本文選題：霍亂弧菌 + 毒素/抗毒素系統(tǒng)　； 參考：《南京農(nóng)業(yè)大學(xué)》2015年博士論文

【摘要】：霍亂弧菌是一種可以引起霍亂傳染病的病原細(xì)菌,人類感染后可導(dǎo)致急性腹瀉、脫水甚至死亡。自18世紀(jì)以來,在世界范圍內(nèi)爆發(fā)了多次霍亂大流行,至今還在威脅人類的健康�；魜y弧菌被人攝入后通過口腔經(jīng)胃液進(jìn)入小腸內(nèi),穿過粘液層后開始定殖。一旦進(jìn)入小腸后,霍亂弧菌的毒力基因被宿主體內(nèi)條件誘導(dǎo)表達(dá),其中毒力共調(diào)節(jié)菌毛(TCP)與霍亂毒素(CT)是導(dǎo)致霍亂弧菌產(chǎn)生毒力的重要致病因子。霍亂弧菌在人體內(nèi)定殖是一個(gè)有序且復(fù)雜的過程,需要特殊的環(huán)境因子以及多個(gè)自身基因的調(diào)控和參與。宿主體內(nèi)對于霍亂弧菌是一個(gè)多樣性的環(huán)境,一方面宿主體內(nèi)因子如膽鹽、低氧濃度等激活毒力系統(tǒng)表達(dá);另一方面,宿主體內(nèi)環(huán)境對病原細(xì)菌生存也是一種逆境。細(xì)菌不僅要面對抗菌物質(zhì)的攻擊,還要與其他腸道土著細(xì)菌發(fā)生競爭性定殖等。在細(xì)菌的定殖過程中,已知的影響細(xì)菌定殖的因素包括毒力共調(diào)節(jié)菌毛、生物膜、毒素/抗毒素系統(tǒng)、抗氧化脅迫等�；魜y孤菌內(nèi)存在至少13套毒素/抗毒素系統(tǒng)(toxin-antitoxin systems,TA)系統(tǒng),被認(rèn)為可以發(fā)揮穩(wěn)定遺傳超級整合子的作用。目前這些系統(tǒng)在致病能力、生物膜形成、耐藥機(jī)制中發(fā)揮的功能尚不清楚。本文以霍亂弧菌中的13組TA系統(tǒng)為研究對象,首先對RelBE家族中7組進(jìn)行了毒素功能鑒定,有6組可以編碼毒素蛋白,對細(xì)菌生長產(chǎn)生抑制。隨后對這13組TA系統(tǒng)的缺失株在乳鼠腸道競爭定殖能力、生物膜形成、耐藥性以及對宿主環(huán)境因子的敏感性等方面進(jìn)行了檢測。發(fā)現(xiàn)其中4組TA系統(tǒng)參與霍亂弧菌的定殖過程,RelBE-4和RelBE-7缺失株在乳鼠體內(nèi)的競爭定殖能力低于野生株,而ParDE-2和ParDE-3缺失株在競爭定殖能力中要高于野生株。TcpA的表達(dá)水平在缺失株與野生株中相比并沒有明顯差異,說明這4組毒素/抗毒素系統(tǒng)可能通過其他途徑影響細(xì)菌的定殖能力。隨后對細(xì)菌抗逆境相關(guān)表型進(jìn)行了檢測,以探究TA系統(tǒng)對細(xì)菌存活能力的影響。在生物膜形成實(shí)驗(yàn)中發(fā)現(xiàn),RelBE-1、RelBE-4、RelBE-7和Phd/Doc 4個(gè)缺失株在培養(yǎng)過程中形成的生物膜較弱,說明了這4組系統(tǒng)參與了生物膜形成,這可能是導(dǎo)致RelBE-4和RelBE-7定殖能力下降的原因之一。在TA系統(tǒng)與細(xì)菌耐藥性關(guān)系的研究中,發(fā)現(xiàn)13株缺失株對氨芐霉素、氯霉素、四環(huán)素、萘丁酮酸以及慶大霉素的最低抑菌濃度與野生株相比并無明顯差異;在對宿主相似因子敏感性檢測中,發(fā)現(xiàn)RelBE-4缺失株對過氧化氫(H202)的耐受性較野生株更為敏感;所有缺失株對膽鹽敏感程度與野生株相比沒有明顯差異;因此推測可能以上實(shí)驗(yàn)條件不能引起TA系統(tǒng)在耐受宿主因子過程中發(fā)揮功能。同時(shí),由于多套TA系統(tǒng)的存在導(dǎo)致基因?qū)用嬲{(diào)控的復(fù)雜性以及功能上的互補(bǔ)性,也提示研究多重缺失株對宿主因子耐受性是目前的趨勢�；魜y弧菌中存在TcpA、MshA和PilA等Ⅳ型菌毛,在細(xì)菌致病過程中起到重要作用。本文在體外條件下發(fā)現(xiàn)了TcpA缺失株對H_2O_2的耐受性降低,回補(bǔ)TcpA后敏感程度可以恢復(fù)到野生株水平;同時(shí)pilA缺失株對H_2O_2較野生株敏感。體外純化的TcpA蛋白并不具有清除H_2O_2、保護(hù)細(xì)胞的作用。對TcpA缺失株中過氧化氫酶KatB的表達(dá)水平進(jìn)行檢測,發(fā)現(xiàn)其表達(dá)量在缺失株中較野生株低,因此提示了TcpA可能起到感受氧壓進(jìn)而將信號傳給過氧化氫酶等作用。通過加入H_2O_2后檢測蛋白的表達(dá)以及定位情況,發(fā)現(xiàn)H_2O_2并不會(huì)影響TcpA蛋白的表達(dá)、分泌以及錨定,在膜蛋白中可以檢測到等量的TcpA蛋白。進(jìn)一步發(fā)現(xiàn)在TcpA蛋白中,兩個(gè)半胱氨酸為關(guān)鍵位點(diǎn),將其突變后導(dǎo)致TcpA蛋白的快速降解,由于這兩個(gè)氨基酸可能會(huì)形成分子內(nèi)二硫鍵,因此破壞二硫鍵的形成可能對蛋白的結(jié)構(gòu)造成影響,也說明這兩個(gè)氨基酸位點(diǎn)對于蛋白結(jié)構(gòu)的穩(wěn)定性至關(guān)重要。但是Ⅳ型菌毛參與細(xì)菌抗氧化性機(jī)制還待進(jìn)一步研究,本文發(fā)現(xiàn)作為毒力蛋白的Ⅳ型菌毛可能參與細(xì)菌抗逆境反應(yīng),對H_2O_2的不耐受性也是導(dǎo)致產(chǎn)生定殖缺陷的一個(gè)原因,提示了毒力因子在定殖過程中發(fā)揮的新功能,為后續(xù)致病機(jī)制的研究奠定了實(shí)驗(yàn)基礎(chǔ)。細(xì)菌胞外多糖與粘液素的互作對霍亂弧菌定殖產(chǎn)生影響。本研究發(fā)現(xiàn)不同血清型的菌株大都表現(xiàn)出在粘液素中游動(dòng)性加快的現(xiàn)象,并且不同的營養(yǎng)環(huán)境會(huì)影響霍亂弧菌在粘液素中的游動(dòng)速率。實(shí)驗(yàn)室前期數(shù)據(jù)表明霍亂弧菌胞外多糖會(huì)減慢霍亂弧菌在粘液素的游動(dòng)性。本文乳鼠競爭定殖實(shí)驗(yàn)證實(shí)胞外多糖產(chǎn)生過量會(huì)影響細(xì)菌在小腸的定殖,造成定殖能力下降,推測其原因就是由于產(chǎn)生過多胞外多糖導(dǎo)致細(xì)菌穿過粘液層速度和擴(kuò)散速度減慢,細(xì)菌穿過粘液層效率下降。這個(gè)結(jié)果是細(xì)菌與宿主因子相互作用的重要體現(xiàn)。同時(shí)細(xì)菌胞外多糖的產(chǎn)生受到粘液素和群體感應(yīng)系統(tǒng)的調(diào)控,細(xì)菌群體可能通過自身和群體感應(yīng)系統(tǒng)調(diào)控胞外多糖合成基因的表達(dá)來改變細(xì)菌穿過粘液層的速度從而達(dá)到高效定殖。本研究進(jìn)一步加深了對霍亂弧菌定殖過程的認(rèn)識。綜上所述,本文發(fā)現(xiàn)霍亂弧菌在宿主內(nèi)成功定殖是一個(gè)復(fù)雜的過程,不同生理系統(tǒng)相互影響形成網(wǎng)絡(luò)調(diào)控系統(tǒng)。一種生理系統(tǒng)通過對霍亂弧菌不同生理行為發(fā)揮作用,從而影響細(xì)菌的定殖能力�；魜y弧菌毒素/抗毒素系統(tǒng)的激活可能依賴于宿主環(huán)境,同時(shí)毒素/抗毒素不僅影響生物膜的形成能力,并且也會(huì)導(dǎo)致產(chǎn)生定殖差異;而毒素共調(diào)節(jié)菌毛蛋白TcpA不僅影響細(xì)菌在宿主體內(nèi)的粘附能力,同時(shí)還通過自身影響細(xì)菌群體的抗氧化脅迫反應(yīng),幫助細(xì)菌在宿主內(nèi)生存以及定殖�；魜y弧菌不僅通過胞外多糖影響生物膜的形成從而影響其在腸道中的吸附能力;還利用胞外多糖與粘液層分子間相互作用影響細(xì)菌通過粘液層的速度,從而影響其在腸道中的定殖能力。本文揭示了霍亂弧菌與宿主相互作用的復(fù)雜性,研究這些因素對霍亂弧菌定殖以及致病性的影響具有重要的意義,為進(jìn)一步深入了解霍亂弧菌在宿主體內(nèi)生存能力以及感染過程的具體細(xì)節(jié)和防治霍亂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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R378

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相關(guān)重要報(bào)紙文章 前10條

1 韋辰;小心霍亂弧菌趁熱作“亂”[N];科技日報(bào);2006年

2 記者 陳靜瑩;汕頭附近海域未被霍亂弧菌污染[N];汕頭日報(bào);2008年

3 記者 王艷紅;霍亂弧菌基因組測序完成[N];人民日報(bào);2000年

4 記者 葉明e，

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