Zhang Weijie, Chen Liangchuan, Feng Haiyun, Wang Junlin, Zeng Fuyuan, Xiao Xing, Jian Jichang, Wang Na, Pang Huanying
Fisheries College, Guangdong Ocean University, Zhanjiang, China.
Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture & Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes, Zhanjiang, China.
Front Vet Sci. 2022 Aug 8;9:938822. doi: 10.3389/fvets.2022.938822. eCollection 2022.
, a Gram-negative bacterium, is an opportunistic pathogen of both marine animals and humans, resulting in significant losses in the aquaculture industry. Type III secretion system (T3SS) is a crucial virulence mechanism of . In this study, the T3SS regulatory gene , which was cloned from wild-type strain HY9901, is 861 bp encoding a protein of 286 amino acids. The Δ was constructed by homologous recombination and Overlap-PCR. Although there was no difference in growth between HY9901 and Δ, the Δ exhibited significantly decreased extracellular protease activity and biofilm formation. Besides, the Δ showed a weakened swarming phenotype and an ~100-fold decrease in virulence to zebrafish. Antibiotic susceptibility testing showed the HY9901Δ was more sensitive to kanamycin, minocycline, tetracycline, gentamicin, doxycycline and neomycin. Compared to HY9901 there were 541 up-regulated genes and 663 down-regulated genes in Δ, screened by transcriptome sequencing. qRT-PCR and β-galactosidase reporter assays were used to analyze the transcription levels of gene revealing that gene could facilitate the expression of gene. Finally, , vaccinated with Δ through intramuscular injection, induced a relative percent survival (RPS) value of 66.7% after challenging with HY9901 wild type strain. qRT-PCR assays showed that vaccination with Δ increased the expression of immune-related genes, including , and α in zebrafish. In summary, these results demonstrate the importance of in and provide a basis for further investigations into the virulence and infection mechanism.
[细菌名称]是一种革兰氏阴性菌，是海洋动物和人类的机会致病菌，给水产养殖业造成重大损失。III型分泌系统（T3SS）是[细菌名称]的关键毒力机制。在本研究中，从[细菌名称]野生型菌株HY9901克隆的T3SS调控基因[基因名称]为861 bp，编码一个286个氨基酸的蛋白质。通过同源重组和重叠PCR构建了Δ[基因名称]突变体。虽然HY9901和Δ[基因名称]在生长方面没有差异，但Δ[基因名称]的细胞外蛋白酶活性和生物膜形成显著降低。此外，Δ[基因名称]表现出较弱的群游表型，对斑马鱼的毒力降低了约100倍。药敏试验表明，HY9901Δ[基因名称]对卡那霉素、米诺环素、四环素、庆大霉素、强力霉素和新霉素更敏感。通过转录组测序筛选，与HY9901相比，Δ[基因名称]中有541个上调基因和663个下调基因。采用qRT-PCR和β-半乳糖苷酶报告基因检测分析[基因名称]基因的转录水平，发现[基因名称]基因可促进[另一基因名称]基因的表达。最后，通过肌肉注射用Δ[基因名称]疫苗接种的斑马鱼，在用HY9901野生型菌株攻毒后诱导的相对存活率（RPS）值为66.7%。qRT-PCR检测表明，用Δ[基因名称]疫苗接种可增加斑马鱼中免疫相关基因的表达，包括[相关基因名称]、[相关基因名称]和α[相关基因名称]。总之，这些结果证明了[基因名称]在[细菌名称]中的重要性，并为进一步研究其毒力和感染机制提供了依据。
