【作者】 张红芝；

【导师】 祁国明； 阚飙；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2009， 博士

【摘要】 霍乱弧菌(Vibrio cholerae)是引起霍乱的病原菌。从1871年至今已经发生过至少七次世界性大流行,其中第七次由O1群El Tor型霍乱弧菌引起,并且现今还处在此次大流行中。我国在霍乱防控中,建立了针对O1群El Tor型霍乱弧菌分型的噬菌体-生物分型方案,将El Tor型霍乱弧菌区分为流行株和非流行株,并进一步分成不同型别。目前研究发现流行株均为产毒株,非流行株几乎全为非产毒株。这一方案对在霍乱防控过程中区别对待这两类菌株、从而采取不同的预防控制措施有重要的意义,至今仍为霍乱弧菌分型的重要方法之一。该分型方案反映了El Tor霍乱弧菌产毒株和非产毒株的遗传学差异,通过对分型方案的机制研究,能够反映出产毒株(流行株)和非产毒株(非流行株)的基因组学差异。山梨醇发酵实验是噬菌体-生物分型方案中一个重要的生化反应,根据山梨醇发酵液pH下降的快慢(表现为发酵液颜色变黄的先后时间)可将El Tor型霍乱弧菌分为快发酵株和慢发酵株,其中流行株均属于慢发酵株,非流行株均属于快发酵株。我们先前通过蛋白质组学和基因转录分析,发现快慢发酵株中存在甘露醇磷酸烯醇式丙酮酸转移酶系统(PTS)操纵子转录水平及其产物的差异,而且在产毒株和非产毒株间甘露醇发酵试验具有与山梨醇发酵试验相似的特征。在本研究中,我们对甘露醇PTS转录及其调控开展了进一步研究。为了解甘露醇诱导El Tor型菌株快发酵株(非产毒株)和慢发酵株(产毒株)的表达差异,我们利用霍乱弧菌全基因组表达谱芯片分析了快慢发酵菌株在甘露醇发酵液中的转录差异基因,发现负责甘露醇的转运的PTS系统成分在快发酵株93097中的转录水平要高于其在慢发酵株N16961的,这可能是快慢菌株代谢甘露醇产酸速度差异的原因之一;另外也发现快发酵株中对甘露醇的利用更多表现为产酸,而在慢发酵株中则表现为更强的能量代谢。我们进一步对快慢菌株中转运甘露醇的PTS系统进行了分析。我们先前的实验已经证实了mtlA、mtlR和mtlD属于甘露醇特异操纵子的成分,mtlA和mtlD在快发酵株中转录水平高于慢发酵株的。在本研究中,我们利用报告基因系统分析了mtl启动子区,发现mtl操纵子上游VCA1044对mtl启动子活性具有很重要的作用,且快发酵株中mtl操纵子的启动子活性要高于慢发酵株,另外在快慢发酵菌株中上调mtlA的转录水平,甘露醇的利用能力均增强了,这些结果说明甘露醇PTS操纵子的转录能力与甘露醇的快慢发酵利用相关,提示是造成快慢发酵株甘露醇发酵速度差异的机制之一。细菌中cAMP-CRP复合物在多个调节通路中发挥作用,包括对糖醇利用的调控。启动子活性收到转录调控因子的调控作用,作为细菌中重要的调控因子,对碳水化合物的代谢具有非常重要的调控作用。因此我们对El Tor型霍乱弧菌中cAMP-CRP复合物对甘露醇PTS操纵子的调节作用进行了研究。利用CRP合成基因crp和cAMP合成基因cya的缺失突变株,证实该复合物对mtl操纵子具有非常重要的正向调控作用。我们发现在快慢发酵株中crp基因的转录水平没有明显差异,但快发酵株中cya的转录水平要高于慢发酵株,且cya表达上调后,也会造成快慢发酵株甘露醇发酵液颜色变黄的时间均提前,提示cAMP-CRP复合物的差异对mtl操纵子的转录水平调控有差别,在快发酵株中,cAMP-CRP复合物活性更高,使mtl操纵子转录水平更高。预测到mtl启动子区有CRP结合的保守位点“TGTGA……TCACA”,通过凝胶迁移实验,证实CRP-cAMP复合物能够与该启动子区结合。以上实验是通过甘露醇发酵进行分析的。我们前期发现El Tor型霍乱弧菌中山梨醇发酵实验与甘露醇发酵实验具有相似的结果,但霍乱弧菌中没有其他细菌中所具有的山梨醇特异的PTS基因簇。山梨醇在很多细菌中属于PTS转运糖醇,我们构建了PTS系统中非特异成分ptsⅠ的缺失突变株,结果显示ptsⅠ缺失后的菌株失去了发酵山梨醇的能力,证实山梨醇在霍乱弧菌中是通过PTS系统转运的。我们发现甘露醇mtl操纵子的基因缺失同样导致山梨醇利用的阻断,而且山梨醇能够诱导甘露醇特异mtl操纵子的高转录表达,因此这些结果说明霍乱弧菌中山梨醇的PTS转运是通过甘露醇特异PTS的。本研究明确了cAMP-CRP复合物在霍乱弧菌中对甘露醇的代谢调节作用,将甘露醇和山梨醇快慢发酵株的调控机制研究进一步延伸,同时明确了霍乱弧菌中山梨醇与甘露醇共同通过一个PTS转运,并且山梨醇是甘露醇PTS的诱导物,显示了霍乱弧菌与其他细菌不同的特征。这些研究更深入了对El Tor霍乱弧菌产毒株和非产毒株在山梨醇和甘露醇发酵速率与利用上的差异机制的理解。在本研究中,也显示山梨醇和甘露醇在霍乱弧菌产毒株和非产毒株中代谢途径的差异,这种差异的原因和作用结果,包括在两种菌株的不同遗传背景下各自对代谢的调节、生长的需求、以及是否导致环境生存能力的差异,另外包括细胞内cAMP水平的差异会带来哪些生长代谢影响等,还需要进一步的研究。更多还原

【Abstract】 Cholera is caused by V. cholerae and it is generally accepted that at least seven distinct pandemics have occured since 1871. In 1961 the seventh and present pandemic began, caused by the O1 E1 Tor biotype. In China, the Phage-biotyping Scheme has been developed to distinguish the O1 E1 Tor biptype strains as "epidemic strains" and "non-epidemic strains", Further studies revealed that all "epidemic strains" were toxigenic and almost "non-epidemic strains" were nontoxigenic. The scheme of Phage-biotype has been used for many years in China and until now, it is also a useful tool for the identification of V. cholerae toxigenic and nontoxigenic strains. Sorbitol fermentation test, one of important tests in the Phage-biotyping scheme, shows different fermentation rate between "epidemic strains" and "non-epidemic strains", therefore are designated slow-fermenting strain and fast-fermenting strain respectively.Firstly, the result obtained from the expression profiles of genes of N16961 and 93097 in 0.2% mannitol fermentation medium revealed that the expression level of the components of the PTS in 93097 was higher than that in N16961, which may cause the different fermentation rate in the two classes of strains. In addition, the result showed that more mannitol was metabolized to produce acid in fast-ferrmenting strain, but more energy was produced by TCA in slow-fermenting strain.We make further analysis of the mannitol-specific PTS in fast-fermenting and slow-fermenting strains. Our previous study has confirmed that mtlA、mtlR and mtlD were the components of mtl operon. The transcription level of mtlA and mtlD in 93097 was higher than that in N16961. In our study, we analyzed the mtl peomoter, and we found that the gene VCA1044 located in the upper part of mtl operon was very important to the activity of mtl promoter. And the activity of mtl promoter from fast-fermenting strain was higher than that in slow-fermenting strain. What’s more, the more mannitol was utilized in toxigenic and nontoxigenic strains in which the expression level of mtlA was upregulated. These results could confirm that transcript level of mtl operon was related to the ability of mannitol in fast-fermenting and slow-fermenting strains, which may cause different fermentation rate.As the important regulator in bacteria, cAMP-CRP complex play a key role in metabolism of carbohydrate. So we analyzed the role of cAMP-CRP complex in regulation of manitole-specific PTS. The results of the gene crp and cya deletion mutants fermenting mannitol confirmed that cAMP-CRP complex could regulate positively the metabolism of mannitol. The transcription level of cya in fast-fermenting strain was higher that that in slow-fermrnting strain. What’s more, the color in mannitol fermentation medium of fast-fermnting and slow-fermenting strains changed to yellow more earlier after the expression level of cya was upregulated. These results suggested that the regulation of cAMP-CPP complex was different in the two classes of strains. Higher activity of cAMP-CRP complex caused higher transcription level of mtl operon in fast-fermentation strain. We predicted the conservative site in mtl promoter, that was "TGTGA.....TCACA", the result of EMSA confirmend that cAMP-CRP complex could bind to the region.Our previous study indicated that the results of mannitol fermentation were similar to those of sorbitol fermentation of V. cholerae E1 Tor. But there was no sorbitol-specific PTS in V. cholerae, which was different from those other bacteria. Sorbitol was PTS sugar in many other bacteria. The ptsI deletion mutant was constructed and the result of sorbitol fermenting indicated that the mutant could not metabolize sorbitol, which confirmed that sorbitol was also transported by PTS in V. cholerae. What’s more, sorbitol could induce the high expression level of mtl operon. Therefore theses results indicated that sorbitol was transported by mannitol-specific PTS in V. cholerae.The study make further analysis of mechanism of mannitol and sorbitol metabolism in V. cholerae, and make clear both that cAMP-CRP complex play a key role in regulation of mannitol and sorbitol metabolism, and sorbitol is transported by manniol-specific PTS as the inducer of mannitol-specific PTS. The characteristic was different from other bacteria. And the study help understand the mechanism of difference of mannitol and sorbitol fermentation rate and metabolism between fast-fermentig and slow-fermenating strains. We also found the different metabolism pathway of mannitol and sorbitol in the two classes of strains. Whether these differences are related to the need of growth and regulation of metabolism? In addition, whether growth of V. cholerae is affected by the level of cAMP? Further study is necessary.更多还原