Abstract
NorV has been known to be an anaerobic nitric oxide reductase associated with nitric oxide (NO) detoxification. Recently, we showed that the norV gene of Aeromonas hydrophila was highly upregulated after co-culturing with Tetrahymena thermophila. Here, we demonstrated that the transcription and expression levels of norV were upregulated in a dose-dependent manner after exposure to NO under aerobic and anaerobic conditions. To investigate the roles of norV in resisting predatory protists and virulence of A. hydrophila, we constructed the norV gene-deletion mutant (ΔnorV). Compared to the wild type, the ΔnorV mutant showed no significant difference in growth at various NO concentrations under aerobic conditions but significantly stronger NO-mediated growth inhibition under anaerobic conditions. The deletion of norV exhibited markedly decreased cytotoxicity, hemolytic and protease activities under aerobic and anaerobic conditions. Also, the hemolysin co-regulated protein (Hcp) in the ΔnorV mutant showed increased secretion under aerobic conditions but decreased secretion under anaerobic conditions as compared to the wild-type. Moreover, the inactivation of norV led to reduced resistance to predation by T. thermophila, decreased survival within macrophages and highly attenuated virulence in zebrafish. Our data indicate a diverse role for norV in the expression of A. hydrophila virulence-associated traits that is not completely dependent on its function as a nitric oxide reductase. This study provides insights into an unexplored area of NorV, which will contribute to our understanding of bacterial pathogenesis and the development of new control strategies for A. hydrophila infection.
Highlights
Aeromonas hydrophila is a Gram-negative bacterium, commonly found in a variety of natural aquatic environments worldwide including seawater, freshwater, sediments and even drinking water [1]
nitric oxide (NO) activates the expression of norV gene To determine how the norV gene in A. hydrophila responds to NO exposure, qRT-RCR analysis and Western blot were preformed
The mRNA level of norV was significantly enhanced under anaerobic conditions compared to that under aerobic conditions (P < 0.001), suggesting that norV expression to some extent may be inhibited by oxygen
Summary
Aeromonas hydrophila is a Gram-negative bacterium, commonly found in a variety of natural aquatic environments worldwide including seawater, freshwater, sediments and even drinking water [1]. This bacterium is responsible for a variety of diseases in amphibians, fish and reptiles. It has been demonstrated that pathogens including Legionella pneumophila [9], Salmonella enteritidis [10, 11], and Mycobacterium avium [12] are able to survive protistan predation, subsequently resulting in resistance to adverse situations such as antibiotics, oxidants and bioacids and increasing virulence. Grazing resistance to protists is an evolutionary precursor of bacterial pathogenicity and promotes bacteria to develop some defensive mechanisms for survival, Liu et al Vet Res (2019) 50:67 such as new gene expression patterns or new proteins which may emerge as virulence determinants in animal and human infections [13, 14]
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