应用基因组学和蛋白组学研究禽致病性大肠杆菌VI型分泌系统效应因子的作用机制

The type VI secretion system of avian pathogenic Escherichia coli (APEC)，possessing a mechanism of needle injection, is considered to closely relate to survival, proliferation and pathogenicity in the host. In this project, based on two distinguishable T6SS clusters which are homologous with neonatal meningitis Escherichia coli have been identified in our early genomics analysis, the molecular epidemiology of T6SS core genes will be surveied in about 500 APEC isolates in China to clarify the correlation between T6SS and pathogenicity of APEC. On this basis, the main funcational gene of T6SS clusters will be selected and sequenced. The possible gene of effect factors will be screened and analysed by comparative genomics. Furthermore, the pinpoint switch protein-VgrG and ATP binding protein-ClpV will be focused. The deletion strains of two focused genes will be constructed and analysed. The possible effectors will be screened through comparing the expression proteins among VgrG, ClpV deletion strains and wild strain by comparative proteomics. These effectors(genes and/or proteins) screened by genomics and proteomics will be verified by transcriptomics, and their deletion strains will be constructed respectively; whereby, their function and conduction pathway will be analysed to clarify the pathogenic mechanism of T6SS. Also it can be looking for a new direction for prevention and control of APEC.

禽致病性大肠杆菌（APEC）的VI型分泌系统（T6SS）具有针形注射机制，被认为与其在宿主体内生存、增殖和致病作用密切相关。本项目在前期基因组学分析已定位出两套与新生儿脑膜炎大肠杆菌同源的T6SS基因簇的基础上，拟对分离保存的约500株APEC菌株进行T6SS核心基因的分子流行病学检测，阐明T6SS基因簇与APEC致病性的相关性。在此基础上，挑选阳性株测定T6SS基因簇全序列并通过比较基因组学分析筛选可能的效应因子；选择T6SS的针尖开关蛋白VgrG和ATP绑定蛋白ClpV两个关键蛋白为研究重点，构建两个蛋白的基因缺失株，分析其功能；采用比较蛋白组学分析两个缺失株与野生株间的表达差异，筛选效应因子；对以上基因组学和蛋白组学筛选的效应子进行转录组学验证并分别构建基因敲除株，分析它们各自的功能以及相互联系的传导通路，从而阐明T6SS的作用机制，并为寻找APEC防控新途径打下基础。

VI型分泌系统（Type VI secretion system, T6SS）是一种革兰阴性菌广泛存在的纳米注射器，通过分泌多样的效应蛋白作为分子武器攻击靶细胞从而参与细菌间竞争及致病过程。本研究证实禽致病性大肠杆菌（APEC）主要编码两套T6SS基因簇，能够促进细菌对上皮细胞粘附及在宿主血液增殖和免疫逃避，在APEC致病过程发挥不同的功能。APEC两套T6SS编码的三个Hcp蛋白分属于三个不同的Hcp亚群，且显示不同的转录调控机制。Hcp蛋白结构的细微差异也是引起其功能差异的重要原因，尤其是Loop L2, 3区域（Vs2）是Hcp1和Hcp2B交付抗菌效应因子和抑制巨噬细胞吞噬所必需的。研究鉴定了一个新的T6SS效应子家族Hcp-ETs，其通过Hcp蛋白携带多样的C端毒性Domain。试验证实Hcp-ET1可通过HNH-DNA酶活性有效地抑制靶细菌的生长，进一步结果显示Hcp-ET2通过Tle1磷脂酶活性破坏靶细胞细胞膜从而介导细菌间对抗。研究发现Hcp-ET3/4模块同时编码两种DNA酶毒素，Pyocin S3和Colicin-Dnase，并证实它们的毒性可被下游重复编码的三个免疫蛋白中和。新鉴定一个T6SS抗菌效应子Rhs-CT1，通过MPTase4金属肽酶活性破坏靶细胞壁完整性，实现杀伤邻近细菌的效应。Rhs-CT1 Domain构筑形式检索发现一系列PAAR-Rhs-CTs蛋白。试验证实Rhs-CT3 RNA酶毒素依赖T6SS装置分泌至靶细胞从而介导细菌间的对抗。进一步分析发现Rhs-CTs模块上游编码的VgrGO1和DUF1795蛋白是其实现抗菌能力所必需的，暗示DUF1795可能是一类重要的T6SS伴侣蛋白。将上述检索策略应用到整个革兰阴性菌，发现PAAR-Rhs-CTs家族总计在超过143个不同的细菌物种中编码36种不同的毒素。