mfpA突变对副溶血性弧菌运动性和生物被膜形成的影响

Influences of mfpA mutation on the motility and biofilm formation of Vibrio parahaemolyticus

摘要:

膜融合蛋白(membrane fusion protein, MFP) vpa1443-vpa1445基因簇(mfpABC)推测可能参与副溶血性弧菌(Vibrio parahaemolyticus, Vp)生物被膜的形成，其中mfpA (vpa1445)编码一种含RTX毒素样结构域的Ca²⁺结合膜外蛋白，但其功能尚未明确。目的 探究mfpA突变对Vp生物被膜形成和运动性的影响。方法 分别构建mfpABC基因簇中3个基因的单突变菌株，比较3个突变株运动性的变化；在此基础上，重点分析mfpA对细菌运动能力和生物被膜形成能力的影响，通过RT-qPCR分析相关基因的表达，并探究其对HeLa细胞毒性的影响。结果 mfpA突变可显著降低Vp的浮游(swimming)和群聚(swarming)运动能力；结晶紫染色和扫描电镜试验表明，ΔmfpA突变可提高菌株生物被膜的形成能力，并增加突变株胞外多糖和胞外蛋白的含量；RT-qPCR证实，ΔmfpA突变株中鞭毛相关基因的表达量下降，而胞外多糖合成相关基因的表达量上升；此外，ΔmfpA突变株可显著减弱Vp的细胞毒性。结论 mfpA突变可通过影响鞭毛和胞外多糖相关基因的表达，降低Vp的运动能力，提高生物被膜的形成能力，削弱其细胞毒性。

Abstract:

The vpa1443-vpa1445 gene cluster (mfpABC) encoding the membrane fusion protein (MFP) is hypothesized to be involved in the biofilm formation of Vibrio parahaemolyticus (Vp). The gene mfpA (vpa1445) encodes a Ca²⁺-binding extracellular protein containing a repeats-in-toxin (RTX) domain, while its function is still under exploration.Objective To study the influences of mfpA mutation on the biofilm formation and motility of Vp.Methods The single mutants of three genes in the mfpABC gene cluster were constructed, and the Vp motility was compared between the three mutants and the wild type. Furthermore, the influences of mfpA mutation on bacterial motility and biofilm formation were analyzed in detail, and the expression of related genes was analyzed by RT-qPCR. Moreover, the cytotoxicity of ΔmfpA to HeLa cells was investigated.Results The mutation of mfpA significantly reduced the swimming and swarming motility of Vp. Crystal violet staining and scanning electron microscopy results showed that the mutation of mfpA enhanced the biofilm formation and increased the content of extracellular polysaccharides and proteins. RT-qPCR confirmed that the expression levels of flagellar genes were downregulated, while those of extracellular polysaccharide synthesis-related genes were upregulated in ΔmfpA. The cytotoxicity of ΔmfpA significantly decreased compared with that of wild type.Conclusion The mutation of mfpA can affect the expression of genes associated with flagella and extracellular polysaccharides to reduce the motility, enhance the biofilm formation, and attenuate the cytotoxicity of Vp.