影响霍乱弧菌在宿主体内定殖的生理调控因素的相关研究

发布时间：2018-05-29 21:49

本文选题：霍乱弧菌 + 毒素/抗毒素系统　；参考：《南京农业大学》2015年博士论文

【摘要】：霍乱弧菌是一种可以引起霍乱传染病的病原细菌,人类感染后可导致急性腹泻、脱水甚至死亡。自18世纪以来,在世界范围内爆发了多次霍乱大流行,至今还在威胁人类的健康。霍乱弧菌被人摄入后通过口腔经胃液进入小肠内,穿过粘液层后开始定殖。一旦进入小肠后,霍乱弧菌的毒力基因被宿主体内条件诱导表达,其中毒力共调节菌毛(TCP)与霍乱毒素(CT)是导致霍乱弧菌产生毒力的重要致病因子。霍乱弧菌在人体内定殖是一个有序且复杂的过程,需要特殊的环境因子以及多个自身基因的调控和参与。宿主体内对于霍乱弧菌是一个多样性的环境,一方面宿主体内因子如胆盐、低氧浓度等激活毒力系统表达;另一方面,宿主体内环境对病原细菌生存也是一种逆境。细菌不仅要面对抗菌物质的攻击,还要与其他肠道土著细菌发生竞争性定殖等。在细菌的定殖过程中,已知的影响细菌定殖的因素包括毒力共调节菌毛、生物膜、毒素/抗毒素系统、抗氧化胁迫等。霍乱孤菌内存在至少13套毒素/抗毒素系统(toxin-antitoxin systems,TA)系统,被认为可以发挥稳定遗传超级整合子的作用。目前这些系统在致病能力、生物膜形成、耐药机制中发挥的功能尚不清楚。本文以霍乱弧菌中的13组TA系统为研究对象,首先对RelBE家族中7组进行了毒素功能鉴定,有6组可以编码毒素蛋白,对细菌生长产生抑制。随后对这13组TA系统的缺失株在乳鼠肠道竞争定殖能力、生物膜形成、耐药性以及对宿主环境因子的敏感性等方面进行了检测。发现其中4组TA系统参与霍乱弧菌的定殖过程,RelBE-4和RelBE-7缺失株在乳鼠体内的竞争定殖能力低于野生株,而ParDE-2和ParDE-3缺失株在竞争定殖能力中要高于野生株。TcpA的表达水平在缺失株与野生株中相比并没有明显差异,说明这4组毒素/抗毒素系统可能通过其他途径影响细菌的定殖能力。随后对细菌抗逆境相关表型进行了检测,以探究TA系统对细菌存活能力的影响。在生物膜形成实验中发现,RelBE-1、RelBE-4、RelBE-7和Phd/Doc 4个缺失株在培养过程中形成的生物膜较弱,说明了这4组系统参与了生物膜形成,这可能是导致RelBE-4和RelBE-7定殖能力下降的原因之一。在TA系统与细菌耐药性关系的研究中,发现13株缺失株对氨苄霉素、氯霉素、四环素、萘丁酮酸以及庆大霉素的最低抑菌浓度与野生株相比并无明显差异;在对宿主相似因子敏感性检测中,发现RelBE-4缺失株对过氧化氢(H202)的耐受性较野生株更为敏感;所有缺失株对胆盐敏感程度与野生株相比没有明显差异;因此推测可能以上实验条件不能引起TA系统在耐受宿主因子过程中发挥功能。同时,由于多套TA系统的存在导致基因层面调控的复杂性以及功能上的互补性,也提示研究多重缺失株对宿主因子耐受性是目前的趋势。霍乱弧菌中存在TcpA、MshA和PilA等Ⅳ型菌毛,在细菌致病过程中起到重要作用。本文在体外条件下发现了TcpA缺失株对H_2O_2的耐受性降低,回补TcpA后敏感程度可以恢复到野生株水平;同时pilA缺失株对H_2O_2较野生株敏感。体外纯化的TcpA蛋白并不具有清除H_2O_2、保护细胞的作用。对TcpA缺失株中过氧化氢酶KatB的表达水平进行检测,发现其表达量在缺失株中较野生株低,因此提示了TcpA可能起到感受氧压进而将信号传给过氧化氢酶等作用。通过加入H_2O_2后检测蛋白的表达以及定位情况,发现H_2O_2并不会影响TcpA蛋白的表达、分泌以及锚定,在膜蛋白中可以检测到等量的TcpA蛋白。进一步发现在TcpA蛋白中,两个半胱氨酸为关键位点,将其突变后导致TcpA蛋白的快速降解,由于这两个氨基酸可能会形成分子内二硫键,因此破坏二硫键的形成可能对蛋白的结构造成影响,也说明这两个氨基酸位点对于蛋白结构的稳定性至关重要。但是Ⅳ型菌毛参与细菌抗氧化性机制还待进一步研究,本文发现作为毒力蛋白的Ⅳ型菌毛可能参与细菌抗逆境反应,对H_2O_2的不耐受性也是导致产生定殖缺陷的一个原因,提示了毒力因子在定殖过程中发挥的新功能,为后续致病机制的研究奠定了实验基础。细菌胞外多糖与粘液素的互作对霍乱弧菌定殖产生影响。本研究发现不同血清型的菌株大都表现出在粘液素中游动性加快的现象,并且不同的营养环境会影响霍乱弧菌在粘液素中的游动速率。实验室前期数据表明霍乱弧菌胞外多糖会减慢霍乱弧菌在粘液素的游动性。本文乳鼠竞争定殖实验证实胞外多糖产生过量会影响细菌在小肠的定殖,造成定殖能力下降,推测其原因就是由于产生过多胞外多糖导致细菌穿过粘液层速度和扩散速度减慢,细菌穿过粘液层效率下降。这个结果是细菌与宿主因子相互作用的重要体现。同时细菌胞外多糖的产生受到粘液素和群体感应系统的调控,细菌群体可能通过自身和群体感应系统调控胞外多糖合成基因的表达来改变细菌穿过粘液层的速度从而达到高效定殖。本研究进一步加深了对霍乱弧菌定殖过程的认识。综上所述,本文发现霍乱弧菌在宿主内成功定殖是一个复杂的过程,不同生理系统相互影响形成网络调控系统。一种生理系统通过对霍乱弧菌不同生理行为发挥作用,从而影响细菌的定殖能力。霍乱弧菌毒素/抗毒素系统的激活可能依赖于宿主环境,同时毒素/抗毒素不仅影响生物膜的形成能力,并且也会导致产生定殖差异;而毒素共调节菌毛蛋白TcpA不仅影响细菌在宿主体内的粘附能力,同时还通过自身影响细菌群体的抗氧化胁迫反应,帮助细菌在宿主内生存以及定殖。霍乱弧菌不仅通过胞外多糖影响生物膜的形成从而影响其在肠道中的吸附能力;还利用胞外多糖与粘液层分子间相互作用影响细菌通过粘液层的速度,从而影响其在肠道中的定殖能力。本文揭示了霍乱弧菌与宿主相互作用的复杂性,研究这些因素对霍乱弧菌定殖以及致病性的影响具有重要的意义,为进一步深入了解霍乱弧菌在宿主体内生存能力以及感染过程的具体细节和防治霍乱提供更有效的手段和方法。
[Abstract]:Vibrio cholerae, a pathogenic bacterium that can cause cholera infectious diseases, can cause acute diarrhea, dehydration and even death after human infection. Since eighteenth Century, a number of cholera pandemics have been broken out worldwide and are still threatening human health. Vibrio cholerae enters the small intestine through the stomatological fluid through the mouth and passes through mucus. The virulence gene of Vibrio cholerae is induced by the host condition once it enters the small intestine. The virulence co regulates TCP and cholera toxin (CT) is an important pathogenic factor causing Vibrio cholerae to produce virulence. The colonization of Vibrio cholerae is an orderly and complex process in human body, and needs special environmental factors. And the regulation and participation of multiple own genes. The host is a diverse environment for Vibrio cholerae in the host. On the one hand, the host factors such as bile salt and hypoxia are activated. On the other hand, the host environment is also a reverse condition for the survival of the pathogenic bacteria. In the process of colonization of bacteria, the known factors affecting the colonization of bacteria include virulence co regulation of pilus, biofilm, toxin / antitoxin system, antioxidant stress, and so on. There are at least 13 toxin-antitoxin systems (TA) systems in cholera isolated bacteria, which are considered to be available. In order to play the role of stabilizing the hereditary superintegrons, the functions of these systems in the pathogenesis, biofilm formation and drug resistance are not yet clear. In this paper, 13 groups of TA systems in Vibrio cholerae were used as the research objects. First of all, 7 groups of toxins were identified, 6 groups of toxin proteins were encoded, and bacteria were grown. Then, the colonization ability of the 13 TA systems in the intestinal competition, biofilm formation, drug resistance and sensitivity to the host environmental factors were detected, and 4 groups of TA systems were found to be involved in the colonization of Vibrio cholerae, and the competitive colonization of RelBE-4 and RelBE-7 missing strains in the milk mice was lower than that of the mice. In the field of wild plant, the expression level of ParDE-2 and ParDE-3 in the competitive colonization was higher than that of wild strain.TcpA, and there was no significant difference between the missing strains and the wild plants. It indicated that the 4 groups of toxins / antitoxin systems may affect the colonization of bacteria by other ways. The effect of the TA system on the viability of the bacteria was explored. In the biofilm formation experiment, it was found that the biofilms formed during the culture process of the 4 missing strains of RelBE-1, RelBE-4, RelBE-7 and Phd/Doc showed that the 4 systems were involved in the formation of biofilm, which may be one of the reasons for the decrease of RelBE-4 and RelBE-7 colonization. In the study of the relationship between bacterial resistance, the minimum inhibitory concentrations of ampicillin, chloramphenicol, tetracycline, naphthoxone and gentamicin were not significantly different from those of the wild strain, and the tolerance of RelBE-4 to H202 was more sensitive than that of the wild strain in the sensitivity test of the host similar factor. There is no obvious difference in the sensitivity of all the missing strains to the wild plants. Therefore, it is presumed that the above experimental conditions may not cause the TA system to play a function in the tolerance of the host factor. At the same time, the complexity of the gene level regulation and the functional complementarity of multiple sets of TA systems also suggest multiple studies. The tolerance of the missing strains to host factors is the current trend. Vibrio cholerae exists in Vibrio cholerae TcpA, MshA and PilA type IVS, which plays an important role in the pathogenic process of bacteria. In vitro, the tolerance of TcpA missing strains to H_2O_2 is reduced, and the sensitivity of TcpA can be restored to the level of the wild strain after the recharge of TcpA; meanwhile, the pilA deletion strain is also found. H_2O_2 was sensitive to the wild plant. The purified TcpA protein in vitro did not have the effect of removing H_2O_2 and protecting the cells. The expression of catalase KatB in the TcpA deletion strain was detected, and its expression was lower in the missing strain than in the wild plant. Therefore, it was suggested that TcpA may play a sense of oxygen pressure and transmit the signal to catalase, etc. After adding H_2O_2 to detect the expression and location of protein, it is found that H_2O_2 does not affect the expression, secretion and anchoring of TcpA protein, and the same amount of TcpA protein can be detected in the membrane protein. It is found that the two cysteine is the key point in the TcpA protein, and the mutation leads to the rapid degradation of TcpA protein. Since the two amino acids may form an intramolecular two sulfur bond, the formation of the two sulfur bonds may affect the structure of the protein, and the two amino acid sites are important for the stability of the protein structure. However, the mechanism of the type IV pilus to participate in the antioxidant activity of bacteria has yet to be further studied. This article is found to be a toxic egg. The type IV pili of white may be involved in the anti adversity reaction of bacteria, and the intolerance of H_2O_2 is also a cause for the production of colonization defects, which indicates the new function of the virulence factor in the colonization process, which lays the foundation for the study of subsequent pathogenesis. The interaction of extracellular polysaccharide and mucin on the colonization of Vibrio cholerae The study found that the strains of different serotypes were mostly shown to travel faster in the mucin, and the different nutritional environment would affect the movement rate of Vibrio cholerae in the mucin. It is found that excessive extracellular polysaccharide may affect the colonization of bacteria in the small intestine and cause a decline in colonization. The reason is that the production of excessive extracellular polysaccharide causes bacteria to slow through the velocity and diffusion of the mucous layer, and the efficiency of bacteria passes through the mucilage layer. This fruit is an important body for the interaction of bacteria and host factors. At the same time, the production of bacterial extracellular polysaccharide is regulated by the mucin and the quorum induction system. The bacterial population may regulate the expression of the extracellular polysaccharide synthesis gene by itself and the quorum induction system to change the velocity of bacteria through the mucous layer to achieve high colonization. This study further deepened the recognition of the colonization of Vibrio cholerae. In summary, this paper shows that the successful colonization of Vibrio cholerae is a complex process in which different physiological systems interact with each other to form a network regulation system. A physiological system plays a role in different physiological behavior of Vibrio cholerae, thus affecting the colonization of bacteria. The activation of Vibrio toxin / antitoxin system may be possible. Depending on the host environment, toxin / antitoxin not only affects the formation ability of the biofilm, but also leads to the production of colonization, and the co regulated bacterial hair protein TcpA not only affects the adhesion of bacteria in the host, but also helps the bacteria to survive in the host by influencing the oxidative stress response of the bacterial colony itself. Vibrio cholerae (Vibrio cholerae) not only affects the formation of biofilm through extracellular polysaccharide, but also affects its adsorption capacity in the intestinal tract. It also affects the colonization of bacteria through the mucus layer by the interaction of extracellular polysaccharide and the mucus layer. This paper reveals the interaction between Vibrio cholerae and its host. It is of great significance to study the effects of these factors on the colonization and pathogenicity of Vibrio cholerae, and to provide more effective means and methods for further understanding the viability of Vibrio cholerae in the host and the specific details of the infection process and the prevention and treatment of cholera.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R378

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