【作者】 王鑫；

【导师】 潘兹书；

【作者基本信息】 武汉大学 ， 微生物学， 2021， 硕士

【摘要】 沙门氏菌作为重要的人畜共患病病原菌,有超过2600种血清型,禽类是沙门氏菌主要的宿主。禽沙门氏菌可造成禽类慢性、急性的传染病,引起肠炎、败血症等。其中鸡白痢沙门氏菌是一种禽特异性病原菌,可垂直传播与水平传播,雏鸡感染后可引起白痢,导致死亡,给养禽业带来极大的经济损失。为适应宿主体内外环境改变的压力,细菌必须要调动相关的适应性机制来适应环境变化,(p)ppGpp介导的严谨反应(stringent response,SR)是细菌重要适应机制之一。本研究对华中地区禽沙门氏菌开展分子流行病学分析,并解析了(p)ppGpp介导的严谨反应在鸡白痢沙门氏菌环境适应与致病中的作用。对华中地区84株沙门氏菌临床分离株的血清型分析显示,沙门氏菌血清D群(鸡白痢沙门氏菌、鸡伤寒沙门氏菌和肠炎沙门氏菌)是主要的亚群,占分离株的84.76%,其中鸡白痢沙门氏菌是优势血清型,占比45.24%。耐药性分析显示,分离株对氨苄青霉素、四环素和粘菌素表现出较高的耐药率。多位点序列分型(Multilocus sequence typing,MLST)分析显示禽沙门氏菌分离株有9个基因型,优势基因型为ST92,沙门氏菌分离株中血清型和基因型之间有较强关联性。为进一步研究(p)ppGpp介导的严谨反应在鸡白痢沙门氏菌适应性和致病性中的作用,本研究构建了鸡白痢沙门氏菌(p)ppGpp合成酶缺失株Δrel AΔspo T,比较不同环境下的生长及其致病能力上的差异。细菌生物学特性研究显示,相较于亲本株,缺失株Δrel AΔspo T在氨基酸饥饿条件下生长更快,对红霉素、氨苄青霉素、环丙沙星和四环素的最小杀菌浓度(MBC)降低,生物被膜形成能力显著降低,在酸性、碱性和高渗透压条件下存活率显著下降。细胞试验表明,粘附和侵袭试验中缺失株Δrel AΔspo T对鸡成纤维细胞DF-1的粘附率比亲本株降低5倍以上,侵袭率比亲本株降低40倍以上;小鼠巨噬细胞RAW264.7中的内化和增殖试验显示,亲本株在巨噬细胞的初始侵入率为0.114%±0.117%,缺失株侵入率是0.037%±0.015%,缺失株胞内存活和复制能力显著减弱,甚至被全部清除,亲本株与互补株的数量显著增加。动物感染试验表明,与亲本株相比,缺失株Δrel AΔspo T在肝脏和盲肠中的定植显著降低,感染第14天肝脏、盲肠中缺失株Δrel AΔspo T几乎不能被检出,缺失株Δrel AΔspo T对雏鸡的致病能力显著减弱,致死率从亲本株的100%显著降低至30%,且更容易被机体清除。转录组分析结果显示,在氨基酸饥饿条件下,865个基因表达发生显著改变,相比亲本株,缺失株中539个基因出现下调表达,326个基因出现上调表达,缺失株中核糖体亚基、DNA复制和转录相关基因的表达上调,与鸡白痢沙门氏菌的生长相关;缺失株中T3SS装置蛋白(inv AEFG、spa PQ、prg H)以及由SPI-1编码的其他的效应蛋白(sip ABCD、sop BDE)显著下调表达,与粘附侵袭能力相关;缺失株中超氧化物歧化酶的(sod1、sod2),T3SS效应蛋白(SPI-2编码的sse ABCDFGJ、sif A、pip B2)显著下调表达,这些基因主要和巨噬细胞中存活与增殖相关,这表明(p)ppGpp合成酶通过调控转录翻译相关基因以及T3SS等毒力因子,在鸡白痢沙门氏菌的环境适应与致病性中发挥重要作用,为解析鸡白痢沙门氏菌的流行与环境适应机制提供了重要的理论数据。更多还原

【Abstract】 Salmonella is an important zoonotic pathogen with more than 2600 Salmonella serotypes,and birds is one of the most crucial hosts of Salmonella.Avian Salmonella can cause poultry chronic or acute infectious diseases,clinical symptoms included enteritis,sepsis,etc.Salmonella Pullorum(S.Pullorum)has strict host specificity for poultry.Transmission of S.Pullorum occurs not only vertically but also horizontally,and the infection causes Pullorum disease in chicks,characterized by white diarrhea resulting in death,which bringing immeasurable economic losses to the poultry industry.Bacteria must activate relevant adaptive mechanisms to adapt to changes of the external environment.Stringent response(SR)is an important adaptation mechanism mediated by the signal molecule(p)ppGpp,which is synthesized by(p)ppGpp synthetases.In this study,molecular epidemiological analysis of avian Salmonella in central China was carried out to analyze the role of(p)ppGpp mediated SR of S.Pullorum in environmental adaptation and pathogenicity.Serotype analysis of 84 isolates of Salmonella isolated from central China showed that Salmonella serogroup D was the main subgroup,accounting for 84.76%,of which S.Pullorum was the primary serotype,accounting for 45.24%.Antimicrobial resistance analysis revealed that all isolates exhibited higher resistance rates to colistin,tetracycline and ampicillin.Multilocus sequence typing(MLST)analysis displayed that there were 9 genotypes in avian Salmonella isolates,of which ST92 was the primary serotype.There was a strong connection between genotypes and serotypes in Salmonella.To investigate the role of(p)ppGpp synthetases in the adaptation and pathogenicity of S.Pullorum,this study constructed(p)ppGpp synthetases mutant Δrel AΔspo T.Bacteria biological characteristics tests shows that comparded with wild-type strain,the growth rate of mutant Δrel AΔspo T was significantly faster under amino acid starvation,and the MBCs of mutant Δrel AΔspo T of erythromycin,ampicillin,tetracycline and ciprofloxacin significantly decreased,the survival rate of mutant Δrel AΔspo T significantly decreased under the condition of acid,alkali and high osmotic pressure.Cell infection tests showed that comparded with wild-type strain,the adhesion and invasion rates of mutant Δrel AΔspo T significantly decreased within DF-1 cells,about 5times and 40 times respectively,and the internalization ability of wild-type strain was0.114%±0.117%,and mutant strain was 0.037%±0.015%,which was significantly reduced.Animal tests have shown that compared with wild-type strain,the contents of mutant Δrel AΔspo T were significantly lower in liver and caecum of infected chickens.For infecton group survived,30% and 100% died in wild-type strain and mutantΔrel AΔspo T infection groups,respectively.Transcription profiling showed the expressing of 865 genes were significantly changed under amino acid starvation.Compared with wild-type strain,539 genes were down-regulated and 326 genes were up-regulated in mutant strain.The expression of genes-related ribosomal subunits,DNA replication and transcription,associated with the growth of S.Pullorum,were significantly up-regulated in mutant strain.Compared with wild-type strain,T3 SS device proteins(inv AEFG,spa PQ,prg H)and other effector proteins(sip ABCD,Sop BDE)encoded by SPI-1 were significantly down-regulated in mutant strain,which are associated with adhesion and invasion.Other down-regulated genes include superoxide dismutase(sod1,sod2)and T3 SS effector proteins(sse ABCDFGJ,sif A,pip B2)encoded by SPI-2,which are mainly related to the survival and proliferation of macrophages.Our study showed(p)ppGpp synthetases play a crucial role in environmental adaptation and pathogenicity of S.Pullorum.This study established foundation for a comprehensive understanding of(p)ppGpp synthetases and regulation mechanism of pathogenicity of S.Pullorum.更多还原