禽病原性大肠杆菌肠菌素相关基因entE、entS、tolC突变株的构建及其致病性评价
发布时间：2019-05-05 09:40
【摘要】：禽致病性大肠杆菌(APEC)属于肠道外致病性大肠杆菌(ExPEC)的成员,能通过呼吸道和生殖道等多种途径感染并引起禽类各种肠道外疾病。目前,该病已成为危害养禽业的重要细菌病之一,给养禽业造成了重大经济损失,尤其是那些养禽业发达的国家,例如,巴西、中国、美国。对于大肠杆菌来说铁离子是一种必需成分,但动物体内游离的铁离子浓度很低,不能满足细菌的生存要求,因此大肠杆菌进化出多种摄铁机制从宿主体内竞争铁离子,肠菌素铁摄取系统就是其中一种。铁离子对于大肠杆菌在宿主体内生存和发挥毒力起着关键作用,这使得铁摄取系统如铁结合性复合物能够成为发展新型药物的靶位点。本研究旨在研究肠菌素相关基因entE、entS、tolC与APEC致病性之间的关系,为揭示APEC的致病机理及进一步构建APEC弱毒疫苗株奠定基础。运用λRed重组系统构建E058△entE、E058△entS、E058△tolC、E058△entE△entS、 E058△entE△entS△tolC基因缺失突变株,同时将带有相应启动子的tolC基因完整阅读框插入低拷贝载体pACYC184中,通过电转化突变株来筛选获得回复株。RT-PCR结果表明entE和tolC基因均不能转录,而其上下游基因的转录不受影响。1日龄SPF鸡致死性试验结果表明,与野生株相比,E058△tolC及E058△entE△entS△tolC突变株的致病力显著降低,而E058△entE、E058△entS、E058△entE△entS突变株致弱不明显。35日龄SPF鸡体内定居与持续试验结果表明,E058△tolC和E058△entE△entS△tolC突变株在各脏器的定居与持续能力均显著低于野生株E058(P0.0001),同时,E058△entE△entS△tolC突变株较E058Ato/C突变株的定居与持续能力显著降低。生长曲线的测定结果显示,在LB培养基中,各突变株生长速度与野生株相似,并未表现出明显差异,但在贫铁培养基中,E058△entE△entS△tolC三基因突变株的生长速度显著低于野生株,而其它突变株生长速度较野生株均没有表现出显著差异。血清杀菌试验显示各突变株和野生株没有表现出显著差异。体外竞争试验结果显示,在LB培养基中,除E058△entS外,突变株均表现为轻度致弱,而在贫铁培养基中,三基因突变株E058△entE△entS△tolC表现为中度致弱。HD-11细胞吞噬试验结果显示,与突变株E058Ato/C相比,突变株E058△entE△entS△tolC对HD-11细胞侵袭率明显增加,差异显著。这些结果表明,参与肠菌素外膜输出基因tolC与APEC E058菌株的致病性高度相关,而参与肠菌素合成基因entE和内膜运输基因entS不影响APEC的致病性。并且三基因缺失株E058△entE△entS△tolC的生长速度在贫铁条件下受到抑制,在鸡的感染模型中,这种生长方面的缺陷或许会在体内有所放大,引起致病性下降。
[Abstract]:Avian pathogenic Escherichia coli (APEC) is a member of extraintestinal pathogenic Escherichia coli (ExPEC). It can be infected by respiratory tract and reproductive tract and can cause all kinds of extraintestinal diseases in poultry. At present, the disease has become one of the most important bacterial diseases in poultry industry, causing great economic losses to poultry industry, especially in countries with advanced poultry industry, such as Brazil, China and the United States. Iron ion is an essential component for Escherichia coli, but the concentration of free iron ion in animals is very low and cannot meet the survival requirements of bacteria. Therefore, E. coli has evolved a variety of iron uptake mechanisms to compete for iron ions from the host body. The enterocin iron uptake system is one of them. Iron ions play a key role in the survival and virulence of Escherichia coli in host, which makes iron uptake system, such as iron-binding complexes, become the target sites for the development of new drugs. The aim of this study was to study the relationship between enterocin-related gene entE,entS,tolC and the pathogenicity of APEC, and to lay a foundation for revealing the pathogenesis of APEC and constructing the attenuated vaccine strain of APEC. The E058 entE,E058 entS,E058 tolC,E058 entE entS,E058 entE entS tolC gene deletion mutant was constructed by 位 Red recombination system, and the complete reading frame of the tolC gene with corresponding promoter was inserted into the low copy vector pACYC184. The results of RT-PCR showed that both entE and tolC genes could not be transcribed, but the transcription of the upstream and downstream genes was not affected. One-day-old SPF chickens showed that compared with wild strains, the results of lethal test showed that the two genes could not be transcribed and the transcription of the upstream and downstream genes was not affected. The pathogenicity of E058 tolC and E058 entE entS tolC mutants was significantly decreased, but that of E058 entE,E058 entS,E058 entE entS mutants was not obvious. 35-day-old SPF chickens were settled in vivo and continuously tested. The colonization and sustainability of E058 tolC and E058 entE entS tolC mutants in various organs were significantly lower than those of wild E058 (P0.0001), and the colonization and sustainability of E058 entE entS tolC mutants were significantly lower than those of E058Ato/C mutants. The results of growth curve showed that the growth rate of mutants in LB medium was similar to that of wild plants, but there was no significant difference in the growth rate of mutants, but in iron-poor medium, the growth rates of mutants were similar to those of wild plants. The growth rate of E058 entE entS tolC trigene mutants was significantly lower than that of wild ones, while the growth rates of other mutants were not significantly different from those of wild ones. Serum bactericidal test showed that there was no significant difference among the mutants and wild strains. The results of in vitro competition test showed that all mutants showed mild weakening in LB medium except E058 entS, but in iron-poor medium, the mutants showed mild weakening in the medium with the exception of E058 entS. The results of phagocytosis test of HD-11 cells showed that the invasiveness of mutant E058 entE entS tolC to HD-11 cells was significantly higher than that of mutant E058 entE entS tolC (P < 0.05), and the difference was significant. These results indicated that the exportation gene tolC of enterocin was highly correlated with the pathogenicity of APEC E058, while entE and entS did not affect the pathogenicity of APEC. The growth rate of the three gene deletion strain E058 entE entS tolC was inhibited under the condition of iron deficiency. In the chicken infection model, the growth deficiency might be magnified in vivo, resulting in a decrease in pathogenicity.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
本文编号：2469464
[Abstract]:Avian pathogenic Escherichia coli (APEC) is a member of extraintestinal pathogenic Escherichia coli (ExPEC). It can be infected by respiratory tract and reproductive tract and can cause all kinds of extraintestinal diseases in poultry. At present, the disease has become one of the most important bacterial diseases in poultry industry, causing great economic losses to poultry industry, especially in countries with advanced poultry industry, such as Brazil, China and the United States. Iron ion is an essential component for Escherichia coli, but the concentration of free iron ion in animals is very low and cannot meet the survival requirements of bacteria. Therefore, E. coli has evolved a variety of iron uptake mechanisms to compete for iron ions from the host body. The enterocin iron uptake system is one of them. Iron ions play a key role in the survival and virulence of Escherichia coli in host, which makes iron uptake system, such as iron-binding complexes, become the target sites for the development of new drugs. The aim of this study was to study the relationship between enterocin-related gene entE,entS,tolC and the pathogenicity of APEC, and to lay a foundation for revealing the pathogenesis of APEC and constructing the attenuated vaccine strain of APEC. The E058 entE,E058 entS,E058 tolC,E058 entE entS,E058 entE entS tolC gene deletion mutant was constructed by 位 Red recombination system, and the complete reading frame of the tolC gene with corresponding promoter was inserted into the low copy vector pACYC184. The results of RT-PCR showed that both entE and tolC genes could not be transcribed, but the transcription of the upstream and downstream genes was not affected. One-day-old SPF chickens showed that compared with wild strains, the results of lethal test showed that the two genes could not be transcribed and the transcription of the upstream and downstream genes was not affected. The pathogenicity of E058 tolC and E058 entE entS tolC mutants was significantly decreased, but that of E058 entE,E058 entS,E058 entE entS mutants was not obvious. 35-day-old SPF chickens were settled in vivo and continuously tested. The colonization and sustainability of E058 tolC and E058 entE entS tolC mutants in various organs were significantly lower than those of wild E058 (P0.0001), and the colonization and sustainability of E058 entE entS tolC mutants were significantly lower than those of E058Ato/C mutants. The results of growth curve showed that the growth rate of mutants in LB medium was similar to that of wild plants, but there was no significant difference in the growth rate of mutants, but in iron-poor medium, the growth rates of mutants were similar to those of wild plants. The growth rate of E058 entE entS tolC trigene mutants was significantly lower than that of wild ones, while the growth rates of other mutants were not significantly different from those of wild ones. Serum bactericidal test showed that there was no significant difference among the mutants and wild strains. The results of in vitro competition test showed that all mutants showed mild weakening in LB medium except E058 entS, but in iron-poor medium, the mutants showed mild weakening in the medium with the exception of E058 entS. The results of phagocytosis test of HD-11 cells showed that the invasiveness of mutant E058 entE entS tolC to HD-11 cells was significantly higher than that of mutant E058 entE entS tolC (P < 0.05), and the difference was significant. These results indicated that the exportation gene tolC of enterocin was highly correlated with the pathogenicity of APEC E058, while entE and entS did not affect the pathogenicity of APEC. The growth rate of the three gene deletion strain E058 entE entS tolC was inhibited under the condition of iron deficiency. In the chicken infection model, the growth deficiency might be magnified in vivo, resulting in a decrease in pathogenicity.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
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