Table_3_The temperature-dependent expression of type II secretion system controls extracellular product secretion and virulence in mesophilic Aeromonas salmonida SRW-OG1.docx

Aeromonas salmonicida is a typical cold water bacterial pathogen that causes furunculosis in many freshwater and marine fish species worldwide. In our previous study, the pathogenic A. salmonicida (SRW-OG1) was isolated from a warm water fish, Epinephelus coioides was genomics and transcriptomics analyzed. Type II secretion system was found in the genome of A. salmonicida SRW-OG1, while the expressions of tatA, tatB, and tatC were significantly affected by temperature stress. Also, sequence alignment analysis, homology analysis and protein secondary structure function analysis showed that tatA, tatB, and tatC were highly conservative, indicating their biological significance. In this study, by constructing the mutants of tatA, tatB, and tatC, we investigated the mechanisms underlying temperature-dependent virulence regulation in mesophilic A. salmonida SRW-OG1. According to our results, tatA, tatB, and tatC mutants presented a distinct reduction in adhesion, hemolysis, biofilm formation and motility. Compared to wild-type strain, inhibition of the expression of tatA, tatB, and tatC resulted in a decrease in biofilm formation by about 23.66%, 19.63% and 40.13%, and a decrease in adhesion ability by approximately 77.69%, 80.41% and 62.14% compared with that of the wild-type strain. Furthermore, tatA, tatB, and tatC mutants also showed evidently reduced extracellular enzymatic activities, including amylase, protease, lipase, hemolysis and lecithinase. The genes affecting amylase, protease, lipase, hemolysis, and lecithinase of A. salmonicida SRW-OG1 were identified as cyoE, ahhh1, lipA, lipB, pulA, HED66_RS01350, HED66_RS19960, aspA, fabD, and gpsA, which were notably affected by temperature stress and mutant of tatA, tatB, and tatC. All above, tatA, tatB and tatC regulate the virulence of A. salmonicida SRW-OG1 by affecting biofilm formation, adhesion, and enzymatic activity of extracellular products, and are simultaneously engaged in temperature-dependent pathogenicity.

嗜水气单胞菌（Aeromonas salmonicida）是一种典型的冷水细菌性病原体，在全球范围内广泛引起多种淡水及海水鱼类发生疖病。在我方先前的研究中，从温水鱼类真鲷（Epinephelus coioides）中分离得到的致病性嗜水气单胞菌（SRW-OG1）进行了基因组学和转录组学分析。在嗜水气单胞菌SRW-OG1的基因组中发现了II型分泌系统，而tatA、tatB和tatC基因的表达受到温度应激的显著影响。此外，序列比对分析、同源性分析和蛋白质二级结构功能分析表明，tatA、tatB和tatC基因高度保守，显示出其生物学重要性。在本研究中，通过构建tatA、tatB和tatC的突变体，我们探讨了嗜水气单胞菌SRW-OG1在中温条件下的温度依赖性致病力调节机制。根据我们的研究结果，tatA、tatB和tatC突变体在粘附、溶血、生物膜形成和运动能力方面表现出显著降低。与野生型菌株相比，tatA、tatB和tatC基因表达的抑制导致生物膜形成减少了约23.66%、19.63%和40.13%，粘附能力分别降低了约77.69%、80.41%和62.14%。此外，tatA、tatB和tatC突变体还显示出明显的胞外酶活性降低，包括淀粉酶、蛋白酶、脂肪酶、溶血和卵磷脂酶等。影响嗜水气单胞菌SRW-OG1中淀粉酶、蛋白酶、脂肪酶、溶血和卵磷脂酶的基因被鉴定为cyoE、ahhh1、lipA、lipB、pulA、HED66_RS01350、HED66_RS19960、aspA、fabD和gpsA，这些基因受温度应激和tatA、tatB、tatC突变体的影响显著。综上所述，tatA、tatB和tatC通过影响生物膜形成、粘附和胞外产物的酶活性来调节嗜水气单胞菌SRW-OG1的致病性，并且同时参与温度依赖性的病原性。