Dual expression profile of type VI secretion system immunity genes protects pandemic Vibrio cholerae

Abstract

The Vibrio cholerae 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 V. cholerae 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 V. cholerae strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill Escherichia coli 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 tsiV1, tsiV2, and tsiV3 that provides pandemic V. cholerae 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 tsiV2. VasX is a secreted, lipid-binding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba Dictyostelium discoideum. Our data suggest the presence of an internal promoter in the open reading frame of vasX that drives expression of the downstream gene tsiV2. 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 tsiV1, tsiV2, and tsiV3 permits V. cholerae to tightly control T6SS gene expression while maintaining immunity to T6SS activity.

Figure 1. Dual regulatory profile of T6SS immunity protein-encoding genes.

(A) Schematic representation of the V. cholerae T6SS gene clusters. Toxin-encoding genes are shown in red and immunity protein-encoding genes in yellow. Promoters upstream of the individual T6SS clusters are indicated by arrows. Regions with promoter activity that drive expression of immunity protein-encoding genes are indicated in grey below arrows. (B) Two V. cholerae strains, C6706 and V52, exemplify the dual regulatory profile of the T6SS system. In the T6SS-off state, C6706 expresses only the T6SS immunity proteins (first panel). In contrast, V52 expresses structural, effector, and immunity T6SS proteins, the latter of which provide protection from T6SS-active kin bacteria (second panel). When T6SS-active V52 come in contact with C6706 in the T6SS-off state, C6706 is protected from a T6SS-mediated attack without engaging in T6SS-mediated virulence (third panel). C6706 succumbs to killing by V52 when immunity genes are removed or not expressed (fourth panel).

Figure 2. Episomal expression of VCA0021, VCA0124, and VC1419 protects respective mutants from T6SS killing.

(A) Survival of rifampicin-resistant prey strains C6706 or C6706ΔVCA0021 harboring empty vector (pBAD24) or pBAD24-VCA0021::FLAG was determined by measuring CFU following exposure to the rifampicin-sensitive predator listed in the legend. V52ΔvasK is a T6SS-null strain that serves as a negative control for T6SS-mediated bacterial killing. Arabinose was included in all samples to drive expression from the P_BAD promoter. These data are representative of three independent experiments, each performed in technical duplicate. Error bars indicate the standard deviation. (B) Survival of rifampicin-resistant prey strains C6706, C6706ΔVCA0124, or C6706ΔVC1419 harboring empty vector (pBAD24), pBAD24-VCA0124::FLAG, or pBAD24-VC1419::FLAG was determined by measuring CFU following exposure to a rifampicin-sensitive predator (listed in the legend) – wild-type V52 or the T6SS-null strain V52ΔvasK (negative control). Arabinose was included in all samples to drive expression from the P_BAD promoter. These data are representative of two independent experiments, each performed in technical duplicate. Error bars indicate the standard deviation.

Figure 3. VasX is a bacterial toxin that is lethal when presented from the periplasm.

(A) VasX is required for killing V. parahaemolyticus RIMD and C6706ΔtsiV2, but not E. coli MG1655. Survival of rifampicin-resistant prey (listed on the x-axis) was determined by counting CFU following exposure to the indicated rifampicin-sensitive predator (legend). V52ΔvasK is a T6SS-null strain and was used as a negative control. Arabinose was included where indicated (“induced”) to drive expression from the P_BAD promoter. These data are representative of three independent experiments, each performed in technical duplicate. Error bars indicate the standard deviation. (B) Episomal expression of vasX does not affect bacterial growth. C6706ΔtsiV2 harboring the plasmids indicated in the legend were grown in liquid culture and the OD₆₀₀ was measured at the time points indicated on the x-axis. Arabinose was included where indicated (“induced”) to drive expression from the P_BAD promoter. These data represent two independent experiments performed in technical triplicate. Error bars indicate the standard deviation. (C) Delivery of VasX to the periplasm is toxic to the producing cell. C6706ΔtsiV2 harboring the plasmids indicated on the x-axis were grown for 8 hours in liquid culture. The recovered CFU/mL were enumerated after harvesting samples at the time points indicated in the legend. Arabinose was included in all samples to drive expression from the P_BAD promoter. These data represent three independent experiments performed in technical duplicate. Error bars indicate the standard deviation.

Figure 4. VasX compromises the integrity of the inner membrane in target cells.

(A) VasX dissipates the target cell's membrane potential. C6706ΔtsiV2 harboring the plasmids indicated on the x-axis were analyzed using the BacLight Membrane Potential Kit and flow cytometry. The red/green fluorescence ratio was calculated for each condition. Carbonyl cyanide m-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.

Figure 5. VasW plays an accessory role in VasX-mediated bacterial killing.

(A) VasW is required for V52 to kill Vibrio parahaemolyticus RIMD. Survival of rifampicin-resistant RIMD was determined by measuring CFU following exposure to the indicated rifampicin-sensitive predator listed on the x-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 C6706ΔtsiV2. Survival of rifampicin-resistant C6706ΔtsiV2 was determined by measuring CFU following exposure to the indicated rifampicin-sensitive predator listed on the x-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) V52ΔvgrG-3 with deletions in vasW or vasX 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) V52ΔvasW 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.

Figure 6. Dual expression profile of the immunity protein-encoding gene tsiV2.

(A) Schematic representation of vasX fragments cloned upstream of lacZ in the plasmid pAH6. (B, C, D) A promoter exists within vasX. β-galactosidase assays were performed using the strains indicated at the top of the graph. Fragments of vasX, or the hcp-2 promoter present in pAH6 are indicated on the x-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.

Figure 7. VasX contains a promoter to drive expression of tsiV2 in several V. cholerae strain backgrounds.

(A) V52 becomes susceptible to killing following deletion of vasH and vasX. Rifampicin-sensitive V52 derivatives (predator) were mixed with rifampicin-resistant V52, V52ΔvasH, V52ΔvasX, V52ΔvasHΔvasX, and E. coli 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 tsiV2 promoter is recognized in several strains. V. cholerae strains indicated on the x-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.