嗜水气单胞菌 tolA 基因生物学功能研究

Biological functions of tolA in Aeromonas hydrophila

摘要:

嗜水气单胞菌( Aeromonas hydrophila)是一种既能感染鱼类，又能感染包括人在内的哺乳动物的病原体。 目的 构建 A. hydrophila ATCC 7966的 tolA基因缺失株，以探究 tolA基因的生物学功能。 方法 通过同源重组技术构建 tolA基因缺失菌株AhΔ tolA，并对AhΔ tolA的生理表型进行测定和分析，利用转录组测序技术探究 tolA缺失导致的相关基因表达变化。 结果 嗜水气单胞菌 tolA缺失菌株的细胞形态发生了明显改变，其对脱氧胆酸钠的敏感度显著增强，氧化应激反应也明显加剧。该缺失菌株的生物被膜形成能力以及多种毒力基因的表达水平显著降低，而外膜囊泡的产量增多。转录组测序及比较分析发现，WT/AhΔ tolA比较组共筛选到300个差异表达基因(differentially expressed genes, DEGs)，其中上调171个，下调129个。GO功能富集分析发现差异表达基因主要富集在氧化还原过程、能量代谢过程、细胞外膜组成和氧化还原酶活性等途径。KEGG富集分析发现，差异表达基因主要富集在次生代谢物生物合成、不同环境下的微生物代谢、辅助因子的生物合成以及氨基酸生物合成等途径。 结论 本研究阐明了 tolA基因在嗜水气单胞菌中的重要生理功能，并探讨了 tolA基因可能参与和影响的多种代谢途径。研究结果为嗜水气单胞菌的防治提供了理论支持。

关键词:嗜水气单胞菌;tolA;生物被膜;外膜囊泡;转录组

Abstract:

Aeromonas hydrophila is a pathogen that can infect both fish and mammals, including humans. Objective To construct the tolA-deleted strain of A. hydrophila ATCC 7966 and use this strain to explore the biological functions of tolA. Methods We constructed the tolA-deleted strain AhΔ tolA by homologous recombination and characterized the physiological phenotype of AhΔ tolA. Transcriptome sequencing was performed to compare the gene expression between the wild type (WT) and AhΔ tolA. Results The cell morphology of AhΔ tolA was changed. The deletion of tolA significantly enhanced the sensitivity to sodium deoxycholate and oxidative stress, while significantly reducing the biofilm formation and the expression levels of several virulence genes. The yield of outer membrane vesicles was significantly increased in AhΔ tolA. Transcriptomic analysis data showed that a total of 300 differentially expressed genes (DEGs) were screened between WT and AhΔ tolA, including 171 genes with up-regulated expression and 129 genes with down-regulated expression. GO enrichment analysis showed that the DEGs were mainly enriched in the oxidation-reduction process, metabolic process, outer membrane, and oxidoreductase activity. KEGG pathway enrichment analysis showed that the DEGs were mainly enriched in the biosynthesis of secondary metabolites, microbial metabolism in diverse environments, biosynthesis of cofactors, and biosynthesis of amino acids. Conclusion This study gives an insight into the roles of tolA in A. hydrophila and provides information about the metabolic pathways involving tolA. These results provide a theoretical reference for the prevention and control of A. hydrophila.

Key words:Aeromonas hydrophila;tolA;biofilm;outer membrane vesicles;transcriptome