Zhang Dunhua, Feng Jun, Wang Yi, Shoemaker Craig A, Wise Allison A, Beck Benjamin H
Aquatic Animal Health Research Unit, USDA-ARS, 990 Wire Road, Auburn, AL 36832, United States.
Department of Biosystems Engineering, Auburn University, Auburn, AL 36849, United States.
FEMS Microbiol Lett. 2025 Jan 10;372. doi: 10.1093/femsle/fnae108.
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.
溶血蛋白是嗜水气单胞菌致病的主要毒力因子组。从导致斑点叉尾鮰严重疾病的强毒嗜水气单胞菌（vAh）基因组中发现了六个编码可能的溶血蛋白的基因。本研究的目的是评估这些溶血蛋白对该细菌毒力的贡献。研究了编码以下六种蛋白质的基因：气溶素（Arl）、21 kDa溶血素（Hly1）、耐热溶血素（Hly2）、磷脂酶/卵磷脂酶相关溶血素（Hly3）、膜相关溶血素III（Hly4）和细胞溶素相关溶血素（Hly5）。使用CRISPR-Cas9介导的方法从细菌中删除单个基因。评估表明，Arl基因缺失（Δarl）的突变体完全丧失溶血活性，而Δhly1-Δhly5突变体与野生型vAh具有相同的活性。与野生型vAh相比，Δarl突变体的细胞外蛋白（ECPs）在体外引起的细胞死亡显著减少（p < 0.01），存活率提高了约20%。Δhly1-Δhly5突变体的ECPs与野生型vAh具有相同的细胞毒性。从Δarl突变体中再次删除hly5进一步降低了该突变体ECP的细胞毒性（Δarl + Δhly5）。体内试验表明，与野生型vAh相比，Δarl和Δhly5突变体导致的鱼类死亡率均降低，分别降低了57%和16%；Δarl + Δhly5突变体导致的死亡率最低，降低了约87%；其他突变体与野生型vAh具有相同的毒力。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹分析明显表明，Arl和Hly5蛋白在从细菌分泌后形成六聚体样稳定结构。Arl和Hly5在细胞毒性和致病方面显然具有协同作用，是本研究分析的六种溶血蛋白中的主要毒力因子。
