Functional analysis of hisJ in Aeromonas veronii reveals a key role in virulence

Aeromonas veronii is an important aquatic zoonotic pathogen in humans and animals. In recent years, extracellular proteins from bacteria have been found to be the major pathogenic factors for aquatic animals. The aim of this study was to systematically analyze the extracellular proteins of nine sources of A. veronii and the effects of hisJ on virulence. We screened only the common proteins from nine different sources of A. veronii by liquid chromatography-tandem mass spectrometry and identified the gene hisJ. We then constructed Delta hisJ (deleted) and C-hisJ (complemented) variants of A. veronii TH0426 to assess the biological function of hisJ. While the Delta hisJ strain did not show altered growth (P > 0.05), we observed that it had reduced colony formation and biofilm formation and reduced adhesion to and invasion of epithelioma papulosum cyprini cells by 2.0-, 1.9-, and 10.8-fold, respectively. Additionally, infection experiments on zebrafish and mouse infection experiments showed that the virulence of the Delta hisJ strain was decreased by 865-fold (P < 0.001) compared with the wild-type strain; virulence of the complemented C-hisJ strain was reduced only 2.8-fold. Furthermore, in the context of hisJ deletion, flagella of A. veronii TH0426 were easily detached and the expression of virulence genes was downregulated. A persistence test (of bacterial colonies in crucian carp) showed that the number of bacteria in the immune organs of the Delta hisJ-infected group was lower than that in the wild-type-infected group. Overall, these results show that hisJ affects flagellar shedding, virulence, biofilm formation, adhesion, and invasion of A. veronii TH0426, and that hisJ is closely associated with virulence and plays a crucial role in its pathogenicity of A. veronii TH0426.