotting with antisera against Hcp and purified antibodies against DnaK are shown. Amino acids of interest form the linker region between the core domain and the ACD. Model of VgrG-1 as part of the tip of the T6SS is shown. The ACD is indicated. Amino acids 642–679 are colored in red. Source data are available online for this figure. Download figure Download PowerPoint Tap-1 is required for the interaction between TseL and VgrG-1 The requirements for both VgrG-1 and Tap-1 to secrete TseL led us to investigate how these proteins contribute to the secretion process of TseL. VgrG-1 is secreted with other VgrG proteins (Pukatzki et al, 2007) and PAAR-domain-containing proteins (Shneider et al, 2013) as part of a macromolecular complex at the tip of the secretion system. We hypothesized that TseL interacts with Tap-1 and VgrG-1 for recruitment to this complex. To test physical interactions among Tap-1, VgrG-1, and TseL, we performed immunoprecipitation experiments followed by Western blot analysis. First, we prepared lysates from V52∆tap-1 cells transformed with empty plasmid, or plasmid allowing expression of Tap-1 with a His-epitope (ptap-1::His) or FLAG-epitope (ptap-1::FLAG). FLAG-tagged Tap-1 was precipitated with anti-FLAG-M2 affinity beads. His-tagged Tap-1 is not expected to bind to the anti-FLAG-M2 affinity gel and allowed us to test for unspecific binding of TseL to the beads, or precipitation of inclusion bodies. Lysates and immunoprecipitates were subjected to SDS–PAGE and analyzed for the presence of TseL, Tap-1::His, and Tap-1::FLAG. TseL was found in the lysates of all three strains, but only in the immunoprecipitate generated with a FLAG epitope (Fig 4A). His-tagged Tap-1 was found in the pellet but not in the supernatant. No band was detected with anti-TseL, anti-His, or anti-FLAG antibodies in precipitated lysates of V52∆tap-1 transformed with empty vector. This analysis established an interaction between Tap-1 and TseL. Figure 4. Tap-1 mediates interaction between TseL and VgrG-1 A, B. Interactions between Tap-1 and TseL (A) or VgrG-1 (B). Shown are immunoblots of lysates (total) and immunoprecipitates with anti-FLAG affinity beads (IP:FLAG) of V52∆tap-1 (A) or V52 vgrG-1::myc (B) transformed with empty vector or a plasmid encoding His-tagged or FLAG-tagged Tap-1. C. Interaction between TseL and VgrG-1. Shown are immunoblots of lysates (total) and immunoprecipitates with an anti-FLAG affinity beads (IP:FLAG) of V52 or V52∆tap-1 transformed with empty vector or a plasmid encoding HIS-tagged or FLAG-tagged VgrG-1. D. Diagrammatic interpretation of (A–C). Arrows indicate interaction. Source data are available online for this figure. Source Data for Figure 4 [embj201591163-sup-0005-SDataFig4.zip] Download figure Download PowerPoint To test whether Tap-1 also interacts with VgrG-1, we analyzed lysates of V52 with a chromosomal myc-tagged version of VgrG-1 that was transformed with either empty vector, ptap-1::His or ptap-1::FLAG. VgrG-1 was found in the lysates of all three strains but only in the immunoprecipitate with Tap-1::FLAG (Fig 4B). No band with the size of VgrG-1 was detected in the immunopr