The potential compensatory mechanism between pmbA and tldD governing the virulence of Aeromonas veronii and its implications on the immune response in freshwater bivalve (Hyriopsis cumingii)

Abstract

Aeromonas veronii is an opportunistic pathogen that causes diseases in aquatic animals, but its key virulence factors remain unclear. In order to evaluate the feasibility of potential compensatory mechanism to be a novel target for developing live attenuated vaccine, virulence-related phenotypes and the immune response of H. cumingii were measured. Then two single-deletion mutants ΔPmbA, ΔTldD and one double-deletion mutant ΔP&T were constructed by homologous recombination. The hemolytic activity of double-deletion mutant ΔP&T showed dramatically lower than wide-type GL2 (P < 0.05), and the two genes could significantly regulate the proteolytic activity on skimmed milk (P < 0.05), except for casein (P > 0.05). All mutants exhibited attenuated capacities to form biofilms (P < 0.05), and pmbA mRNA was upregulated dramatically (P < 0.05) under exogenous threats modeled by osmotic stress, in which tldD mRNA was overlapped. Besides, the absence of one gene resulted in significantly higher (P < 0.05) mRNA level of another gene. ftsH mRNA was restrained dramatically (P < 0.05) in all mutants, and aerA mRNA was suppressed remarkably (P < 0.05) in ΔP&T. Challenge tests showed that the pathogenicity of ΔP&T on Hyriopsis cumingii was further reduced compared with the two single-deletion mutants, and its LD50 was 6.31-fold than that of GL2. In addition, enzymatic activities in the hemolymph and hepatopancreas of ΔP&T group were significantly lower (P < 0.05) than those in GL2 group at different time points, and their peaks in hepatopancreas were found almost at 12 h post challenge, which were supported by the qPCR results of HcIL-17 and TR. Besides, histopathological examination displayed that the degree of tissue damage in ΔP&T group was alleviated remarkably compared with GL2 group. Finally, bacterial loads in the hemolymph and hepatopancreas of ΔP&T group were lower significantly (P < 0.05) than other groups. The above results demonstrate that the compensatory mechanism between the genes pmbA and tldD is a novel target to attenuate A. veronii, and the double-deletion mutant ΔP&T is helpful for the development of live attenuated vaccine.