Contribution of hemagglutinin/protease and motility to the pathogenesis of El Tor biotype cholera

Abstract

Vibrio cholerae is a highly motile organism that secretes a Zn-dependent metalloprotease, hemagglutinin/protease (HapA). HapA has been shown to have mucinase activity and contribute to the reactogenicity of live vaccine candidates, but its role in cholera pathogenesis is not yet clear. The contribution of motility to pathogenesis is not fully understood, since conflicting results have been obtained with different strains, mutants, and animal models. The objective of this work was to determine the contribution of HapA and motility to the pathogenesis of El Tor biotype cholera. To this end we constructed isogenic motility (motY) and mucinase (hapA) single and double mutants of an El Tor biotype V. cholerae strain. Mutants were characterized for the expression of major virulence factors in vitro and in vivo. The motility mutant showed a remarkable increase in cholera toxin (CT), toxin coregulated pilus major subunit (TcpA), and HapA production in vitro. Increased TcpA and CT production could be explained by increased transcription of tcpA, ctxA, and toxT. No effect was detected on the transcription of hapA in the motility mutant. The sodium ionophore monensin diminished production of HapA in the parent but not in the motility mutant. Phenamil, a specific inhibitor of the flagellar motor, diminished CT production in the wild-type and motY strains. The hapA mutant showed increased binding to mucin. In contrast, the motY mutation diminished adherence to biotic and abiotic surfaces including mucin. Lack of HapA did not affect colonization in the suckling mouse model. The motility and mucinase defects did not prevent induction of ctxA and tcpA in the mouse intestine as measured by recombinase-based in vivo expression technology. Analysis of mutants in the rabbit ileal loop model showed that both V. cholerae motility and HapA were necessary for full expression of enterotoxicity.

FIG. 1.

Production of CT, TcpA, and HapA. Strains AC-V66 (wild type), AJB31 (hapA), AJB32 (motY), and AJB35 (hapA motY) were grown in TSB for 16 h at 37°C with shaking to determine HapA production. For CT (supernatant) and cell-associated TcpA (pellet), strains were grown in AKI medium for 18 h at 30°C. Each value is the mean of six independent cultures. Error bars indicate the standard deviations. *, Significantly different from the wild type (t test, α = 0.05). **, Significantly different from the wild type (α = 0.01). OD₆₀₀, optical density at 600 nm.

FIG. 2.

Relative expression of ctxA, tcpA, toxT, and hapA mRNA. Production of mRNA was measured by real-time RT-PCR with SYBR Green. The recA mRNA was used as a reference. For ctxA, tcpA, and toxT mRNA, AC-V66 (wild type) and AJB32 (motY) were grown in AKI medium for 8 h at 30°C. For hapA mRNA, strains were grown in TSB at 37°C to an optical density at 600 nm of 2. Each bar reflects the average of six independent cultures. Error bars indicate the standard deviations. *, Significantly different from the wild type (t test, α = 0.05). **, Significantly different from the wild type (α = 0.01).

FIG. 3.

Effect of phenamil and monensin on production of HapA and CT. For HapA production (top panel), six cultures of AC-V66 (wild type, open bar) and AJB32 (motY, filled bar) were grown with and without phenamil or monensin in TSB for 18 h at 37°C. For CT production (lower panel), strains were grown with and without phenamil or monensin in AKI cultures for 18 h at 30°C. *, Significantly different from untreated control (t test, α = 0.05). **, Significantly different from untreated control (α = 0.01). OD₆₀₀, optical density at 600 nm.

FIG. 4.

Adherence properties of the wild type, mucinase, and motility isogenic mutants. Strains AC-V66 (wild type), AJB31 (hapA), AJB32 (motY), and AJB35 (hapA motY) were grown in LB medium for 24 h at 30°C in 96-well polystyrene microtiter plates and in polystyrene microtiter plates coated with 1 mg/ml gastric porcine mucin in PBS. V. cholerae attachment to these surfaces is expressed as the optical density at 570 nm (OD₅₇₀) (attached)/optical density at 600 nm (OD₆₀₀) (growth) ratio. V. cholerae strains were grown in LB at 37°C for 16 h with shaking and approximately 10⁸ vibrios in DMEM-F12 medium inoculated to T84 cells grown in 24-well tissue cultures plates to 95% confluence. Results are expressed as the fraction of V. cholerae cells bound to T84 cells. Each value is the mean of three independent cultures and six wells per culture. The error bars indicate the standard deviations. *, Significantly different from untreated control (t test, α = 0.05). **, Significantly different from untreated control (α = 0.01).

FIG. 5.

Analysis of mucinase and motility mutants in the rabbit ileal loop model. A. Rabbit ileal loops were inoculated with 10⁸ CFU of each strain AC-V66 (wild type), AJB31 (hapA), AJB32 (motY), and AJB35 (hapA motY) in PBS and incubated for 9 h. Results are expressed as fluid accumulation (FA) (in milliliters) per loop length (in centimeters). Shown are means ± standard deviations; n = 5. *, Different from the wild type (α = 0.01); **, different from hapA (α = 0.05) and motY (α = 0.01). B. Cell pellets and fluids recovered from three loops per strain were analyzed for production of TcpA and CT. ‡, Different from the wild type (α = 0.01).