罗非鱼源无乳链球菌VII型分泌系统膜蛋白EssC 的生物学功能研究

Biological function of type VII secretion system membrane protein EssC of Streptococcus agalactiae from Tilapia

摘要

[目的]本文旨在探究罗非鱼源无乳链球菌VII型分泌系统（type VII secretion system，T7SS）膜蛋白 EssC 的生物学功能。[方法]以 GD201008-001菌株为研究对象，通过生物信息学分析、预测该菌株的 T7SS 基因簇及其潜在底物，并构建基因缺失株 ΔessC 和互补株 C ΔessC ，测定菌株的生长曲线，制备 T7SS 底物多克隆抗体并检测 T7SS 底物的分泌，同时测定菌株抗巨噬细胞吞噬能力以及对罗非鱼脑细胞的毒性和对小鼠的毒力。[结果]成功构建了 T7SS 膜蛋白 EssC 的基因缺失株 ΔessC和互补株 CΔessC ，并制备了 4 种预测的 WXG 家族底物多克隆抗体，通过蛋白免疫印迹法用多克隆抗体对提取的野生株和缺失株分泌蛋白进行检测。结果显示：野生株的分泌蛋白中能检到预测的 T7SS 底物，说明该菌株的 T7SS 是具有功能性的，而 ΔessC 株的分泌蛋白中未检测到 T7SS 底物，说明 essC 基因影响了 T7SS 底物的分泌；细胞感染试验显示，与野生株相比，ΔessC 株的抗巨噬细胞吞噬能力显著下降（ P<0.01 ），对罗非鱼脑细胞的毒性也极显著降低（ P<0.001 ），而基因互补株又能恢复至野生株水平；动物攻毒试验结果显示，essC 基因缺失能延缓小鼠死亡。[结论]essC 基因在无乳链球菌 GD201008-001的 T7SS 底物分泌中发挥重要作用，并对细菌的毒力产生影响。

Abstract

[Objectives] Exploring the biological function of the type VII secretion system（T7SS）EssC component of Streptococcus agalactiae from tilapia.[Methods] This study predicted the T7SS gene cluster and potential substrates of the GD201008-001 strain through bioinformatics analysis ，and constructed the gene deficient strain ΔessC and the gene complementary strain CΔessC .The growth curve of the strain was determined，substrate polyclonal antibodies were prepared，and the secretion of T7SS substrate was detected.At the same time，the strain＇s ability to resist macrophage phagocytosis，toxicity to tilapia brain cells，and toxicity to mice were also measured. [Results] The gene deletion strain ΔessC and complementary strain CΔessC of T7SS membrane protein EssC were successfully constructed，and four predicted WXG family substrate polyclonal antibodies were prepared.The extracted wild-type and deletion strain secreted proteins were detected by Western blot using polyclonal antibodies.The results showed that the predicted T7SS substrate could be detected in the secreted proteins of the wild-type strain，indicating that the T7SS in this strain was functional. However，T7SS substrate wasn＇t detected in the secretion protein of ΔessC ，indicating that ess C affects the secretion of T7SS substrate. Cell infection experiments showed that the anti-macrophage phagocytic ability (P<0.01) and the toxicity (P<0.001) to tilapia brain cells of ΔessC were also significantly reduced，while the gene complementary strain was able to recover to the level of the wild strain.Animal experiments showed that the deletion of the essC gene could delay death in mouse. [Conclusions] The essC gene played an important role in the secretion of T7SS substrate and also had impact on the virulence of the bacteria.

关键词

无乳链球菌 / VII型分泌系统 / essC 基因 / 细胞毒性

Key words

Streptococcus agalactiae / type VII secretion system / essC gene / cytotoxicity

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基金资助

国家自然科学基金-区域创新发展联合基金项目(U23a20254)

江苏省重点行业领域人才项目