本文關鍵詞： 霍亂弧菌 定殖能力 膽鹽 競爭黏附 多重抗藥性　出處：《南京農(nóng)業(yè)大學》2012年碩士論文　論文類型：學位論文

【摘要】：烈性傳染病霍亂由革蘭氏陰性細菌霍亂弧菌所引起,該細菌主要毒力基因CTXΦ和VPI(霍亂弧菌毒力島)受AphB-ToxR級聯(lián)調(diào)控系統(tǒng)調(diào)控,該系統(tǒng)中LysR家族基因aphB直接調(diào)控霍亂弧菌毒力基因的表達。LysR家族基因廣泛存在于細菌各種屬中,為原核生物中目前已知最大的轉(zhuǎn)錄因子家族,其調(diào)控的基因功能多樣,涉及合成代謝、群體感應、毒力等眾多方面。目前發(fā)現(xiàn)霍亂弧菌基因組中約有40個LysR型基因,其中大多尚未被研究。 本文選取其中4個未被研究的LysR型基因分別構(gòu)建其缺失突變株,通過比較突變株與野生株在定殖能力、毒力、生物膜形成能力等方面的差異,發(fā)現(xiàn)了一個參與霍亂弧菌定殖的LysR型基因vc2324。檢測其相鄰上游基因vc2323的轉(zhuǎn)錄情況,證實vc2324正調(diào)控vc2323的轉(zhuǎn)錄。宿主腸道競爭定殖實驗發(fā)現(xiàn)vc2323缺失株定殖能力顯著下降且較vc2324下降幅度更大。結(jié)合LysR家族基因經(jīng)典調(diào)控模式,推測vc2323為影響霍亂弧菌定殖的功能基因。 蛋白質(zhì)同源性分析發(fā)現(xiàn),假設蛋白VC2323與大腸桿菌中碲抗性蛋白TehA.流感嗜血桿菌HiTehA和擬南芥中SLAC1通道蛋白屬于同源蛋白,推測該蛋白可能具有亞碲酸鹽抗性、多重耐藥性等多種功能。通過TcpA western blot和tcpA轉(zhuǎn)錄水平的檢測,發(fā)現(xiàn)vc2323、vc2324均不影響霍亂弧菌毒力蛋白TCP的表達和形成。檢測不同物質(zhì)對vc2323表達的影響,并比較vc2323缺失株和野生株對KNO2、H2O2的敏感性差異,發(fā)現(xiàn)CO2/HCO3-不影響vc2323的表達,vc2323與霍亂弧菌NO/NO2抗性、活性氧抗性無明顯關系。同時還發(fā)現(xiàn)vc2323、vc2324均不影響霍亂弧菌在LB培養(yǎng)基中生物膜的形成,也與霍亂弧菌對H+耐受力無關。 體外競爭黏附實驗發(fā)現(xiàn)：粗膽鹽存在時,vc2323缺失株較野生株對小鼠小腸的黏附力明顯下降,且隨黏附時間的延長差異更加顯著；而當無粗膽鹽存在時,二者對腸道的黏附力無明顯差異。且體外實驗表明,野生株與vc2323缺失株對粗膽鹽的敏感性相同。綜合以上結(jié)果,分析vc2323對霍亂弧菌定殖能力的影響可能是因為在粗膽鹽存在時其能夠影響霍亂弧菌對宿主腸道的黏附能力,進而導致vc2323缺失株較野生株定殖能力顯著下降,詳細的分子機制尚待更深入的研究。 同時,比較不同抗菌物質(zhì)對缺失株與野生株最小殺菌濃度的差異,發(fā)現(xiàn)vc2323缺失株對氯霉素和萘啶酮酸的耐受性分別下降了約64%和74%,對亞碲酸鉀的抗性下降了約33%,表明vc2323還參與霍亂弧菌的抗藥性和亞碲酸鹽抗性。 綜上所述,本研究發(fā)現(xiàn)LysR家族基因vc2324及其靶基因vc2323參與霍亂弧菌的定殖和抗藥性及一定的亞碲酸鹽抗性,為霍亂的防控提拱了更多的參考。
[Abstract]:Cholera is caused by Vibrio cholerae, a gram-negative bacterium. The main virulence genes CTX 桅 and virulence island of Vibrio cholerae (Vibrio cholerae) are regulated by AphB-ToxR cascade control system. In this system, LysR family gene aphB directly regulates the expression of virulence gene of Vibrio cholerae. LysR family gene exists widely in various genera of bacteria, and it is the largest known transcription factor family in prokaryotes, and its gene function is diverse. About 40 LysR genes have been found in the genome of Vibrio cholerae, most of which have not been studied. In this paper, four of the unstudied LysR type genes were selected to construct their deletion mutants, and the difference of colonization ability, virulence and biofilm formation ability between the mutant and wild strain was compared. A LysR type gene vc2324was found to be involved in the colonization of Vibrio cholerae. The transcription of its adjacent upstream gene vc2323 was detected. It was confirmed that vc2324 was regulating the transcription of vc2323. The host intestinal competitive colonization assay showed that the colonization ability of vc2323 deficient strains was significantly decreased and the decrease was greater than that of vc2324. Combined with the classical regulation model of LysR family genes, vc2323 was supposed to be the functional gene affecting colonization of Vibrio cholerae. Protein homology analysis showed that the protein VC2323 was assumed to be homologous with the tellurium resistant protein Teha.Haemophilus influenzae HiTehA and Arabidopsis thaliana SLAC1 channel protein, which suggested that the protein might be tellurite resistant. By detecting the transcription level of TcpA western blot and tcpA, it was found that vc2323 and vc2324 did not affect the expression and formation of virulence protein TCP of Vibrio cholerae. The sensitivity of vc2323 deletion strain and wild strain to KNO2H _ 2O _ 2 was compared. It was found that CO _ 2 / HCO _ 3- had no effect on the expression of vc2323 and NO/NO2 resistance of Vibrio cholerae (Vibrio cholerae). It was also found that vc2323 Vc2324 did not affect the biofilm formation of Vibrio cholerae in LB medium and had nothing to do with the tolerance of Vibrio cholerae to H. The results of in vitro competitive adhesion test showed that the adhesion of VC2323 deficient strain to the small intestine of mice was significantly lower than that of the wild strain, and the difference was more significant with the prolongation of the adhesion time, but when there was no crude bile salt in the presence of crude bile salt, the adhesion force of VC2323 strain was significantly lower than that of the wild strain. The results showed that the sensitivity of wild strain to crude bile salt was the same as that of vc2323 deficient strain. The analysis of the effect of vc2323 on colonization ability of Vibrio cholerae may be due to its ability to affect the adhesion of Vibrio cholerae to the intestinal tract of the host in the presence of crude bile salt, which leads to a significant decrease in colonization ability of the vc2323 deficient strain than that of the wild strain. Detailed molecular mechanisms need to be further studied. At the same time, the difference of minimal bactericidal concentration of different antimicrobial substances to the missing strain and the wild strain was compared. It was found that the tolerance to chloramphenicol and naphthalic acid of the vc2323 deletion strain decreased by about 64% and 74, respectively, and the resistance to potassium tellurite decreased by about 33, indicating that vc2323 was also involved in the resistance of Vibrio cholerae and tellurite resistance. In conclusion, this study found that LysR family gene vc2324 and its target gene vc2323 were involved in colonization and drug resistance of Vibrio cholerae and tellurite resistance to some extent, which provided more references for the prevention and control of cholera.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378

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