肠出血型大肠埃希菌VI型分泌系统分泌蛋白鉴定及功能研究

Enterohemorrhagic Esherichia coli (EHEC) is a bacterium that can cause severe foodborne disease, and E. coli O157:H7 is the most important EHEC serotype in relation to public health. It is highly virulent, and an inoculation of fewer than 50 to 100 CFU of E. coli O157:H7 is sufficient to cause infection. EHEC is transmitted to humans primarily through consumption of contaminated foods, such as raw or undercooked ground meat products and raw milk. EHEC may lead to a life-threatening disease including haemolytic uraemic syndrome (HUS), especially in young children and the elderly..Based on the genome sequence of EHEC strain EDL933, we identified a 30-kb fragment harboring 28 ORFs named sequentially from Z0243 to Z0270, which are homologus to those of the type VI secretion system (T6SS) in Vibiro cholerae. We found that the locus is absent in most of non-pathogenic E. coli strains and present in the pathogenic E. coli strains inculding neonatal meningitis-associated E. coli (NMEC) strain RS218, avian pathogenic E. coli (APEC) strain O1, enteroaggregative E. coli (EAEC) strain 042, Uropathogenic E. coli (UPEC) strain UTI89 and others. The role of this locus was confirmed as a secretion system by preliminary experiments including gene knockout, secretion protein isolation and Western blot. We further used the mass spectrometry (MS) to identify the T6SS related effector proteins by comparing the spuernatants from wild type strain and the deletion mutant of the locus in strain EDL933. One of the effectors identified by MS, Hcp1(Z0264) was confirmed by Western blot using Hcp1 specific antiserum. Z0254 encoding an AAA+ ATPase, is required for the secretion function of T6SS and overexpression of Z0254 increased the secterion of Hcp1. Moreover, the deletion mutant of the locus showed defects in the bacterial binding to and invasion of Caco-2 cell. Therefore, we proposed that the 30-kb locus of EHEC strain EDL933 encoded a T6SS and use secreted effector proteins to mediate the bacterial interaction and host cells..In this application, therefore, our goal is to identify the effectors secreted by the T6SS of EHEC and examine whether these effectors alert the host cell morphology and subvert host cell functions. Further, we will explore their molecular mechanisms including target molecules and singling pathways in the interaction between EHEC and host cell. Finally, the mouse model will be employed to evaluate the role played by these effectors during infection. The information derived from this application will help us characterize the newly identified T6SS and be beneficial in understanding the pathogenesis of EHEC and developing novel strategies to prevent EHEC such as vaccines.

肠出血型大肠埃希菌（EHEC，典型血清型O157:H7）能引起散发性或爆发性出血性结肠炎和溶血性尿毒综合征等疾病，属严重威胁人类健康的病原体。本研究组根据基因组序列信息分析发现EHEC基因组中含有一长约30 kb的片段，与新近发现的霍乱弧菌VI型分泌系统高度同源，且此片段在非致病性大肠埃希菌中不存在。预实验发现此片段编码一个新的分泌系统，并利用质谱技术初步鉴定了一些分泌蛋白。该片段的缺失使得EHEC黏附和侵袭真核宿主细胞的能力明显下降。因此我们提出假说：EHEC的VI型分泌系统负责一些效应蛋白的分泌，对EHEC毒力具有重要作用。本课题拟利用细胞和动物模型，确定EHEC的VI型分泌系统分泌蛋白，研究分泌蛋白和VI型分泌系统在EHEC与宿主相互作用过程中的功能。研究结果有助于进一步认识VI型分泌系统的分子生物学特征和功能，对揭示EHEC致病机制、疾病治疗以及疫苗研发都具有重要的意义。

通过四年的努力，我们按照原计划，对肠出血型大肠埃希菌VI型分泌系统（T6SS）进行研究，进一步阐明了大肠埃希菌VI型分泌系统的功能以及在肠出血型大肠埃希菌致病过程中的作用。本课题按照既定研究计划和时间节点进行，顺利完成了原定研究内容，并延着课题研究方向拓展了研究内容，完成了预定目标。.我们通过生物信息学手段分析发现，EHEC O157:H7的参考菌株EDL933中存在T6SS的核心组分，并界定出T6SS编码基因簇，通过实验验证，我们发现T6SS 是有活性的，且其活性受转录因子H-NS负调控。通过体外实验验证，我们发现EHEC 的T6SS并没有杀菌作用。通过收集WT和ΔT6SS的分泌蛋白，并采用串联质谱检测样品中的蛋白组分，我们找到了T6SS的3个候选的效应蛋白：Z5583、Z0873和Z1921。随后的Western blotting实验和β-内酰胺酶实验表明，Z1921为T6SS的效应蛋白。通过序列比对和文献检索，我们发现Z1921为Mn-过氧化氢酶。我们随后将Z1921命名为KatN，并测定了Z1921的酶活性，明确了KatN对于H2O2压力的拮抗能力。我们发现，在EHEC 中，在对数期，katN的表达受RpoS和OxyR的正调控，与是否外加H2O2的压力无关。实验表明，在被巨噬细胞吞噬后，EHEC 的T6SS相关基因和katN的表达水平明显提高。这证明在EHEC 与RAW264.7相互作用过程中，T6SS处于激活状态，且可能通过向细菌胞外转运KatN，维持细菌在胞内一定的生存水平。我们通过进一步的免疫荧光实验和TEM-1 β-lactamase实验，证明当EHEC 被巨噬细胞吞噬后，KatN可以被分泌到巨噬细胞中。通过对被EHEC 感染后的巨噬细胞进行活性氧（ROS）水平检测，发现KatN对降低巨噬细胞内活性氧水平有显著作用。最后，我们通过EHEC 感染小鼠模型实验，发现T6SS在EHEC 对小鼠的致病性这一过程中发挥重要作用。.综上所述，我们发现在EHEC 中存在一个有功能的T6SS，该分泌系统有助于细菌对小鼠的毒力。当感染宿主并被宿主天然免疫系统中的巨噬细胞吞噬后，EHEC 的T6SS被更大程度地激活，通过T6SS的介导，向细菌胞外分泌KatN，来降低细菌所面临的氧化压力，维持着细菌在巨噬细胞内的生存水