Contributions of hemolytic proteins in virulent <i>Aeromonas hydrophila</i> to motile <i>Aeromonas</i> septicemia disease of channel catfish (<i>Ictalurus punctatus</i>)

Hemolytic proteins are a major group of virulence factors in pathogenic Aeromonas hydrophila. Six genes encoding presumable hemolytic proteins were revealed from the genome of virulent A. hydrophila (vAh) that caused severe disease in channel catfish. The aim of this study was to assess the contribution of these hemolytic proteins to the virulence of this bacterium. Genes coding for following six proteins were investigated: aerolysin (Arl), 21-kDa hemolysin (Hly1), thermostable hemolysin (Hly2), phospholipase/lecithinase-related hemolysin (Hly3), membrane-associated hemolysin III (Hly4), and cytolysin-associated hemolysin (Hly5). Individual genes were deleted from the bacterium using CRISPR-Cas9 mediated methods. Assessment showed that deletion of Arl gene (Δarl) completely abolished hemolytic activity of this mutant while Δhly1-Δhly5 mutants had the same activity as the wild vAh. Extracellular proteins (ECPs) of the Δarl mutant caused significantly (p < 0.01) less cell death in vitro with viability increased by approximately 20%, compared to the wild vAh. ECPs of mutants Δhly1-Δhly5 remained the same cell toxicity as the wild vAh. A second deletion of hly5 from the Δarl mutant further lowered the cell toxicity of the ECP of the mutant (Δarl + Δhly5). Assays in vivo showed that both Δarl and Δhly5 mutants caused less fish mortality with reduction of 57% and 16%, respectively, compared to the wild vAh; the Δarl + Δhly5 mutant caused the least mortality with approximately 87% of reduction; and other mutants had the same virulence as the wild vAh. Analyses of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western blotting evidently indicate that both Arl and Hly5 proteins formed hexamer-like stable structures post secretion from the bacterium. Arl and Hly5 apparently had synergistic action in cytotoxicity and causing disease and were the major virulence factors among the six hemolytic proteins analyzed in this study.