禽致病性大肠杆菌中Ⅲ型分泌系统2的分布及其组分EivC功能研究

发布时间：2018-04-20 18:51

本文选题：禽致病性大肠杆菌 + Ⅲ型分泌系统2　；参考：《安徽农业大学》2017年硕士论文

【摘要】：细菌通过分泌系统将蛋白质转运到细菌胞外或直接注入宿主细胞内,与细菌的生存及致病性密切相关。Ⅲ型分泌系统(TypeⅢsecretion system,T3SS)存在于致病性大肠杆菌、沙门菌等细菌中,与毒力因子的分泌及致病力有关。研究人员发现EHEC O157:H7存在与沙门菌SPI-1类似的毒力岛,将其命名为E.coli TypeⅢsecretion system 2(ETT2)。调查发现,不同致病性大肠杆菌中均含有ETT2毒力岛。尽管大部分ETT2毒力岛存在基因突变和缺失,其在黏附入侵、胞内生存、生物被膜形成等方面发挥重要作用。APEC(Avian pathogenic E.coli,APEC)属于肠道外致病性大肠杆菌(Extraintestinal Pathogenic E.coli,ExPEC),可以引起禽大肠杆菌病,给养禽业造成巨大经济损失。研究表明,APEC与人ExPEC基因组结构相似,具有许多相同的毒力因子和相似的致病机制。因此,APEC是人ExPEC的毒力基因贮库,对人类健康造成潜在威胁。为了阐明APEC中ETT2的分布及其致病机制,本研究对APEC中ETT2进行流行病学调查,并分析ETT2 ATPase EivC关键活性位点及其对APEC致病性的影响,为进一步研究ETT2在APEC中的致病机制提供参考。1.ETT2在APEC中的亚型及流行病学调查为了检测ETT2在APEC中的分布,本研究根据EHEC O157:H7的ETT2毒力岛序列设计8对重叠PCR引物,检测ETT2在APEC临床分离株中的分布。然后,对ETT2目的条带进行测序,分析ETT2亚型。同时,通过PCR方法鉴定APEC的血清型、进化分群,并分析ETT2与血清型、进化分群之间的关联性。结果发现57.6%(141/245)APEC分离株含有ETT2毒力岛。测序结果显示APEC中ETT2分为完整型ETT2(命名为A亚型)和缺失突变型ETT2(命名为B、C、D、E亚型)。血清型分析表明ETT2主要存在于O1、O2和O78血清型APEC中。此外,O78血清型APEC仅含有缺失突变型ETT2,而完整型ETT2主要存在于O1和O2血清型APEC中。分析发现,T3SS相关基因簇(eip)仅存在于完整型ETT2阳性菌株中。大肠杆菌进化分群分析显示,66.7%(32/48)B1、64.8%(35/54)D、59.6%(31/52)A、44.9%(35/78)B2进化分群APEC含有ETT2。然而,ETT2毒力岛和其他毒力基因分布没有关联性。本研究表明,APEC中ETT2的亚型及分布均显著高于人的ExPEC,进一步证明APEC对人类健康造成潜在威胁。2.ETT2毒力岛组分EivC克隆表达及其ATPase活性分析为了分析APEC ETT2毒力岛组分EivC的ATPase活性及其关键活性位点,本研究比对分析病原菌ATPase序列,分别设计克隆表达引物及点突变引物,然后构建重组表达质粒。经IPTG诱导表达后纯化相应蛋白,并检测其ATPase活性。结果显示,EivC蛋白包含F0F1 ATPase的保守序列Walker Box A、Walker Box B及DCCD Box,因此EivC可能属于ATPase。酶活试验结果显示,His-EivC融合蛋白具有ATPase活性,磷酸盐释放速率为0.28±0.08mmol/min/mg。根据病原菌ATPase的关键位点,选择EivC蛋白第175位赖氨酸、第199位精氨酸、第201位精氨酸、第233位精氨酸进行点突变,然而点突变与野生型EivC的ATPase活性无显著差异。故本研究表明ETT2毒力岛组分EivC为ATPase,但是第175、199、201、233位氨基酸不影响其ATPase活性。3.禽致病性大肠杆菌APCE94 eivC基因缺失株、互补株的构建及特性分析为了分析ETT2 ATPase EivC对APEC表型和毒力的影响,本研究利用Red同源重组系统构建APCE94的eivC基因缺失株,并利用低拷贝质粒pSTV28构建eivC基因互补株。然后比较分析野生株、缺失株与互补株的生长特性、运动性、抗血清杀菌能力、黏附/侵袭能力、HD-11细胞内存活能力、动物致病力等差异。通过荧光定量方法检测APEC鞭毛、菌毛、毒力基因及HD-11细胞的细胞因子的表达水平;并通过透射电子显微镜观察细菌表面形态变化。结果显示,与野生株相比,eivC缺失株的生长特性无显著变化,但其运动性显著降低。电镜检测结果表明eivC缺失株表面的鞭毛减少,而菌毛增加,从而导致细菌运动性降低,荧光定量PCR结果显示鞭毛和毒力基因表达下调。另外,eivC缺失导致其抗血清杀菌能力、HD-11胞内存活能力显著下降。动物试验结果表明APEC94ΔeivC对雏鸭的致病力、体内存活和定殖能力显著降低。细胞炎症因子荧光定量分析发现缺失株APEC94ΔeivC感染HD-11细胞后IL-1β和IL-8显著上调,其可能与ETT2及其效应因子相关。研究结果表明,ETT2毒力岛ATPase EivC参与APEC的运动性和致病性。
[Abstract]:Bacteria are transported to the extracellular or directly injected into the host cells through the secretory system, which is closely related to the survival and pathogenicity of the bacteria. The type III secretory system (Type III secretion system, T3SS) exists in pathogenic Escherichia coli, Salmonella and other bacteria, related to the secretion of virulence factors and pathogenicity. Researchers found EHEC O 157:H7 has a virulence Island similar to Salmonella SPI-1, named E.coli Type III secretion system 2 (ETT2). It was found that ETT2 virulence island was found in different pathogenic Escherichia coli. Although most of the ETT2 virulence island has genetic mutation and deletion, it plays an important role in adhesion invasion, intracellular survival, and biofilm formation. .APEC (Avian pathogenic E.coli, APEC) belongs to the intestinal pathogenic Escherichia coli (Extraintestinal Pathogenic E.coli, ExPEC), which can cause avian colibacillosis and cause huge economic loss to poultry industry. The study shows that APEC is similar to the human ExPEC genomic structure, and has many similar virulence factors and similar pathogenic mechanisms. APEC is the toxic gene storage of human ExPEC, which poses a potential threat to human health. In order to clarify the distribution and pathogenesis of ETT2 in APEC, this study conducted an epidemiological investigation on ETT2 in APEC, and analyzed the key active sites of ETT2 ATPase EivC and its effect on the pathogenicity of APEC, in order to further study the pathogenesis of ETT2 in APEC. Referring to the subtype and epidemiological investigation of.1.ETT2 in APEC in order to detect the distribution of ETT2 in APEC, this study was designed to detect the distribution of ETT2 in APEC clinical isolates by designing 8 overlapping PCR primers based on the ETT2 virulence Island sequence of EHEC O157:H7. Then, the ETT2 strip was sequenced and the ETT2 subtype was analyzed. The correlation between the ETT2 and the serotypes and the evolution groups was analyzed. The results showed that the 57.6% (141/245) APEC isolates contained ETT2 virulence island. The sequencing results showed that ETT2 in APEC was divided into complete ETT2 (named A subtype) and deletion mutant ETT2 (named B, C, D, and subtype). 2 and O78 serotype APEC. In addition, O78 serotype APEC only contains missing mutant ETT2, while intact ETT2 mainly exists in O1 and O2 serotype APEC. It is found that T3SS related gene clusters (EIP) exist only in the intact type ETT2 positive strains. 8) B2 evolution group APEC contains ETT2., however, the distribution of ETT2 virulence island and other virulence genes is not related. This study shows that the subtypes and distribution of ETT2 in APEC are significantly higher than those of human ExPEC. It is further proved that APEC has a potential threat to human health as a potential threat to.2.ETT2 virulence island component EivC clon expression and ATPase activity analysis in order to analyze APEC The ATPase activity of T2 virulence island component EivC and its key active site. This study designed the cloning and expression primers and point mutation primers for the analysis of the pathogenic bacteria ATPase sequence. Then the recombinant expression plasmid was constructed. The expression of the corresponding protein was purified after IPTG induction, and the ATPase viability was detected. The results showed that the EivC protein contained the conservatism of F0F1 ATPase. Sequence Walker Box A, Walker Box B and DCCD Box, so EivC may belong to the ATPase. enzyme activity test results, and His-EivC fusion protein has ATPase activity, phosphate release rate is 0.28 + according to the key site of pathogenic bacteria, select 175th lysine, 199th arginine, 201st arginine, 233rd. There is no significant difference between the point mutation and the ATPase activity of the wild type EivC. Therefore, this study shows that the EivC of ETT2 virulence island is ATPase, but the 175199201233rd bit amino acid does not affect the ATPase activity of the.3. avian pathogenic Escherichia coli APCE94 eivC gene deletion strain, and the construction and characteristics of the complementary strains are analyzed in order to distinguish them. The effects of ETT2 ATPase EivC on APEC phenotypes and virulence were analyzed. The eivC gene deletion strain of APCE94 was constructed using Red homologous recombination system and a eivC gene complementary strain was constructed with low copy plasmid pSTV28, and the growth characteristics, mobility, antisera bactericidal ability, adhesion / invasion ability, HD-1 of the wild strain and the complementary strain were compared and analyzed in the wild plant, HD-1. 1 cell memory live ability and animal pathogenicity difference. The expression level of APEC flagellum, pilus, virulence gene and HD-11 cell factor was detected by fluorescence quantitative method; and the morphological changes of bacterial surface were observed by transmission electron microscope. The results showed that the growth characteristics of eivC deletion plants were not significantly different from those of wild plants, but they were transported. The results showed that the flagellum on the surface of the eivC deletion strain decreased and the pilus increased, which resulted in the decrease of the bacterial motility. The fluorescence quantitative PCR results showed that the expression of flagellum and virulence genes decreased. In addition, the deletion of eivC led to its antiserum bactericidal ability and the memory living ability of HD-11 cells decreased significantly. Animal test results showed AP EC94 Delta eivC significantly decreased the virulence of ducks, in vivo survival and colonization. The fluorescence quantitative analysis of cell inflammatory factors found that IL-1 beta and IL-8 were significantly up-regulated after the deletion of APEC94 Delta eivC infected HD-11 cells, which may be related to ETT2 and its effect factors. The results showed that ETT2 poison island ATPase EivC participated in the motion and pathogenicity of APEC.

【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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