The phospholipase effector Tle1^Vc promotes Vibrio cholerae virulence by killing competitors and impacting gene expression

Figures & data

Figure 1. Tle1^Vc is a lipase effector with antibacterial activity.

(a) Comparison of genetic organization of T6SS Aux1 cluster between V. cholerae E1 and N16961. Genes were annotated in the genome database. (b) Functional domain of novel effector was predicted using pfam database. (c) The distribution of Tle1^Vc homologs in V. cholerae. The percentage of Tle1^Vc-positive strain among non-toxigenic (CT-) and toxigenic (CT+) V. cholerae strains was 60.89% and 2.89%, respectively. Related data are available in Data Set S1. (d) Phylogenetic analyses of Tle1^Vc in V. cholerae E1 and representative members of the Tle1 family from different bacteria using MEGA6.0. Maximum-likelihood phylogeny constructed with a bootstrap of 1000. Sequence logo of conservative motifs of Tle1^Vc were shown by WebLogo. (e) Toxicity of expressing Tle1^Vc and its catalytic mutants in E. coli. BL21 cells expressing Tle1^Vc and its variants in cytoplasm (Cyto-Tle1^Vc) and periplasmic space (Tat-Tle1^Vc, Tat-Tle1^VcS356A, Tat-Tle1^VcD417A and Tat-Tle1^VcD496A) were serially diluted onto LB agar plates containing either 0.2% glucose or 0.2% arabinose. The expression of Tle1^Vc and its variants in BL21 cells were detected through western blot (below). RpoB served as an internal reference. The representative image from three independent biological replicates was displayed. (f) The lipase activity assay of Tle1^Vc proteins. Purified Tle1^Vc and its inactivated mutants were incubated with Tween 20 catalytic substrate at 37°C, the OD₅₀₀ was monitored at indicated time points. Error bars represent the mean ±SD of three independent biological replicates. (g and h) The measurement of phospholipase activity of Tle1^Vc. Enzymatic activity of the Tle1^Vc and its protein variants against vesicles containing phospholipid derivatives with fluorescent signal at the sn1 or sn2 positions. (i) Schematic of a phospholipid indicating the activities defined in the effector protein Tle1^Vc.

Figure 2. Tle1^Vc effector impairs the integrity of bacterial membrane.

(a-e) Time-lapse microscopy of E. coli BL21 cells expressing pBAD24 empty plasmid (a), Tat-Tle1^Vc (b), Tat-Tle1^VcS356A (c), Tat-Tle1^VcD417A (d) or Tat-Tle1^VcD496A (e) grown on LB agar pads containing DiBAC₄(3) and SYTOX blue dyes and 0.2% arabinose (induction). Scale bar, 5 μm; The representative image from two independent biological replicates was shown. (f) Quantitative analysis of percentage of green fluorescence of cells expressing Tle1^Vc and its catalytic variants. Statistical significance is indicated (unpaired, Student’s t-test: ***P < .001).

Figure 3. Tli1^tox- specifically neutralizes the toxicity of Tle1^Vc.

(a) Survival of the Δtle1^Vc/tli1^tox- mutant in competition against a panel of predator strains. The Δtle1^Vc/tli1^tox- mutant containing pSRKKm vector complemented with or without Tli1^tox- as prey competed with predators indicated at 1:1 ratio. Both 50 μM IPTG and 0.1% arabinose were included in LB agar plate to induce corresponding genes expression. Surviving prey cells were serially diluted and determined on the LB plate. Error bars represent the mean ±SD of at least three independent biological replicates. Statistical significance is indicated (unpaired, Student’s t-test: ns, not significant; **P < .01). (b) The effect of homologous immunity proteins on Tle1^Vc toxicity. BL21 cells co-expressed an arabinose-inducible pBAD24 vector or its variants of pBAD24 with Tat-Tle1^Vc and either an IPTG-inducible pSRKKm vector or its derivatives of pSRKKm with Tli1^tox+ or Tli1^tox- were grown in LB medium in presence of 0.1% arabinose and 30 μM IPTG. The OD₆₀₀ was recorded in time points indicated. Error bars represent the mean ±SD of three independent biological replicates. Statistical significance is indicated (unpaired, Student’s t-test: ns, not significant; ****P < .0001). (c) SEC analysis of complex formation between the proteins indicated. Protein sample (50 μM) were mixed at a 1:1 ratio and were separated on a Superdex 200 Increase 10/300 GL column and Molar mass was obtained using an AKTA FPLC system coupled with a static light scattering detector. (d) ITC analysis of the interaction between Tle1^Vc and Tli1^tox-. Proteins with high concentrations (100 μM) were loaded into the syringe and titrated into the cells containing corresponding interactors with low concentrations (10 μM).

Figure 4. Tle1^Vc delivery requires functional T6SS and VgrG1^E1.

(a) The role of T6SS on Tle1^Vc secretion. Wild-type and ΔvasK mutant were grown to OD₆₀₀ 1.2; the pellets and supernatant were collected for western blot analysis. The Tle1^Vc polyclonal antibody was used to detect the Tle1^Vc secretion and RpoB served as a loading control. (b and c) The effect of VgrG1^E1 on Tle1^Vc-mediated killing activity. The E. coli MG1655 (b) and Δtle1^Vc/tli1^tox- mutant (c) as prey was co-incubated with predator indicated at 1:1 ratio. When needed, 0.1% arabinose were included in LB medium to induce vgrG1^E1 expression. Error bars represent the mean ±SD of three independent biological replicates. Statistical significance is indicated (unpaired, Student’s t-test: ns, not significant; *P < 0.05; **P < .01). (d) The interaction analysis of Tle1^Vc and VgrG1^E1. The protein Tle1^Vc was immobilized on a CM5 sensor chip. After coupling, ethanolamine was used to block unreacted NHS ester groups. Indicated gradient concentrations of VgrG1^E1 and T6SS effector TseV (unpublished data) as negative control were serially injected into the channel to evaluate the binding kinetic parameters. A reference channel was only activated and blocked to normalize results.

Figure 5. Tle1^Vc contributes to Vibrio cholerae intra- and interspecies antagonism.

(a – f) V. cholerae C6706 (a), V. vulnificus (b), V. parahaemolyticus 1360 (c), E. coli MG1655 (d), A. hydrophila (e), E. aerogenes (f) as prey competed with V. cholerae E1 predator indicated at the ratio of 5:1 (predator: prey). Surviving prey cells were serially diluted and determined on the LB plate containing antibiotics. Error bars represent the mean ±SEM of at least three independent biological replicates. Statistical significance is indicated (unpaired, Student’s t-test: *P < .05; **P < .01; ***P < .001; ****P < .0001).

Figure 6. Tle1^vc-induced strong tit-for-tat response requires the T6SS-H1 of PAO1, catalytic activity of effector and the physical puncture of T6SS.

(a) Surviving CFU of V. cholerae strains indicated after a T6SS attack by PAO1. V. cholerae strains wild type, Δtle1^Vc, Δtle1^Vc+pTle1^Vc (complemented with tle1^Vc), and ΔvasK were co-incubated either with P. aeruginosa PAO1 WT or ΔtssB1 (inactivated T6SS-H1 cluster) mutant at the ratio of 5:1 (P. a: V. c) on LB agar plate for 4 h. Surviving V. cholerae cells were serially diluted and determined on the LB plate containing antibiotics. Error bars represent the mean ±SD of three independent biological replicates. Statistical significance is indicated (unpaired, Student’s t-test: ns, not significant; *P < .05). (b) Growth of P. aeruginosa cells expressing the V. cholerae Tle1^Vc or its inactivated mutant in LB media supplemented with a final concentration of 100 μM IPTG. Cells expressing Tat-sfGFP were used as control. (c) Survival of P. aeruginosa periplasmically expressing the indicated effector. Cells of serial-dilution were used to compare. Representative image of plates is shown. (d) Quantification of surviving V. cholerae E1 ΔvasK after a T6SS attack by P. aeruginosa periplasmically expressing pPSV37 empty vector or its variants with effectors indicated. Statistical significance is indicated (unpaired, Student’s t-test: ns, not significant). (e) Surviving V. cholerae E1 (4eff or ΔvasK) after a T6SS attack by P. aeruginosa expressing the indicated effector. 4eff (maintained physical puncture) and ΔvasK (lost physical puncture) as prey. Statistical significance is indicated (unpaired, Mann-Whitney test: **P < .01; ***P < .001).

Figure 7. Phospholipase effector Tle1^Vc mediates bacterial motility through modulating flagellar gene expression.

(a) The effect of Tle1^Vc on V. cholerae motility. Overnight cultures of V. cholerae E1 strains wild type, tle1^VcD417A (Tle1^Vc catalytic mutation on genome), Δtle1^Vc, Δtle1^Vc+pTle1^Vc (complemented with tle1^Vc) and ΔvasK were blotted on 0.3% agar LB plate at room temperature. When needed, 0.2% arabinose was added to induce Tle1^Vc expression. Diameter was measured after 36 h. Error bars represent the mean ±SD of at least three independent biological replicates. Representative image of plates was shown in Figure 7a below. Statistical significance is indicated (unpaired, Student’s t-test: ns, not significant; **P < .01; ***P < .001; ****P < .0001). (b) Schematic regulation of the V. cholerae flagellar transcription hierarchy. (c) Quantitative analysis of flagellar gene expression. Cells grown in Figure 7a were collected after 16 h and total RNA was extracted and quantified using indicated primers (Table S2). Error bars represent the mean ±SD of three independent biological replicates. (d) Transmission electron microscopy of V. cholerae E1 strains indicated with 3% uranyl acetate staining. (e) The impact of flagellar genes on V. cholerae Δtle1^Vc motility was investigated. Cell cultures of V. cholerae E1 strains, including the wild type, Δtle1^Vc, Δtle1^Vc+pFlaA (complemented with flaA), and Δtle1^Vc+pFlaE (complemented with flaE), were spotted onto 0.3% agar LB plates at room temperature. In cases where flagellar gene expression was required, 0.2% arabinose was added as an inducer. The diameter of the motility zone was measured after 48 hours. A representative image of the plates can be seen in Figure 7d below. Statistical significance is indicated (unpaired, Student’s t-test: ***P < .001; ****P < .0001). (F) Transmission electron microscopy of V. cholerae E1 WT and its mutants complementing with flagellar genes indicated.

Figure 8. The effect of Tle1^Vc on E1’s in vivo fitness and survival of immunity mutant.

(a) The mice pretreated with streptomycin were infected by intragastric inoculation with the WT strain (10⁸ CFU) or the Δtle1^Vc mutant (10⁸ CFU). After 3 days post-infection, colonized bacteria in the cecum were enumerated by serial plating on selective media. Error bars represent the mean ± SD of four independent replicates. Significance was calculated using unpaired, Student’s t-test, * P < .05. (b) Motility assays of V. cholerae E1 were conducted by spotting cultures of the wild type (WT) and ΔflaA mutant onto 0.3% agar LB plates at room temperature. The diameter of the motility zone was measured after 48 hours. Error bars indicate the mean ± SD of at least three independent biological replicates. A representative image of the plates is displayed in Figure 8b below. Statistical significance is denoted (unpaired Student’s t-test: ****P < .0001). (c) The effect of motility on V. cholerae E1 colonization was assessed using an orally infected mouse model following the procedure described in Figure 8a. The mice were pretreated accordingly and subsequently infected with the WT strain and the ΔflaA mutant. Error bars represent the mean ± SD of eight independent replicates. Statistical significance was determined using an unpaired Student’s t-test (ns). (d and e) The competitive colonization of V. cholerae. The mice treated with continuous streptomycin were infected with the 1:1 mixture of the WT strain (lacZ⁻) and the sensitive Δtle1^Vc/tli1^tox- mutant (lacZ⁺). Output cells in feces (d) were detected at day 1 to 3 post-infection; after 3 days post-infection, colonized bacteria in the cecum (e) were counted by serial plating on selective media containing X-Gal. Error bars represent the mean ± SD of six independent replicates. Significance was calculated using unpaired, Student’s t-test, * P < .05; *** P < .001; **** P < .0001. (f) The competitive index (CI) of the Δtle1^Vc/tli1^tox- mutant versus the WT strain in the cecum from Figure 8c. Horizontal line represents mean of the CI.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.