The type VI secretion system (T6SS) and hemolysin HlyA are important virulence factors in Vibrio cholerae. The forkhead-associated (FHA) domain is a conserved phosphopeptide binding domain that exists in many regulatory modules. The FHA domain protein-encoding gene is conserved in the T6SS gene cluster and regulates the assembly and secretion of the T6SS. This study shows for the first time that the FHA domain protein TagH plays a role in controlling the hemolytic activity of V. cholerae, in addition to regulating the T6SS. TagH negatively regulates HlyA expression at the transcriptional and post-translational levels. The phosphopeptide binding sites of the FHA domain of TagH play a key role in the regulation of hemolytic activity. The deletion of tagH enhances the intestinal pathogenicity and extraintestinal invasion ability of V. cholerae, which mainly depend on the expression of HlyA. This study provides evidence that helps unravel the novel regulatory role of TagH in HlyA and provides critical insights which will aid in the development of strategies to manage HlyA.

The spread of multidrug-resistant strains of Neisseria gonorrhoeae poses a great challenge in gonorrhea treatment. At present, vaccination is the best strategy for gonorrhea control. However, given the extensive antigenic variability of N. gonorrhoeae, the effectiveness of monovalent vaccines is limited. Therefore, increasing the coverage of vaccination by using a multivalent vaccine may be more effective. In this study, a trivalent vaccine comprising three conserved antigens, namely, the App passenger domain, MetQ, and neisserial heparin binding antigen (NHBA), was constructed, and its protective effect was evaluated. Trivalent vaccines induced stronger circulating IgG and IgA antibody responses in mice than monovalent vaccines, in addition to eliciting Th1, Th2, and Th17 immune responses. Antiserum generated by the trivalent vaccine killed N. gonorrhoeae strains (homologous FA1090 and heterologous FA19), exhibiting superior bactericidal capacity than NHBA and MetQ vaccine antisera against N. gonorrhoeae, but similar capacities to those of the App vaccine antiserum. In addition, the trivalent vaccine antiserum achieved greater inhibition of N. gonorrhoeae FA1090 strain adherence to ME-180 cells compared to that elicited by the monovalent vaccine antiserum. In a mouse vaginal infection model, the trivalent vaccine was modestly effective (9.2% decrease in mean area under curve compared to the pCold-TF control mice), which was somewhat better than the protection seen with the monovalent vaccines. Our findings suggest that recombinant multivalent vaccines targeting N. gonorrhoeae exhibit advantages in protective efficacy compared to monovalent vaccines, and future research on multivalent vaccines should focus on optimizing different antigen combinations.