Table 1.
Response of Vibrio cholerae strains to ZAC-3 IgG.
Fig 1.
ECM production by V. cholerae upon treatment with ZAC-3 IgG.
V. cholerae strains at an OD600 of 0.4 were treated with ZAC-3 IgG (9 μg/mL) or an isotype control, SyH7 IgG, at 37°C for either 1, 2 or 4h in static conditions, followed by CV staining (Materials and Methods). Statistical significance between treatment groups at each time point was determined by two-way ANOVA followed by Tukey multiple comparison test. *; P< 0.05. ns; not significant. Each bar represents at least three biological replicates with three technical replicates each.
Fig 2.
Dose and time dependent production of ECM by V. cholerae in response to ZAC-3 IgG treatment.
(A) V. cholerae O395 was treated with indicated amounts of ZAC-3 IgG (1.6–50 μg/mL), or an isotype control (50 μg/mL) for 2.5 h before being assessed for ECM production using CV. Visible macroagglutination (Agglut.) is indicated on the right:—no agglutination; +, agglutination within 3 h; ++, agglutination within 2 h. Statistical significance was determined by one-way ANOVA followed by Tukey multiple comparison test: *, P< 0.05; ns, not significant. (B) Representative image of CV staining for a single biological replicate (done in triplicate) from Panel A. C; control. (C) CV production as a function of time (hours) following treatment with ZAC-3 IgG (9 μg/mL) or an isotype control. At each time point CV staining was significantly higher (P< 0.05) in wells that had been treated with ZAC-3 compared to corresponding controls, as determined by Student’s t-Test. (D) A representative image of CV staining from Panel C. The results presented in Panels A and C constitute at least three biological replicates with three technical replicates each. CV staining shown in Panels B and D appears blue rather than purple due to image processing.
Fig 3.
SEM analysis of V. cholerae following treatment with ZAC-3 IgG.
SEM images of V. cholerae O395 treated with (A) medium control, or (B) ZAC-3 IgG (9 μg/mL) for 1 h. The insets in Panel B are shown as panels C and D. Highlighted is evidence of extracellular debris (yellow arrows; panels B) and web-like extensions between cells (red arrows; panels B-D). Scale bars, 1 μm.
Fig 4.
Relationship between agglutination and ECM production by V. cholerae O395.
CV staining induced following treatment (2.5 h) with (A) ZAC-3 Fab fragments, (B) 2D6 IgA, (C) 2D6 IgG and (D) ZAC-3 IgG at the indicated concentrations (y-axis). Note that panel (D) is reproduced from Fig 2A for the sake of comparison. Appropriate isotype and Fab controls (50 μg/mL) were included in each experiment and are shown by solid black bars. Vertical symbols indicate macroagglutination (Agglut.);—no agglutination; +++ agglutination within 1 h; ++ agglutination within 2 h, and + agglutination within 3 h. Each graph constitutes at least three biological replicates with three technical replicates each. Statistical significance across treatments was determined by one way ANOVA, followed by a Tukey multiple comparison test, *; P< 0.05. ns; not significant.
Fig 5.
Non-motile V. cholerae O395 mutants do not produce ECM in response to ZAC-3.
Relative CV levels produced by V. cholerae O395 ΔmotX (non-motile) and ΔflaA (aflagellate) mutants, as compared to wild-type V. cholerae O395, in response to ZAC-3 IgG or an isotype control (9 μg/mL) after 2.5 h. For wild type V. cholerae O395 (but not the motility mutants), CV staining levels were significantly higher in the ZAC-3 treated group as compared to the control (P< 0.05). The graph constitutes three biological replicates with three technical replicates each. Statistical significance was determined by two-way ANOVA, followed by a Tukey multiple comparison test. Macroagglutination (Agglut.) scale is as follows;—no agglutination; ++, agglutination within 2 h; +, agglutination within 3 h.
Fig 6.
The ECM produced by V. cholerae O395 in response to ZAC-3 IgG is enriched in LPS, but not VPS.
(A) VPS-specific ELISA of V. cholerae strains C6706, C6706 ΔvpsL, O395, and O395 ΔvieA following 1 h treatment with SyH7 (an isotype control) or ZAC-3 IgG (9 μg/mL). (B) Microtiter wells cultured with strains O395 and O395 ΔvieA treated for 1 h with SyH7 or ZAC-3 IgG, as described in Panel A, were subjected to straining with CV to detect relative ECM production. Stain O395 ΔvieA produced elevated levels of ECM compared to the wild type strain in the absence and presence of ZAC-3, as determined by two-way ANOVA followed by a Tukey multiple comparison test. *; P< 0.05. The graph constitutes three biological replicates with three technical replicates each. Anti-OSP (C) and anti-LPS (D) ELISA probed with 2D6 IgA or rabbit polyclonal antisera (BD Difco). The ZAC-3 treated cells exhibited higher OSP and LPS signals as compared to control cells, as determined by a one-way ANOVA followed by a Tukey multiple comparison test. The ELISAs are representative of three biological replicates with two technical replicates each.
Fig 7.
ECM production by V. cholerae C6706 in response to ZAC-3 IgG is VPS independent.
Mid-log phase cultures of either V. cholerae C6706 El Tor or an isogenic vpsL mutant were treated for 1 h at 37°C in static conditions with ZAC-3 (9μg/mL) or an isotype control MAb. Both strains displayed, as compared to isotype controls, increased levels of CV straining in response to ZAC-3 IgG, as determined by a two-way ANOVA followed by a Tukey multiple comparison test. *; P< 0.05. The results presented are the average of at least three biological replicates with three technical replicates each. (B) ECM-ELISA of the same treatment groups described above, with no significant difference in VPS signal seen between the control and ZAC-3 treated groups in either strain, as determined by two-way ANOVA followed by a Tukey multiple comparison test. Graph is composed of data from two biological replicates with 2 technical replicates each.
Fig 8.
Pre-treatment of V. cholerae with ZAC-3 IgG results in resistance to secondary complement-mediated lysis.
(A) CV staining of mid-log phase V. cholerae O395 following treatment for 1 h with SyH7 F(ab’)2, 2D6 F(ab’)2, ZAC-3 F(ab’)2 at (9 μg/mL), or 2D6 IgA (36μg/mL). (B) Complement-mediated lysis of V. cholerae O395 following the pre-treatments described in Panel A. Each bar constitutes the combined results from at least three biological replicates. In both panels, statistical significance across treatments was determined by one way ANOVA, followed by a Tukey multiple comparison test. ns, not significant. *; P< 0.05.
Table 2.
V. cholerae strains used in this study.
Table 3.
Antibodies used in this study.