无钙环境对副溶血弧菌功能表型及基因表达的影响

Effects of calcium-free conditions on the functional phenotypes and gene expression of Vibrio parahaemolyticus

摘要:

目的 分析无钙离子(Ca²⁺)环境对副溶血弧菌生物膜形成、毒力及基因表达的影响。方法 向培养基中添加乙二醇双(2-氨基乙基醚)四乙酸(ethylene glycol-tetraacetic acid, EGTA)以螯合Ca²⁺，从而构建无Ca²⁺环境。采用结晶紫染色实验、泳动及群集运动实验检测副溶血弧菌的生物膜形成能力和运动能力；通过神奈川现象检测、HeLa细胞黏附及细胞毒性实验分析副溶血弧菌的毒力表型；利用RNA-seq技术分析无Ca²⁺环境对副溶血弧菌基因表达的影响。结果 无Ca²⁺环境可抑制副溶血弧菌的生长，显著降低其生物膜形成能力、胞内c-di-GMP水平、泳动及群集运动能力。同时，该环境还能抑制副溶血弧菌的溶血活性及细胞黏附活性，但增强其细胞毒性。转录组分析显示，无Ca²⁺环境下有359个基因呈现显著差异表达(differentially expressed genes, DEGs)，这些基因涉及生物膜形成、运动、毒力及调控子相关基因；其中，参与侧鞭毛和极鞭毛合成的基因表达下调，多数与毒力相关的基因表达上调，推定的调控子基因表达下调。结论 无Ca²⁺环境对副溶血弧菌的生物膜形成、运动能力、毒力以及基因表达均具有显著影响。

Abstract:

Objective To investigate the effects of calcium ion-free (Ca²⁺-free) conditions on the gene expression, biofilm formation, and virulence of Vibrio parahaemolyticus.Methods Ethylene glycol-tetraacetic acid was used to chelate Ca²⁺ in culture media to create Ca²⁺-free conditions. Crystal violet staining was employed to evaluate the biofilm formation of V. parahaemolyticus. Swimming and swarming assays were performed to assess the motility. Additionally, the Kanagawa phenomenon test, HeLa cell adhesion assay, and cytotoxicity experiment were conducted to analyze the virulence phenotypes of V. parahaemolyticus. By comparing the expression profiles, we analyzed the effect of Ca²⁺-free conditions on the gene expression in V. parahaemolyticus.Results Ca²⁺-free conditions inhibited the growth and significantly reduced the biofilm formation, intracellular c-di-GMP levels, and motility of V. parahaemolyticus. Furthermore, Ca²⁺-free conditions suppressed the hemolytic activity and reduced the bacterial adhesion to HeLa cells, while enhancing the cytotoxicity of V. parahaemolyticus. Transcriptomic analysis revealed 359 differentially expressed genes (DEGs) under Ca²⁺-free conditions. These DEGs were mainly associated with biofilm formation, virulence factors, and regulators. Notably, the genes involved in lateral flagella and polar flagellum were downregulated, while most virulence genes were upregulated. The majority of putative regulator genes were downregulated.Conclusion Ca²⁺-free conditions significantly affect the biofilm formation, motility, virulence, and gene expression of V. parahaemolyticus.