A functional VipA-VipB interaction is required for the type VI secretion system activity of Vibrio cholerae O1 strain A1552
© Bröms et al.; licensee BioMed Central Ltd. 2013
Received: 24 October 2012
Accepted: 30 April 2013
Published: 3 May 2013
Many Gram-negative bacteria rely on a type VI secretion system (T6SS) to infect eukaryotic cells or to compete against other microbes. Common to these systems is the presence of two conserved proteins, in Vibrio cholerae denoted VipA and VipB, which have been shown to interact in many clinically relevant pathogens. In this study, mutagenesis of a defined region within the VipA protein was used to identify residues important for VipB binding in V. cholerae O1 strain A1552.
A dramatically diminished interaction was shown to correlate with a decrease in VipB stability and a loss of hemolysin co-regulated protein (Hcp) secretion and rendered the bacterium unable to compete with Escherichia coli in a competition assay.
This confirms the biological relevance of the VipA-VipB interaction, which is essential for the T6SS activity of many important human pathogens.
KeywordsVibrio cholerae Type VI secretion VipA VipB ClpV Hcp
The type VI secretion system (T6SS) is a recently discovered mechanism in Gram-negative bacteria that targets secreted proteins to eukaryotic as well as prokaryotic cells [1, 2]. Like type III and type IV secretion systems (T3SS and T4SS), the T6SS mediates the contact-dependent translocation of effector substrates directly into the recipient cell . Although the genetic contents and organization may vary, 13 core subunits of T6SSs have been recognized . Two of these are highly conserved , and we have demonstrated that the interaction between these proteins occurs in a range of clinically important pathogens, including Vibrio cholerae, Francisella tularensis, Salmonella enterica, Escherichia coli, Pseudomonas aeruginosa, and Yersinia pseudotuberculosis. Since many of these proteins could also bind to cognate partners from other bacteria, the mechanism behind complex formation appears highly conserved. Moreover, a region encompassing a putative and conserved alpha-helix present in all of the VipA homologues of the 6 aforementioned bacteria was shown to be important for binding to their cognate partner protein . Even subtle amino acid substitutions within this domain were found to result in essentially null mutant phenotypes for F. tularensis, neutralizing its ability to escape from the phagosomes and, thus, its ability to replicate within the cytosol of infected macrophages and rendering it avirulent . The VipA-binding domain of VipB proteins has been less characterized, but may reside within the N-terminus based on recent work in Burkholderia cenocepacia. The same region was also shown to be necessary for the T6SS activity of B. cenocepacia.
In V. cholerae, VipA/VipB have been shown to form filaments that structurally resemble bacteriophage T4 contractile tail sheaths and these were quickly disassembled by ClpV, an AAA+ traffic ATPase family protein [8–10]. The tubules were shown to cycle between assembly, quick contraction, disassembly, and re-assembly, suggesting that the sheath may energize the translocation of substrates by a phage tail-like contraction mechanism . The importance of ClpV for secretion of hemolysin co-regulated protein (Hcp) has been demonstrated in both V. cholerae V52 and P. aeruginosa[9, 11].
In most T6SSs, Hcp and valine-glycine repeat protein G (VgrG) are exported by the secretion machinery under normal laboratory cultural conditions. This is not the case for V. cholerae O1 strain N16961, and therefore it was suggested that the T6SS of V. cholerae O1 strains was functionally inactive . Our recent studies showed, however, that the T6SS of V. cholerae O1 strains can be activated when the bacteria are grown under high osmolarity conditions, resulting in the secretion of Hcp into the culture medium . In the same study, Hcp secretion was shown to require the presence of VipA .
Here, residues within the previously identified VipB-binding domain of VipA (aa 104–113)  were exchanged to alanine as a means to identify key residues important for the interaction. To determine the biological consequences of a diminished VipA-VipB interaction in V. cholerae O1 strain A1552, the mutants were assessed for their ability to bind to and stabilize VipB, promote secretion of Hcp, and compete against E. coli in a competition assay.
Substitutions within the large α-helix of VipA negatively impacts on VipA/VipB complex formation
Protein-protein interactions in the yeast two-hybrid assay
Relative β-gal activity
0.5 ± 0.1% ***
100.0 ± 5.8%
1.0 ± 0.2% ***
92.7 ± 4.1%
92.4 ± 3.4% *
74.6 ± 3.4% ***
64.1 ± 10.7% *
1.1 ± 0.3% ***
48.8 ± 2.0% ***
1.0 ± 0.2% ***
Mutating the VipB-interaction site of VipA leads to unstable VipB and essentially abolishes Hcp secretion
VipA/VipB complex formation influences the ability of V. cholerae to compete with E. coli
VipA interacts with the N-terminus of ClpV in the yeast two-hybrid assay
Recently, VipA/VipB was shown to form tubular, cogwheel-like structures that are converted by a threading activity of ClpV into small complexes [9, 10]. The N-domain of ClpV (residues 1–178) was shown to mediate the binding to the VipA/VipB complex, and it was suggested that the primary contact between this complex and the N-domain is mediated by VipB . Recently, Pietrosiuk et al. identified a ClpV recognition site within VipB and showed that productive ClpV-VipB interactions require the oligomeric state of both proteins .
V. cholerae depends on virulence factors like toxin co-regulated pili (TCP) and cholera toxin (CT), to cause the severe, life-threatening diarrheal disease, cholera [22, 23]. A T6SS was recently implicated as an additional virulence determinant of V. cholerae that is required for Hcp secretion , for killing of amoeba and bacteria [12, 20], and also contributes to the inflammatory diarrhea in infant mice and rabbits [24, 25]. The large majority of T6SS genes (12 out of 17), including VipA, VipB, ClpV, VasF and VasK, are required for Hcp secretion, killing of amoeba and bacteria and are predicted to encode structural T6SS components [9, 12, 20]. In addition, regulatory proteins, VasH and VCA0122 [12, 20], as well as effector proteins, VgrG-1 and possibly VCA0118, have also been identified [20, 24, 26, 27].
By using an in silico approach analyzing the F. tularensis VipA-VipB homologues, we previously identified four distinct α-helices (H1 to H4) in the VipA homologue, IglA . Interestingly, all of these helices were found to be essential for growth of F. tularensis in macrophages. While H3 and H4 were located in regions of little importance for VipB binding, H1 and H2 overlapped with regions crucial for the interaction. Although the F. tularensis T6SS is phylogenetically only distantly related to other T6SSs, domains structurally very similar to the four helices with the same specific locations were predicted in an extensive number of homologues of other Gram-negative bacteria. These structural similarities also correlated to a functional relationship, as evidenced by our demonstration of both native and heterologous interactions between the A-B homologues of 6 Gram-negative bacteria, including Vibrio, despite rather low levels of amino acid identities. Thus, the evidence indicates that the H2, and possibly also the H1, helices are essential for the formation of the A/B complex due to the strong preservation of these structures despite different evolutionary origins.
In view of this background, we wanted to further characterize the previously identified interaction of the H2 helix of VipA using a targeted mutagenesis approach. Residues within the conserved α-helix of VipA were exchanged to alanine and the resulting mutants tested in a B2H system. By this approach, several residues important for the VipB interaction were identified, i.e. D104, V106, V110, P111 and L113. Interestingly, out of these, V106, V110 and L113 were homologous to the residues V105, V109 and I112 respectively of the F. tularensis homologue IglA, which when mutated resulted in diminished IglB binding . This confirms that the mechanism behind A/B complex formation is conserved in distantly related pathogens. The small but consistent defect in VipB-binding, however, had no visible effect on VipB expression/stability or Hcp secretion in vitro, although mutants D104A, V110A and L113A were all less efficient at competing with E. coli when tested in a bacterial competition assay. These results resemble those obtained with IglA, for which mutants V109A and L115A showed a defect in IglB binding, but not on IglB stability, yet both mutants were completely unable to grow within host cells and were also avirulent in mice . Thus, even subtle defects in the A-B interaction have drastic impact on the competitive ability of T6S-containing pathogens, as well as on their ability to successfully infect host cells. By combining two or more of the single substitutions that resulted in a defect in VipB-binding, an additive effect was apparent; the ability to interact with VipB binding was poor or abolished in both B2H and Y2H systems, and similarly to a vipA null mutant, these multiple substitution mutants were unable to support stable VipB, Hcp secretion, and to compete with E. coli in a bacterial competition assay. This is the first time that this type of systematic mapping has been carried out in Vibrio. Importantly, the mutants provide a powerful tool for further dissecting the functional role of VipA/VipB in the V. cholerae T6SS.
The protein stability assay utilizing chloramphenicol to stop de novo protein synthesis revealed that VipB was very rapidly degraded in the absence of VipA. This indicates that VipB degradation may be a potent mechanism used by T6SS-containing bacteria to regulate the activity of the secretion system in response to distinct environmental stimuli. In further support of an important role of environmental stimuli for the VipA-VipB interaction and thereby control of T6S, we observed that a high concentration of salt appeared beneficial for the stability of the complex. High salt (340 mM) is also an important trigger for the activity of the T6SS of V. cholerae O1 strain A1552 , which is a concentration not far from that found in the normal ocean habitat of Vibrio, i.e. around 500 mM.
Overall, the results on the VipA-VipB interaction agreed between the B2H and Y2H methods. The multiple alanine substitution mutants that failed to interact with VipB, or exhibited intermediate binding, showed unstable expression of VipB in V. cholerae and E. coli, indicating a lack of proper interaction with the latter. Importantly, the failure to interact was not due to protein instability, since the mutant alleles were shown to be expressed at wild-type levels in V. cholerae as well as in the E. coli B2H system.
The exact role of the VipA/VipB complex is still elusive, but our data indicate that the functional VipA/VipB complex is a prerequisite for the normal function of the T6SS. It has been suggested to guide effector proteins to the secretion channel, analogous to what has been suggested for chaperones of type III secretion systems [28, 29]. However, a study aimed to elucidate the essential function of ClpV for T6S, identified a direct interaction with VipB and revealed a remodeling of the VipA/VipB complex upon interaction with ClpV . The complex alone appeared as large, tubular, cogwheel-like structures but these were dissolved when interacting with ClpV into small complexes. Moreover, no direct interaction was observed between the VipA/VipB complex and the secreted substrates Hcp or VgrG2. Thus, these findings suggest that the complex does not direct the secretory proteins for export, but instead it was proposed that the ClpV-mediated remodeling of VipA/VipB controls the dynamics of VipA/VipB tubules by regulating the number and size of the complexes and ultimately the activity of the T6S apparatus .
A follow-up study utilized an immobilized library of 15-mer peptides of VipA and VipB to identify the binding site between the N-terminus of ClpV and VipA/VipB . While no VipA binding was identified by this approach, a few VipB peptides appeared to interact and two located in the N-terminus of VipB were subjected to further analysis. The binding was shown to involve a hydrophobic groove of ClpV, but the interaction was weak and it was hypothesized that ATP-driven ClpV hexamerization is important for coupling multiple weak interactions and to ensure a rather selective binding of ClpV to the macromolecular complex. These findings might be reconciled with those we obtained using the yeast two-hybrid interaction assay. The binding to VipB may simply be too weak to be revealed by this assay. Interestingly, the two-hybrid assay did detect binding between the N-terminus of ClpV and VipA as well as two VipA homologues encoded by P. aeruginosa and Y. pseudotuberculosis. This may be a reflection of that the peptide library used by Pietrosiuk et al. may not be sufficient to reveal an interaction present between the ClpV N-terminus and intact VipA proteins, since there may be secondary structures of VipA that allow its binding to ClpV. Our finding also implies that the VipA-VipB interaction with ClpV may be more complicated than previously anticipated. Although the study by Pietrosiuk et al. did not detect VipA degradation in a cell-free context, levels were significantly reduced when intact V. cholerae bacteria were analyzed, indicating that there may be direct interaction between ClpV and VipA .
Altogether, our findings indicate that the VipA/VipB complex has unique functional constraints and our previous findings indicate that the constraints are shared by the homologous complexes in other Gram-negative bacteria. Since VipA-VipB homologues are present in such a wide variety of pathogens, this interaction offers a unique and attractive target for the development of novel antibacterial agents. Future investigations to identify drugs that block the VipA-VipB interaction could lead to the development of therapeutics effective against a wide range of infectious diseases.
VipA and VipB homologues are known to interact in many Gram-negative pathogens. In V. cholerae, their essential role in the secretion of T6S substrates has been demonstrated previously. Using site-directed mutagenesis within VipA, we demonstrated that a dramatically diminished interaction to VipB was shown to correlate with a decrease in VipB stability and a loss of Hcp secretion and rendered the bacterium unable to compete with Escherichia coli in a competition assay. This confirms the biological relevance of the VipA-VipB interaction, which is a prerequisite also for the T6S activity of intracellular pathogens like Francisella tularensis and Burkholderia cenocepacia. Thus, this conserved interaction offers an attractive target for the development of novel antibacterials.
Bacterial strains, plasmids and growth conditions
Bacterial strains and plasmids used in this study are listed in a table [see Additional file 1]. E. coli and V. cholerae were cultivated on Luria Bertani (LB) agar or broth at 37°C unless stated otherwise. When necessary, carbenicillin (Cb; 100 μg/ml), kanamycin (Km; 50 μg/ml), chloramphenicol (Cm; 25 μg/ml), rifampicin (Rif; 100 μg/ml), streptomycin (Strp; 50 μg/ml) or tetracycline (Tet; 10 μg/ml) were used.
Construction of expression plasmids
Primer combinations and restriction sites used for vector construction are listed in a table [see Additional file 2]. All PCR amplified fragments were first cloned into the pCR4-TOPO TA cloning vector (Invitrogen AB) to facilitate sequencing (Eurofins MWG Operon) before proceeding with the cloning. Mutated vipA alleles containing in-frame deletions or codon-usage adapted alanine substitutions were constructed by overlap PCR . V. cholerae A1552 chromosomal DNA was used as template in the PCR reactions, with the exception of the multiple substitution mutants which were constructed sequentially using previously generated substitution mutants as template. Thus, the double mutants D104A/V106A and V110A/L113A were generated using D104A and V110A respectively as template, the triple mutant D104A/V106A/V110A was generated using D104A/V106A as template and the quadruple mutant D104A/V106A/V110A/L113A was generated using D104A/V106A/ V110A as template.
For trans-complementation studies, PCR amplified 6 × HisC tagged vipB or vipA mutants were introduced into plasmid pMMB66EH  to allow expression from the ptac promoter and transferred into V. cholerae by conjugation using S17-1λ pir as donor.
To investigate protein-protein interactions in E. coli, PCR amplified fragments encoding VipA or mutants thereof, VipB, full-length or truncated ClpV (first 178 residues), were ligated into plasmids pBRGPω (directs the synthesis of a Gal11P-ω fusion protein and can be used to create fusions to the N-terminus of the ω subunit of E. coli RNAP) and pACTR-AP-Zif (directs the synthesis of the zinc finger DNA-binding domain of the murine Zif268 protein and can be used to create fusions to the N-terminus of Zif268) . Plasmids were introduced into the reporter strain KDZif1ΔZ by electroporation.
To perform protein-protein interactions studies in yeast, PCR amplified fragments encoding mutant derivatives of VipA, full-length or truncated ClpV (first 178 residues), were ligated into the GAL4 activation domain plasmid pGADT7 or the GAL4 DNA-binding domain plasmid pGBKT7 (Clontech Laboratories, Palo Alto, CA, USA). To construct pGADT7 variants encoding YPTB1483 Δ105-114 and PA2365 Δ109-118, the corresponding alleles were lifted by NdeI/BamHI and NdeI/EcoRI digestion from vectors pJEB582 and pJEB584  respectively, and introduced into pGADT7. Plasmids were transferred into strain AH109 or Y187 as described previously .
Analysis of T6S protein production and secretion
To induce type VI secretion in V. cholerae A1552 derivatives, bacterial strains were grown in LB medium containing 340 mM NaCl and samples were taken at OD600 = 2.0 as described previously . At OD600 = 1.0, IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added at a final concentration of 0.5 mM to induce expression from the ptac promoter. To assess protein secretion, TCA precipitated supernatants were analyzed, while intrabacterial protein levels were determined using total samples or pelleted bacteria. Protein samples were separated by SDS-PAGE and analyzed by Western blot using polyclonal antibodies recognizing VipB (Agrisera, Vännäs, Sweden) or Hcp , while VipA (His6-tagged) was visualized using monoclonal Anti-His antibodies (Qiagen, Sollentuna, Sweden). Proteins were visualized using the Enhanced Chemiluminescence system (ECL) (Amersham Biosciences, Uppsala, Sweden).
The intrabacterial protein stability assay was adapted from Feldman and colleagues  with some modifications. In short, V. cholerae was grown overnight at 37°C in LB, diluted 200 × in fresh medium and grown for 1.5 h before addition of 0.5 mM IPTG. After 2 h, protein synthesis was stopped by addition of 50 μg/ml chloramphenicol (corresponds to time zero). Samples were taken out at different time points and analyzed by Western blot using antisera recognizing 6 × His or VipB (above) in combination with ECL.
RNA extraction and qRT-PCR
RNA extraction, qRT-PCR and the sequence of the primers used have been described elsewhere . For each sample, the mean cycle threshold of the test transcript was normalized to that of tmRNA . Results were analysed using the delta delta Ct method of analysis and converted to relative expression ratio (2-ΔΔCt) for statistical analysis , using a paired two-tailed t-test to compare means. Data is presented as the mean N-fold change ± standard deviation of 2 independent experiments where triplicate samples were used.
Bacterial two-hybrid assay (B2H)
KDZif1ΔZ reporter cells were grown overnight at 37°C in LB with appropriate antibiotics, diluted 100 × in fresh medium supplemented with antibiotics and 0.5 mM IPTG. At OD600 = 0.5-0.7, cells were harvested, permeabilized with SDS-CHCl3 and assayed for β-galactosidase activity as described . To determine levels of VipA mutants or VipB, protein samples were separated by SDS-PAGE and subjected to Western blot analysis using polyclonal antibodies recognizing VipA (kind gift from Professor Axel Mogk)  or VipB in combination with ECL. B2H assays were performed at least three times in duplicates on separate occasions. A two-sided t-test with equal variance was used to determine statistical significance.
Yeast two-hybrid assay (Y2H)
Protein expression analysis of Saccharomyces cerevisiae lysates and analysis of protein-protein interactions were performed according to established methods . Specifically, interactions were determined by growth of yeast on synthetic dropout minimal agar (Clontech Laboratories) devoid of tryptophan, leucine (SD-LT) and adenine resulting from ADE2 reporter gene activation. The interactive potential was confirmed by comparative growth at 25°C, 30°C and 37°C to provide an insight into the relative energy required for each interaction, and by induction of two independent reporter genes, HIS3 and lacZ, by growing yeast on SD-LT agar lacking histidine and in liquid culture using ONPG (o-Nitrophenyl-beta-D-Galactopyranoside (Sigma-Aldrich, St. Louis, MO, USA) as substrate respectively. Due to an intrinsic leakiness with the HIS3 reporter, 1.5 mM 3-aminotriazole was added to histidine dropout media to suppress false positives . To monitor MEL1 expression directly on SD-LT plates containing X-α-Gal (Sigma-Aldrich), yeast was spotted and grown for 2 days before the degree of blue colour development indicative of α-galactsidase activity and X-α-Gal hydrolysis was scored. Protein expression was verified using antibodies recognizing the activation or DNA-binding domain of GAL4 (Clontech Laboratories).
E. colicompetition assay
Vibrio and E. coli MC4100 (all containing empty pMMB66EH or vipA-expressing derivates thereof) were grown overnight at 37°C in LB medium containing 340 mM NaCl medium and Cb. Next day, strains were subcultured 1/100 in fresh medium. IPTG was added to a final concentration of 0.5 mM to V. cholerae strains at OD600 = 1.0, and upon reaching OD600 = 2.0, Vibrio was mixed at a 3 to 1 ratio with E. coli of OD600 = 0.2, followed by rigorous vortexing for 1 min. As controls, E. coli was also mixed with LB (LB control and inoculum control). The inoculum control, which was used to estimate the original numbers of E. coli in the assay, was diluted and spread immediately as described below, while 100 μL of the LB control or the V. cholerae - E. coli mixtures were incubated on 0.22 μM nitrocellulose filters (Millipore) placed on well-dried LA plates supplemented with 340 mM NaCl, Cb and IPTG. After 5 h of incubation at 37°C, bacterial cells were harvested from the filter and serial dilutions generated and spread on LA plates containing Strp (selects for E. coli only) in triplicates. Next day, the number of surviving E. coli was counted. The ability of Δhcp, ΔvipA and ΔvipA expressing wild-type or mutated VipA in trans to compete with E. coli was compared.
Type VI secretion
Type VI secretion system
Type III secretion system
Type IV secretion system
Hemolysin co-regulated protein
Valine-glycine repeat protein G
Bacterial 2-hybrid assay
Yeast 2-hybrid assay
Toxin co-regulated pili
Enhanced chemiluminescence system.
This work was supported by grants 2006–3426 (to JEB), 2006–2877 and 2009–5026 (to AS) and 2010–3073 (to SNW) from the Swedish Research Council and a grant from the Medical Faculty, Umeå University, Umeå, Sweden. The work was performed in part at the Umeå Centre for Microbial Research (UCMR).
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