El Tor型霍乱弧菌中甘露醇和山梨醇代谢机制研究

发布时间：2018-06-04 08:02

本文选题：霍乱弧菌 + 山梨醇　；参考：《中国疾病预防控制中心》2009年博士论文

【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。
[Abstract]:Vibrio cholerae (Vibrio cholerae) is a pathogenic bacteria causing cholera. Since 1871, there have been at least seven worldwide pandemics, seventh of which are caused by the O1 group El Tor Vibrio cholerae, and are still in this pandemic. In cholera prevention and control, the phage biotype of the O1 group El Tor type cholera vibrio type is established in China. The El Tor type of Vibrio cholerae was divided into epidemic and non epidemic strains. The current study found that all the epidemic strains were all strains, and that the non epidemic strains were almost all non producing strains. This scheme treats these two types of bacteria differently in the prevention and control of cholera, thus taking different preventive and controlling measures to be heavy. The significance of it is still one of the important methods for the classification of Vibrio cholerae. The scheme reflects the genetic differences between the El Tor and non producing strains of Vibrio cholerae. Through the study of the mechanism of the classification scheme, it can reflect the genomics differences of the producing strains (epidemic strains) and non producing strains (non epidemic strains).
The experiment of sorbitol fermentation is an important biochemical reaction in the phage biotyping scheme. According to the slow decline of pH in the sorbitol fermentation broth, El Tor Vibrio cholerae can be divided into fast fermenting and slow fermenting strains, all of which belong to slow fermenting strains and non epidemic strains belong to fast fermentation. By proteomics and gene transcription analysis, we found that the transcriptional level of the mannitol phosphoenolpyruvate transferase system (PTS) and the differences in the product were found in the fast and slow fermented plants, and the mannitol fermentation test was similar to that of the sorbitol fermentation test between the producing and non producing strains. In this study, we further studied the transcription and regulation of mannitol PTS. In order to understand the difference in expression of mannitol induced El Tor strain fast fermenting strain (non producing strain) and slow fermenting strain (strain producing strain), we used the whole genome expression chip of Vibrio cholerae to analyze the transcriptional difference of fast and slow fermenting strains in mannitol fermentation broth. It was found that the transcriptional level of the PTS system components responsible for the mannitol transport in the fast fermenting strain 93097 was higher than that in the slow fermenting strain N16961, which may be one of the reasons for the difference in the acid production rate of the fast and slow strains of mannitol, and also found that the use of mannitol in the fast fermented strain is more characterized by acid production and in the slow fermented plant. It shows stronger energy metabolism.
We further analyzed the PTS system of the mannitol in fast and slow strains. Our previous experiments have confirmed that mtlA, mtlR and mtlD belong to the mannitol specific operon, and mtlA and mtlD have higher transcriptional levels in the fast fermented plants than the slow fermenting strains. In this study, we used the report gene system to analyze the MTL promoter. The upstream VCA1044 of the MTL operon was found to play an important role in the activity of MTL promoter, and the promoter activity of the MTL operon in the fast fermenting strain was higher than that of the slow fermentation plant. In addition, the transcriptional level of mtlA was up-regulated in the fast and slow fermentation strains, and the utilization of mannitol increased. These results indicate the transcriptional ability of the mannitol PTS operon. It is related to mannitol fast fermentation utilization, suggesting that it is one of the mechanisms leading to the difference of mannitol fermentation speed between fast and slow fermentation strains.
The cAMP-CRP complex in bacteria plays a role in multiple regulatory pathways, including regulation of the use of sugar alcohols. Promoter activity receives the regulatory role of transcriptional regulators. As an important regulator in bacteria, it has a very important regulatory role in the metabolism of carbohydrates. Therefore, we have a cAMP-CRP complex in El Tor Vibrio cholerae. The regulatory effect of the compound on the mannitol PTS operon was studied. The deletion mutant strain of the gene CRP and cAMP synthesis gene CyA was synthesized by CRP. It was proved that the complex had a very important positive regulation on the MTL operon. We found that there was no significant difference in the transcription of the CRP gene in the fast and slow fermenting strains, but the CyA in the fast fermented strain was CyA. The transcriptional level was higher than that of the slow fermentation plant, and after the expression of CyA was up-regulated, the time of the color change of the mannitol fermentation broth in the fast and slow fermenting strain was early, suggesting that the difference in the cAMP-CRP complex was different to the regulation of the transcription level of the MTL operon. In the fast fermenting strain, the cAMP-CRP complex was more active and the MTL operon transcriptional level was higher. The CRP binding site "TGTGA.TCACA" was found in the promoter region of MTL, which confirmed that CRP-cAMP complex could be combined with the promoter region by gel mobility test.
The above experiments were analyzed by mannitol fermentation. We found that the El Tor Vibrio cholerae in Zhongshan was similar to the mannitol fermentation experiment, but there were no sorbitol specific PTS gene clusters in Vibrio cholerae, and sorbitol was PTS transporter in many bacteria. The deletion mutant of the non specific component PTS I in the PTS system was constructed. The results showed that the strain after PTS I loss lost the ability to ferment the sorbitol, and confirmed that sorbitol was transported through the PTS system in Vibrio cholerae. We found that the deletion of the gene of the mannitol MTL operon also leads to the blocking of sorbitol utilization, and sorbitol can be used. The results suggest that the high transcription of mannitol specific MTL operon is responsible for the PTS transport of Vibrio cholerae Zhongshan, which is mannitol specific PTS.
In this study, the metabolic regulation of cAMP-CRP complex in Vibrio cholerae was clarified, and the regulation mechanism of mannitol and sorbitol was further extended. At the same time, it was clear that Vibrio cholerae Zhongshan sorbitol and mannitol were transported through a PTS, and sorbitol was the inducer of mannitol PTS. The difference between Vibrio and other bacteria. These studies have further understood the differences in the mechanism of the difference in the fermentation rate and utilization of sorbitol and mannitol of Vibrio cholerae El Tor and non producing strains of Vibrio cholerae. In this study, the metabolic pathways of sorbitol and mannitol in Vibrio cholerae and non producing strains of Vibrio cholerae were also shown in this study. The causes and results of the differences include the regulation of metabolism, the demand for growth, and the differences in the survival ability of the two strains, as well as the effects of differences in intracellular cAMP levels on growth and metabolism, and further research.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378

【引证文献】