Role of msbB Gene in Physiology and Pathogenicity of Vibrio parahaemolyticus

Abstract

The msbB gene, encoding a lipid A phosphatease, is crucial for lipopolysaccharide (LPS) synthesis in Gram-negative bacteria and plays a critical role in their virulence. This study investigated the role of msbB in Vibrio parahaemolyticus, a significant marine pathogen causing gastroenteritis in humans and infections in aquatic animals. We constructed an msbB deletion mutant (ΔmsbB) and a complementary strain (CΔmsbB) using homologous recombination. The growth, outer membrane permeability, stress and antibiotic sensitivity, biofilm formation, swarming motility, and virulence of the wild-type (WT), ΔmsbB, and CΔmsbB strains were assessed. Additionally, the pathogenicity of ΔmsbB was evaluated using L. vannamei shrimp models. The results showed that the msbB gene was successfully deleted and complemented, and its deletion did not impair bacterial growth. However, the ΔmsbB strain exhibited an increased outer membrane permeability, reduced resistance to stresses and antibiotics, defective biofilm formation, and a reduced swarming motility. In a Tetrahymena co-culture, the ΔmsbB strain showed attenuated virulence. In shrimp infected with the ΔmsbB strain, the cumulative mortality rate was 22%, significantly lower than the 62% observed in the WT strain. Moreover, the expression levels of immune-related genes in the shrimp hepatopancreas were significantly lower in the ΔmsbB group, indicating a significant reduction in infection capability and pathogenicity. These findings indicate that the msbB gene is critical for the virulence of V. parahaemolyticus and suggest that msbB is a potential target for therapeutic interventions and vaccine development against V. parahaemolyticus infections.

Keywords: V. parahaemolyticus; msbB gene; pathogenicity; shrimp infection; virulence.

Figure 1

(A) The strategy of msbB gene was knocked out and complemented by allele exchange in V. parahaemolyticus; (B) agarose gel electrophoresis of PCR products amplified by ΔmsbB T1/T2 and CΔmsbB T1/T2 separately. Left: WT (1160 bp) and ΔmsbB mutant (200 bp); right: ΔmsbB mutant (1352 bp) and its complementary strain CΔmsbB (2313 bp); and (C) the growth curves of WT, ΔmsbB, and CΔmsbB strains. n = 3.

Figure 2

(A) Comparison of outer membrane permeability of WT, ∆msbB and C∆msbB. The fluorescence intensity was determined with excitation at 360 nm and emission at 428 nm. Statistical comparisons were performed using one-way ANOVA analyses followed by a Dunnett’s multiple comparison test; each bar represents as the mean  ±  SD. n = 6; (B) comparison of resistance to 13 antibiotics among WT, ∆msbB and C∆msbB. The fold change is calculated by log2 (the MIC of mutants/MIC of WT); (C) survival of V. parahaemolyticus and ΔmsbB in the presence of varying different ions and antibiotics. Error bars represent the standard deviation of the mean from at least three (n = 3) independent replicates. Statistical comparisons were performed using two-way ANOVA analyses followed by a Dunnett’s multiple comparison test; and (D) analysis of biofilm formation ability of WT, ∆msbB and C∆msbB strains. Statistical comparisons were performed using one-way ANOVA analyses followed by a Dunnett’s multiple comparison test. The data are presented as the mean ± SD (n = 3). Columns have been marked with an asterisk (* p < 0.05; *** p < 0.001). ns: not significant.

Figure 3

(A) Visualization of bacterial flagellar with negative-staining electron microscopy. Scale bar = 1 μm; (B) swimming motility assay of WT, ∆msbB, and C∆msbB on LB plates with 0.3% agar cultured for 6 h and 12 h at 37 °C; (C) analysis of motility ability of WT, ∆msbB, and C∆msbB strains. The diameters of swimming zone reflect bacterial migration on the 0.3% agar. Statistical comparisons were performed using two-way ANOVA analyses followed by a Dunnett’s multiple comparison test; (D) qRT-PCR analysis of the transcription levels of polar flagellar genes (flgB, flgK, flgM, fliC, and fliE) in ∆msbB compared to WT. *** p < 0.001; and (E) qRT-PCR analysis of the transcription levels of lateral flagellar genes (fliK, fliR, flgB, lafA, and motY) in ∆msbB compared to WT. Statistical comparisons were performed using two-way ANOVA analyses followed by a Dunnett’s multiple comparison test; The data are presented as the mean  ±  SD (n  =  3). * p < 0.05; ** p < 0.01; and *** p < 0.001. ns: not significant.

Figure 4

Virulence assessment of WT, ∆msbB, and C∆msbB using the Tetrahymena model. (A–C) Growth dynamics of ∆msbB, C∆msbB, and WT strains co-cultured with or without Tetrahymena. Tetra represents Tetrahymena. (D) Relative survival of ∆msbB, C∆msbB, and WT strains co-cultured with Tetrahymena. Relative survival was calculated as the OD₆₀₀ of strains co-cultured with Tetrahymena divided by the OD₆₀₀ of bacteria grown alone at the end of the experiment. Data are presented as mean ± SD from three independent measurements. *** p < 0.001. ns: not significant. (E) Viability of Tetrahymena cells treated with lysates from the three strains after 6 h, assessed using CCK8 reagents. Statistical comparisons were performed using one-way ANOVA analyses followed by a Dunnett’s multiple comparison test; Data are presented as the mean ± SD (n = 3). The significant difference in the results was analyzed. ** p < 0.01. ns: not significant.

Figure 5

Assessment of ∆msbB and WT strains’ pathogenicity against shrimp. (A) Relative survival curve of shrimp infected by ∆msbB and WT strains with immersion in concentration 4 × 10⁷ CFU/mL; (B) clinical sign of diseased shrimps during infection at 10 d. The arrows indicate hepatopancreas, foregut, and uropod. Scale bar: 1 cm; (C) hepatopancreas of shrimp challenged with ∆msbB and WT strains at 7 d stained with H&E. HpB, B cells; HpF, F cell; HpE, E cell; HpR, R cell; karyomegaly, enlargement of nucleus; Nec, necrosis; Atr, atrophy; Slo, sloughing of epithelial cells. Scale bar: 100 μm; and (D) comparative analysis of the expression of immune-related genes and inflammatory cytokine interleukin-1β (IL-1β) gene in hepatopancreas of shrimp infected with ∆msbB strain vs. WT group. Statistical comparisons were performed using two-way ANOVA analyses followed by a Dunnett’s multiple comparison test; Columns have been marked with an asterisk (* p < 0.05; *** p < 0.001).