RovM和RovA调控鼠疫耶尔森氏菌生物被膜形成和毒力的分子机制研究

发布时间：2018-05-07 05:33

本文选题：鼠疫耶尔森氏菌 + RovM　；参考：《中国人民解放军军事医学科学院》2016年博士论文

【摘要】：鼠疫耶尔森氏菌是(Yersinia pestis,以下简称“鼠疫菌”)是引发烈性传染病鼠疫的病原菌,是一种革兰氏染色阴性的短小杆菌。而且,鼠疫菌在不同的培养条件或宿主环境下会呈现不同的形态。鼠疫菌染色体基因组大小约为4.65 Mb,包含约4000个基因。大多数的鼠疫菌菌株都包含3种质粒:p PCP1,p CD1和p MT1,3种质粒上包含有大量重要的毒力因子,对于鼠疫菌在宿主体内生存、传播以及致病有着重要意义。鼠疫菌进入人体后,感染初期会在几天内迅速扩散至人体的淋巴结形成腺鼠疫(bubonic plague);一旦进入血液经大量繁殖后容易引发败血症鼠疫(septicemic plague),并进一步扩散至脾、肝、肺等器官和组织;最终,一部分患者会发展为肺鼠疫(pneumonic plague),病原菌可以直接通过空气传播给其他人,易传播且致死率高。鼠疫菌引发的烈性传染病曾在世界范围内引发3次大流行,给人类带来了深重的灾难,并在一定程度上影响了各民族的政治、经济和文化。而且,近些年来世界卫生组织(WHO)将鼠疫列为重新抬头的传染病。鼠疫防治的研究工作仍然显得十分重要。鼠疫菌由其祖先假结核耶尔森氏菌(Yersinia pseudotuberculosis,以下简称“假结核菌”)进化而来,然而完全区别于假结核菌的传播方式,在自然界中,鼠疫菌通过节肢动物跳蚤的叮咬在宿主动物间进行循环传播,携带病原菌的跳蚤偶然间叮咬到人类会引发人间鼠疫。鼠疫菌在跳蚤体内传播时,会形成致密的生物被膜堵塞跳蚤的前胃,形成所谓的“菌栓”,使得跳蚤吸食的血液无法进入胃内消化,跳蚤时刻处于饥饿状态,反复叮咬宿主从而促进了鼠疫菌的传播。因此,鼠疫菌生物被膜的产生对于其经蚤传播的能力是具有重要意义的。鼠疫菌拥有复杂而精确的调控网络控制着各个生物被膜形成决定性因子以及毒力因子的激活和抑制,这其中各级调控子的协同作用对于鼠疫菌的致病和传播有着重要意义。目前的研究表明,许多重要的调控子以及它们调控的靶标基因在鼠疫菌生物被膜形成以及病原菌致病性中扮演着重要角色。鼠疫菌中hms HFRS操纵子坐落于染色体上的pgm位点内,负责合成和运输多聚β-1,6-N-乙酰-D-氨基葡糖(poly-β-1,6-N-acetylglucosamine exopolysaccharide)类似的多糖化合物,是生物被膜基质的主要成分,与鼠疫菌菌落能否被刚果红染料染色有直接关系。环二鸟苷单磷酸(c-di-GMP)是一个在细菌内广泛存在的第二信使分子,影响细菌毒力因子的表达和生物被膜形成。在鼠疫菌内,Hms T和Hms D是仅有的两个鸟苷酸环化酶能够催化c-di-GMP的合成,从而促进生物被膜的形成。然而,Hms P是鼠疫菌中唯一催化降解c-di-GMP的磷酸二酯酶,从而抑制鼠疫菌生物被膜的产生。在革兰氏阴性菌中,脂多糖(Lipopolysaccharide)是构成外膜必不可少的主要成分。一般来说,脂多糖由三部分组成:脂质A,核心多糖(Kdo)和O抗原,而鼠疫菌的脂多糖仅由脂质A在糖基转移酶的催化下与Kdo连接。在鼠疫菌中,waa A,waa E和coa D构成一个三基因的操纵子waa AE-coa D。waa A编码的糖基转移酶催化Kdo连接到脂质A上,而waa A缺失后的鼠疫菌也表现出明显的生物被膜形成缺陷。waa E编码的蛋白对于核心多糖链内庚糖的修饰起到关键作用,而研究表明waa E缺失后体外培养细菌时生物被膜形成约有40%左右的降低。这说明了鼠疫菌生物被膜的形成和脂多糖的合成有着重要联系。在鼠疫菌中,p H6抗原是重要的黏附因子以及毒力因子。p H6抗原的合成和分泌需要两个相邻的操纵子基因簇的参与,即psa ABC和psa EF。psa EF可以编码调控子蛋白促进psa ABC的表达。重要的毒力调控子Rov A和Pho P可以通过直接结合psa ABC和psa EF的启动子区结构,分别激活和抑制它们的表达。p H6抗原可以通过介导鼠疫菌黏附到肺泡上皮细胞,对于鼠疫菌引发小鼠腺鼠疫和肺鼠疫的发生都有重要的贡献。Rov A是Mar R/Sly A家族的调控子,影响着许多细菌大量毒力基因的表达,在三种致病型耶尔森氏菌的毒力基因调控中都扮演重要角色。许多Mar R家族的转录调控子,例如表皮葡萄球菌(Staphylococcus epidermidis)的Tca R,屎肠球菌(Enterococcus faecium)的Asr R,变异链球菌(Streptococcus mutans)的Rca R以及金黄色葡萄球菌(Staphylococcus aureus)的Sar Z/Sar A等,都有研究报道影响细菌生物被膜的形成,然而Rov A对于鼠疫菌生物被膜形成的调控机制还是未知的。Rov M是Lys R家族的一个转录调控子,它首次被发现是在假结核菌中,研究表明Rov M可以直接结合到rov A基因的启动子区并抑制其转录,而且Rov M影响假结核菌的侵袭能力、毒力及运动性等。许多Lys R家族的转录调控子,例如菊欧文氏菌(Erwinia chrysanthemi)的Pec T,胡萝卜软腐欧文氏菌(Erwinia carotovora)以及大肠杆菌(Escherichia coli)的Lrh A,都在细菌毒力及生物被膜基因调控方面影响重大。那么,Rov M在鼠疫菌毒力及生物被膜形成的基因调控网络中又扮演怎样的角色呢?本研究中,以Rov M和Rov A两个调控子作为主要的研究对象,通过生物被膜形成检测的相关表型实验如:结晶紫染色半定量实验、线虫虫卵发育实验、菌落表面形态观察等实验方法探究Rov M和Rov A对鼠疫菌生物被膜形成的影响。结果表明:Rov M能够促进鼠疫菌生物被膜的形成,而Rov A却抑制鼠疫菌生物被膜的形成。通过检测并比较鼠疫菌不同菌株之间细菌胞内c-di-GMP的含量探究Rov M和Rov A是否影响第二信使分子c-di-GMP的合成。结果表明:Rov M显著地激活c-di-GMP的合成,而Rov A却强烈地抑制c-di-GMP的合成。通过皮下感染以及静脉感染两种方式给小鼠注射鼠疫菌,绘制小鼠生存曲线来探究Rov M对鼠疫菌毒力的影响。结果表明:Rov M抑制鼠疫菌的毒力。在获得了表型实验结果后,进一步通过精细的基因调控实验如:引物延伸实验、实时定量荧光PCR实验、Lac Z报告基因融合实验以及凝胶阻滞实验等方法探究Rov M和Rov A对鼠疫菌生物被膜形成以及毒力相关的靶基因和调控子基因调控的分子机制。结果表明,Rov M通过激活hms HFRS、hms T、hms CDE的表达,并同时抑制hms P的表达促进鼠疫菌生物被膜的形成。然而,Rov M间接地抑制YPO1635-pho PQ-YPO1632的表达,却不影响waa AE-coa D、pho PQ-YPO1632和fur的表达。Rov A通过抑制hms T、waa AE-coa D、YPO1635-pho PQ-YPO1632和pho PQ-YPO1632的表达阻碍鼠疫菌生物被膜的形成。然而,Rov A却不影响hms HFRS、hms CDE、hms P、fur和rov M的表达。并且,Rov M通过直接抑制rov A的表达,且同时间接抑制psa ABC和psa EF的表达来抑制鼠疫菌毒力。同时,Rov M可以激活自身的表达,且在26°C下高表达而37°C下低表达。我们汇总本研究中的实验结果以及前期的研究成果,大胆的提出了Rov M和Rov A在鼠疫菌致病和传播中可能存在的调控通路。在跳蚤体内环境温度(26°C)下,Rov M处于激活状态,它可以通过调控生物被膜形成相关决定性因子来促进鼠疫菌生物被膜的形成,并且同时抑制Rov A的表达,使得Rov A作用的大量毒力因子处于被抑制状态,从而有利于增强鼠疫菌经蚤传播的能力。当鼠疫菌由跳蚤传播到哺乳动物体内后,其生存环境由26°C转变为37°C,此时Rov M的表达明显降低,导致生物被膜形成的相关决定性因子处于抑制状态,而原本表达处于抑制状态的Rov A得到激活,通过调控大量的毒力因子来增强鼠疫菌的致病性。因此,我们可以看出Rov M和Rov A在鼠疫菌调控网络中的重要地位,Rov M和Rov A分别通过激活和抑制鼠疫菌生物被膜的形成,并同时分别抑制和激活鼠疫菌的毒力来影响鼠疫菌经跳蚤的传播能力以及对哺乳动物或人类的致病性。当生长环境温度由26°C转变为37°C时,假结核菌中的Rov M表达水平无明显变化,而鼠疫菌中Rov M的表达水平却显著降低,这可能是由假结核菌进化到鼠疫菌过程中的一个适应性变化。
[Abstract]:The plague Jerson Prand (Yersinia pestis, hereinafter referred to as "Yersinia pestis") is the pathogenic bacteria causing the strong infectious disease of the plague, and is a gram-negative bacilli. Moreover, the Yersinia pestis will present different forms in different culture conditions or host environment. The genome size of the pestis is about 4.65 Mb, including about 40. 00 genes. Most of the strains of Yersinia pestis contain 3 plasmids: P PCP1, P CD1 and P MT1,3 contain a large number of important virulence factors, which are important for the survival, transmission and pathogenesis of Yersinia pestis in the host. Plague (bubonic plague); once the blood is brought into the blood, it can easily cause the septicemia plague (septicemic plague), and further spread to the spleen, liver, lung and other organs and tissues; finally, a part of the patients will develop into the lung plague (pneumonic plague), the pathogen can be transmitted directly through the air to other people, easy to spread and high lethal rate. The strong infectious disease caused by Phytophthora has triggered 3 pandemics worldwide, which has brought serious disasters to human beings, and to some extent affected the politics, economy and culture of all ethnic groups. Moreover, in recent years, the WHO (WHO) has listed the plague as a resurgence of the infectious disease. The research on the prevention and control of plague still appears to be very important. Yersinia pestis is evolved from its ancestor false tuberculosis Jerson Prand (Yersinia pseudotuberculosis, hereinafter referred to as "pseudo tuberculosis"), but it is completely different from the mode of transmission of pseudo tuberculosis bacteria. In nature, Yersinia pestis is circulated through the bite of the arthropod flea in the host animal, and the flea of the pathogen is accidental. In the flea body, Yersinia pestis will form a dense biological membrane that clog the flea's front stomach, and form a so-called "fungus bolt", which makes the flea sucking blood unable to enter the stomach, the flea is at the moment of starvation, repeatedly bites the host and promotes the spread of the Yersinia pestis. Therefore, the rat The production of the biofilm of the Phytophthora is of great significance to its ability to spread through the fleas. Yersinia pestis has a complex and accurate control network that controls the decisive factors of the biofilm formation and the activation and inhibition of the virulence factors. The synergism of the regulators at all levels has important implications for the pathogenesis and spread of the Yersinia pestis. The current research shows that many important regulators and their target genes play an important role in the formation of Yersinia pestis biofilm and pathogenicity of the pathogen. The HMS HFRS operon in Yersinia pestis is located in the PGM site on the chromosome and is responsible for the synthesis and transport of poly beta -1,6-N- acetyl -D- glucosamine (poly- beta -1,6-N). -acetylglucosamine exopolysaccharide) a similar polysaccharide compound, the main component of the biofilm matrix, is directly related to whether the bacterial colony of Yersinia pestis can be dyed by Congo red dye. C-di-GMP is a widely existing second messenger in bacteria, affecting the expression of bacterial virulence factors and biofilm. Formation. In Yersinia pestis, Hms T and Hms D are the only two guanosine cyclase catalyzing the synthesis of c-di-GMP, thus promoting the formation of biofilm. However, Hms P is the only phosphodiesterase that catalyzes the degradation of c-di-GMP in Yersinia pestis, thus inhibiting the production of the biofilm of the Yersinia pestis. In Gram-negative bacteria, the lipopolysaccharide (Lipopolysacch) Aride) is an essential component of the outer membrane. Generally speaking, lipopolysaccharide consists of three parts: lipid A, core polysaccharide (Kdo) and O antigen, while LPS are connected to Kdo with the lipid A only under the catalysis of glycosyltransferase. In Yersinia pestis, WAA A, WAA E and COA D constitute a operon encoding of the three gene. The glycosyltransferase catalyzes Kdo to connect to the lipid A, and the Yersinia pestis after the deletion of WAA A also shows a distinct biofilm formation defect, the protein encoded by.Waa E plays a key role in the modification of the core polysaccharide chain, and the study shows that the biofilm formation is about 40% reduced when the WAA E is missing in vitro. There is an important link between the formation of the biofilm of Yersinia pestis and the synthesis of lipopolysaccharide. In Yersinia pestis, the synthesis and secretion of P H6 antigen, an important adhesion factor and the.P H6 antigen of virulence factor, requires the participation of two adjacent operon genes, that is, PSA ABC and PSA EF.psa EF can promote the expression of PSA ABC by encoding the regulatory subproteins. Important virulence regulators, Rov A and Pho P, can activate and inhibit their expression of.P H6 antigen by directly binding to the promoter region of PSA ABC and PSA EF, and can be used to mediate the adhesion of Yersinia pestis to alveolar epithelial cells. Family regulator, affecting the expression of a large number of bacterial virulence genes, plays an important role in the regulation of three pathogenic Jerson Prand's virulence genes. Many transcriptional regulators of the Mar R family, such as Tca R of Staphylococcus epidermidis (Staphylococcus epidermidis), Asr R for Enterococcus faecium (Enterococcus faecium), and variant chain balls The Rca R of Streptococcus mutans and Sar Z/Sar A of Staphylococcus aureus (Staphylococcus aureus) have been reported to affect the formation of bacterial biofilm. However, Rov A on the regulation mechanism of the biofilm formation of Yersinia pestis is an unknown transcriptional regulator. It was first found to be false. In tuberculosis, studies have shown that Rov M can directly bind to the promoter region of the ROV A gene and inhibit its transcription, and Rov M affects the invasiveness, virulence and motility of the Mycobacterium tuberculosis. Many Lys R family transcriptional regulators, such as the Pec T of the chrysanthemum (Erwinia Chrysanthemi), and the soft rot of carrots. And Lrh A of Escherichia coli (Escherichia coli) are all important in the effects of bacterial virulence and biofilm gene regulation. Then, what is the role of Rov M in the virulence of Yersinia pestis and the gene regulatory network formed by biofilm? In this study, the two regulators of Rov M and Rov A were used as the main research objects, through biological being The related phenotypic experiments of membrane formation detection, such as the semi quantitative experiment of crystal violet staining, the egg development experiment of nematode worm, and the morphology observation of the colony surface, explored the effect of Rov M and Rov A on the formation of the biofilm of Yersinia pestis. The results showed that Rov M could promote the formation of the biofilm of Yersinia pestis, while Rov A inhibited the shape of the biofilm of Yersinia pestis. Explore whether Rov M and Rov A affect the synthesis of second messenger c-di-GMP by detecting and comparing the intracellular c-di-GMP content of bacteria between the different strains of Yersinia. The results show that Rov M activates the synthesis of c-di-GMP significantly, while Rov A strongly inhibits the synthesis of c-di-GMP. Two ways are given by subcutaneous infection and venous infection. Mice injected with Yersinia pestis and plotted the survival curve of mice to explore the effect of Rov M on the virulence of Yersinia pestis. The results showed that Rov M inhibited the virulence of Yersinia pestis. After obtaining the results of the phenotypic experiment, the fine gene regulation experiments, such as primer extension experiment, real-time quantitative fluorescence PCR experiment, Lac Z report gene fusion experiment and gel resistance, were further studied. The molecular mechanism of Rov M and Rov A on the formation of Yersinia pestis biofilm and the regulation of virulence related target genes and regulatory subgenes was investigated. The results showed that Rov M expressed the expression of HMS HFRS, HMS T, HMS CDE, and inhibited the formation of the Yersinia pestis biofilm at the same time. The expression of -pho PQ-YPO1632 does not affect WAA AE-coa D, Pho PQ-YPO1632 and fur's expression.Rov A through inhibition of HMS T, which obstruct the formation of the membrane of the Yersinia pestis. The expression of ROV A and the inhibition of the expression of PSA ABC and PSA EF to inhibit the virulence of Yersinia pestis. At the same time, Rov M can activate its own expression and low expression under 26 degree C and low expression under 37 degree C. The possible regulatory pathway. Under the environment temperature of the flea (26 C), Rov M is in the active state. It can promote the formation of Yersinia pestis biofilm by regulating the biofilm formation, and inhibit the expression of Rov A at the same time, so that a large number of virulence factors of Rov A are in a state of inhibition, which is beneficial to it. Increasing the ability of Yersinia pestis to spread through fleas. When the Yersinia pestis is transmitted to the mammalian body, the survival environment changes from 26 C to 37 C, and the expression of Rov M decreases obviously, resulting in the inhibition of the related decisive factors of the biofilm formation, and the Rov A in the original expression at the inhibitory state is activated by the regulation of a large number of factors. The virulence of Yersinia pestis is enhanced by the virulence factors. Therefore, we can see the important position of Rov M and Rov A in the control network of Yersinia pestis. Rov M and Rov A inhibit and activate the virulence of Yersinia pestis by activating and inhibiting the formation of the biofilm of Yersinia pestis, respectively. The pathogenicity of mammalian and human beings. When the temperature of the growth environment changed from 26 C to 37 C, the expression level of Rov M in the Mycobacterium tuberculosis was not obviously changed, but the expression level of Rov M in Yersinia pestis was significantly reduced, which may be a suitable change in the process of evolution from pseudo tuberculosis bacteria to Yersinia pestis.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378

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