Contributions of hemolytic proteins in virulent Aeromonas hydrophila to motile aeromonas septicemia disease of channel catfish (ictalurus punctatus)

Authors: Zhang, Dunhua; WANG, YI - Auburn University; Shoemaker, Craig; Wise, Allison; Beck, Benjamin

Submitted to: FEMS Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/31/2024
Publication Date: 12/10/2024
Citation: Zhang, D., Wang, Y., Shoemaker, C.A., Wise, A.L., Beck, B.H. 2024. Contributions of hemolytic proteins in virulent Aeromonas hydrophila to motile aeromonas septicemia disease of channel catfish (ictalurus punctatus). FEMS Microbiology Letters. 372:108. https://doi.org/10.1093/femsle/fnae108.
DOI: https://doi.org/10.1093/femsle/fnae108

Interpretive Summary: Virulent Aeromonas hydrophila (vAh) is a group of extremely versatile bacteria, which are well-adapted in aquatic environments with potential to cause severe disease in both fish and humans. In aquaculture, many farmed fish species are susceptible to vAh. Infected fish typically developed severe hemorrhage or motile Aeromonas septicemia (MAS), resulting in high rates of mortality. Numerous studies found that pathogenic A. hydrophila possesses many virulence factors, among which hemolytic proteins produced by the pathogen play important roles in disrupting the host cell membrane, lysing red blood cells, and provoking disease. The aim of this study was to assess the contribution of hemolytic proteins to the virulence of pathogenic A. hydrophila by deleting individual coding genes from the bacterial genome with CRISP-Cas9 based editing methods. Results of this study showed that aerolysin and cytolytic hemolysin, among six hemolytic proteins studied, were the most virulent factors in terms of cytotoxicity and fish mortality. These two proteins apparently interacted in the augmentation of pathogenicity. Whether the attenuated mutant, with aerolysin and cytolytic hemolysin encoding genes deleted from the bacterial genome, can be used as a live vaccine against vAh infection will be investigated in the future.

Technical Abstract: Aims: 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 proteins to the virulence of this bacterium. Methods and Results: 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 ('arl) completely abolished hemolytic activity of this mutant while 'hly1 – 'hly5 mutants had the same activity as the wild vAh; only 'arl mutant had reduced cytotoxicity though a double deletion mutant, 'arl+'hly5, had significantly less cytotoxicity than the 'arl mutant; only 'arl (significantly) and 'hly5 (not significantly) mutants caused less fish mortality though 'arl+'hly5 mutant had significantly lower pathogenic potential than the 'arl mutant. Conclusions: Aerolysin (Arl) and cytolytic hemolysin (Hly5), both forming heptamer-like stable structures post secretion, are major virulence factors and had synergistic action in cytotoxicity and causing disease. Impact Statement: The 'arl+'hly5 mutant may be reformed as a live vaccine against vAh infection.