Dual Expression Profile of Type VI Secretion System Immunity Genes Protects Pandemic <i>Vibrio cholerae</i>

<div><p>The <i>Vibrio cholerae</i> type VI secretion system (T6SS) assembles as a molecular syringe that injects toxic protein effectors into both eukaryotic and prokaryotic cells. We previously reported that the <i>V. cholerae</i> O37 serogroup strain V52 maintains a constitutively active T6SS to kill other Gram-negative bacteria while being immune to attack by kin bacteria. The pandemic O1 El Tor <i>V. cholerae</i> strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill <i>Escherichia coli</i> prey. Yet, C6706 exhibits full resistance when approached by T6SS-active V52. These findings suggested that an active T6SS is not required for immunity against T6SS-mediated virulence. Here, we describe a dual expression profile of the T6SS immunity protein-encoding genes <i>tsiV1</i>, <i>tsiV2</i>, and <i>tsiV3</i> that provides pandemic <i>V. cholerae</i> strains with T6SS immunity and allows T6SS-silent strains to maintain immunity against attacks by T6SS-active bacterial neighbors. The dual expression profile allows transcription of the three genes encoding immunity proteins independently of other T6SS proteins encoded within the same operon. One of these immunity proteins, TsiV2, protects against the T6SS effector VasX which is encoded immediately upstream of <i>tsiV2</i>. VasX is a secreted, lipid-binding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba <i>Dictyostelium discoideum</i>. Our data suggest the presence of an internal promoter in the open reading frame of <i>vasX</i> that drives expression of the downstream gene <i>tsiV2</i>. Furthermore, VasX is shown to act in conjunction with VasW, an accessory protein to VasX, to compromise the inner membrane of prokaryotic target cells. The dual regulatory profile of the T6SS immunity protein-encoding genes <i>tsiV1</i>, <i>tsiV2</i>, and <i>tsiV3</i> permits <i>V. cholerae</i> to tightly control T6SS gene expression while maintaining immunity to T6SS activity.</p></div

VasW plays an accessory role in VasX-mediated bacterial killing.

<p>(A) VasW is required for V52 to kill <i>Vibrio parahaemolyticus</i> RIMD. Survival of rifampicin-resistant RIMD was determined by measuring CFU following exposure to the indicated rifampicin-sensitive predator listed on the <i>x</i>-axis. Arabinose was included where indicated (“induced”) to drive expression from the P_BAD promoter. These data represent three independent experiments. Error bars indicate the standard deviation. (B) VasW is required for V52 to kill C6706Δ<i>tsiV2</i>. Survival of rifampicin-resistant C6706Δ<i>tsiV2</i> was determined by measuring CFU following exposure to the indicated rifampicin-sensitive predator listed on the <i>x</i>-axis. Arabinose was included where indicated (“induced”) to drive expression from the P_BAD promoter. These data represent three independent experiments. Error bars indicate the standard deviation. (C) V52Δ<i>vgrG-3</i> with deletions in <i>vasW</i> or <i>vasX</i> does not secrete Hcp. Bacterial pellet and supernatant samples from the strains indicated at the top of the blot were subjected to western blotting with α-Hcp and α-DnaK (loading and lysis control) antibodies. (D) V52Δ<i>vasW</i> does not secrete VasX. Bacterial pellet and supernatant samples from the strains indicated at the top of the blot were subjected to western blotting with α-VasX and α-DnaK (loading and lysis control) antibodies.</p

Dual expression profile of the immunity protein-encoding gene <i>tsiV2</i>.

<p>(A) Schematic representation of <i>vasX</i> fragments cloned upstream of <i>lacZ</i> in the plasmid pAH6. (B, C, D) A promoter exists within <i>vasX</i>. β-galactosidase assays were performed using the strains indicated at the top of the graph. Fragments of <i>vasX</i>, or the <i>hcp-2</i> promoter present in pAH6 are indicated on the <i>x</i>-axis. Data are representative of two independent experiments performed in triplicate and the error bars indicate the standard deviation. *** = p<0.001, ** = p<0.005, * = p<0.01 relative to the empty vector control. p-values were calculated based on the Student's one-tailed, paired t-test.</p

<i>VasX</i> contains a promoter to drive expression of <i>tsiV2</i> in several <i>V. cholerae</i> strain backgrounds.

<p>(A) V52 becomes susceptible to killing following deletion of <i>vasH</i> and <i>vasX</i>. Rifampicin-sensitive V52 derivatives (predator) were mixed with rifampicin-resistant V52, V52Δ<i>vasH</i>, V52Δ<i>vasX</i>, V52Δ<i>vasH</i>Δ<i>vasX</i>, and <i>E. coli</i> MG1655 (prey). Surviving prey were enumerated by selection on LB agar containing rifampicin and the results were plotted. Data are representative of three independent experiments. Error bars indicate the standard deviation. (B) The internal <i>tsiV2</i> promoter is recognized in several strains. <i>V. cholerae</i> strains indicated on the <i>x</i>-axis were transformed with pAH6-vasX(2208-3258) or plasmid control. Transformed strains were subjected to β-galactosidase assays and the Miller units were calculated and plotted. Data represent two independent experiments performed in triplicate; error bars indicate the standard deviation. *** = p<0.001, ** = p<0.005, * = p<0.01 relative to the empty vector control. n.s; not significant. p-values were calculated based on the Student's one-tailed, paired t-test.</p

Bacterial strains.

<p>Bacterial strains.</p

VasX compromises the integrity of the inner membrane in target cells.

<p>(A) VasX dissipates the target cell's membrane potential. C6706Δ<i>tsiV2</i> harboring the plasmids indicated on the <i>x</i>-axis were analyzed using the BacLight Membrane Potential Kit and flow cytometry. The red/green fluorescence ratio was calculated for each condition. Carbonyl cyanide <i>m</i>-chlorophenyl hydrazone (CCCP) is a chemical that uncouples the proton gradient and was used as a positive control for dissipation of membrane potential in this experiment. Arabinose was included in all samples (except in the sample noted as “not induced”) to drive expression from the P_BAD promoter. These data are representative of three independent experiments. Error bars indicate standard deviation. *** = p<0.001, ** = p<0.005 relative to SecP::vasX (induced, -CCCP). p-values were calculated using the Student's one-tailed, paired t-test. (B) Cells producing periplasmic VasX are permeable to propidium iodide (PI). The strain indicated at the top of each histogram (living or ethanol-killed) was incubated in the presence of PI and analyzed by flow cytometry. P2 represents cells not permeable to PI and P3 represents cells permeable to PI. The percentage of cells represented in P2 and P3 populations is indicated. These data represent three independent experiments.</p

Genomic and Functional Analysis of the Type VI Secretion System in <em>Acinetobacter</em>

<div><p>The genus <em>Acinetobacter</em> is comprised of a diverse group of species, several of which have raised interest due to potential applications in bioremediation and agricultural purposes. In this work, we show that many species within the genus <em>Acinetobacter</em> possess the genetic requirements to assemble a functional type VI secretion system (T6SS). This secretion system is widespread among Gram negative bacteria, and can be used for toxicity against other bacteria and eukaryotic cells. The most studied species within this genus is <em>A. baumannii,</em> an emerging nosocomial pathogen that has become a significant threat to healthcare systems worldwide. The ability of <em>A. baumannii</em> to develop multidrug resistance has severely reduced treatment options, and strains resistant to most clinically useful antibiotics are frequently being isolated. Despite the widespread dissemination of <em>A. baumannii</em>, little is known about the virulence factors this bacterium utilizes to cause infection. We determined that the T6SS is conserved and syntenic among <em>A. baumannii</em> strains, although expression and secretion of the hallmark protein Hcp varies between strains, and is dependent on TssM, a known structural protein required for T6SS function. Unlike other bacteria, <em>A. baumannii</em> ATCC 17978 does not appear to use its T6SS to kill <em>Escherichia coli</em> or other <em>Acinetobacter</em> species. Deletion of <em>tssM</em> does not affect virulence in several infection models, including mice, and did not alter biofilm formation. These results suggest that the T6SS fulfils an important but as-yet-unidentified role in the various lifestyles of the <em>Acinetobacter</em> spp.</p> </div

The T6SS of 17978 is not used for killing of <i>E. coli</i> MG1655.

<p>Survival of <i>E. coli</i> was determined by plate counts after exposure to wild type17978, 17978 with vector control (17978/pWH1266), the 17978 Δ<i>tssM</i> T6SS mutant, and its complemented (pTssM) and vector control (pWH1266) derivatives. Wild type <i>V. cholerae (</i>V52), and the isogenic <i>tssM</i> mutant derivative (V52 Δ<i>tssM</i>), were used as positive and negative controls for bacterial killing, respectively. The data presented correspond to three independent experiments and are plotted as means ± SD. Comparison of the 17978 strains shows no significant differences in killing (n.s.; p>0.05; Tukey’s multiple comparison post-test).</p

Identification of conserved T6SS components in selected <i>Acinetobacter</i> spp.

<p>Locus tag identifiers are shown for the conserved <i>tss</i> components of several T6SS-containing <i>Acinetobacters</i>, as well as their homologs in <i>V. cholerae</i>, <i>P. aeruginosa</i>, and <i>B. pseudomallei</i>.</p

The T6SS is active in several species of <i>Acinetobacter</i>.

<p> A) Whole cell and supernatant samples prepared from cultures of several <i>A. baumannii</i> strains were probed with anti-Hcp (top panels) and the lysis control anti-RNA polymerase (RNAP; bottom panels). B) Whole cell and supernatant samples prepared from cultures of different species within the genus <i>Acinetobacter</i> probed as described above. C) Summary of growth and Hcp secretion characteristics, determined by Western blot and ELISA, of all T6SS-positive strains analyzed in this study. “Fast” growing strains (++) and “slow” growing strains (+) were defined as those which reached a high or low optical density, respectively, and set arbitrarily by the indicated line in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055142#pone.0055142.s002" target="_blank">Figure S2</a>. Hcp secretion is summarized as high (↑) or low (↓) based on Western blots and ELISA assays (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055142#pone.0055142.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055142#pone.0055142.s002" target="_blank">S2</a>).</p