T3SS1和T3SS2影响副溶血弧菌生物学特性及细胞致病性的比较

Comparison of biological characteristics and cytopathogenicities between T3SS1 and T3SS2 in Vibrio parahaemolyticus

摘要:

[目的]探究两套Ⅲ型分泌系统T3SS1和T3SS2影响副溶血弧菌生物学特性及细胞致病性的差异和相关性。[方法]以T3SS1和T3SS2主要结构基因vcrD1和vcrD2为研究对象，利用同源重组技术分别构建单基因和双基因缺失株ΔvcrD1、ΔvcrD2、ΔvcrD1-vcrD2，以及互补株CΔvcrD1和CΔvcrD2；分析各菌株的生长特性、生物被膜形成能力、运动性的差异；比较各菌株对细胞毒性以及对细胞炎性因子转录水平的影响。[结果]与野生株相比，各缺失株的生长速度无显著差异。缺失株ΔvcrD1生物被膜形成能力、运动性和细胞毒性均极显著下降；缺失株ΔvcrD2主要表现为细胞炎性因子IL-1β和IL-6转录水平的显著上调，同时对细胞毒性作用下降。双基因缺失株ΔvcrD1-vcrD2在缺失株ΔvcrD1的基础上，生物被膜形成能力、运动性、细胞毒性均进一步显著下降，但在细胞炎性因子的转录水平上，则与ΔvcrD1一致，与野生株相比均无显著差异。[结论]T3SS1和T3SS2对副溶血弧菌生物学特性和细胞致病性的影响存在差异。T3SS1主要影响细菌的生物被膜形成、运动性及细胞毒性作用；T3SS2不影响生物被膜形成、运动性等生物学特性，参与细菌对细胞炎性反应中的负调控作用，同时具有一定的细胞毒性作用。T3SS1有助于副溶血弧菌在环境中的生存，而T3SS2可有利于细菌在宿主体内免疫逃避的过程。T3SS1和T3SS2对副溶血弧菌生物学特性和细胞致病性的影响可能存在一定的相关作用，具体机制有待进一步研究。

Abstract:

[Objective] To elucidate differences of biological characteristics and cytopathogenicities between T3SS1 and T3SS2 in Vibrio parahaemolyticus. [Methods] With the main structural protein genes vcrD1 and vcrD2 as the research object, we used the homologous recombination technique to construct a series of deletion mutants ΔvcrD1, ΔvcrD2, ΔvcrD1-vcrD2, and complementary strains CΔvcrD1, CΔvcrD2 of V. parahaemolyticus SH112 strain, then analyzed the differences of growth, biofilm, motility, cytotoxicity to RAW264.7 macrophages and Caco-2 cells, and transcriptional regulation of pro-inflammatory cytokine IL-1β, IL-6 in RAW264.7 macrophages between mutant strain and wild strain. [Results] Compared to the wild strain, the mutant strains had no difference on growth rate, however, the vcrD1 mutant obviously weakened biofilm formation, motility, cytotoxicity of V. parahaemolyticus; the vcrD2 mutant positively upregulated the transcriptional levels of pro-inflammatory cytokines, and significantly attenuated cytotoxicity to cells. The ΔvcrD1-vcrD2 double deletion mutant showed weaker biofilm, motility, cell toxicity than vcrD1 mutant strain, and as similar to the vcrD1 mutant, in the mRNA levels of inflammatory cytokines when compared with the wild strain. [Conclusion] Contributions of two T3SSs to biological characteristics and cytopathogenicities of V. parahaemolyticus are different. The T3SS1 was mainly involved in biofilm formation and motility of V. parahaemolyticus, but also has a significant cytotoxic effect; T3SS2 did not affect bacterial biofilm formation and motility, but played a negative regulation on cell inflammatory reaction, potentially contributing to bacterial immune evasion in host. T3SS1 contributed to bacterial survival in the environment; T3SS2 may help V. parahaemolyticus to evade the host immune response. Moreover, T3SS1 and T3SS2 may play related functions on biological characteristics and cytopathogenicities of V. parahaemolyticus, but the specific mechanism still needs further study.