致腹泻奇异变形杆菌毒力因子研究及应用

发布时间：2018-05-20 08:05

本文选题：奇异变形杆菌 + 毒力因子　；参考：《南方医科大学》2016年博士论文

【摘要】：奇异变形杆菌(Proteus mirabilis)是一种能引起泌尿系统感染的常见致病菌,但该菌已知的毒力基因与腹泻关系尚不明确,如何引起腹泻尚无定论。目前国外针对奇异变形杆菌的研究主要集中于引起泌尿系统感染的菌株中,对耐药研究较为多,但对奇异变形杆菌致腹泻的相关机制研究,Pubmed收录的论文很少。我们在部分食物中毒事件中,从病人腹泻标本和剩余食品中都分离到奇异变形杆菌,未分离到其他致病菌。同一批次分离株皆具有相同的脉冲场凝胶电泳(PFGE)型别,感染小鼠后能造成腹泻或死亡。而从健康人群与外环境分离的菌株不引起小鼠出现以上症状,其PFGE图谱间差异较大。由此提出假设,除了已知的毒力基因外,致腹泻的奇异变形杆菌的基因组中可能还存在着其它特异的毒力因子,是造成食物中毒的主要原因。围绕以上线索,本文主要开展以下几方面的研究：(1)致腹泻奇异变形杆菌病原学和分子生物学特征分析；(2)全基因组测序分析比较获得仅存在于致腹泻菌株中的特异基因；(3)在致腹泻菌株C02011中新发现的Ⅳ型分泌系统(T4SS)毒力岛编码基因virBl-11基本特性分析；(4)构建奇异变形杆菌C02011的virB9基因缺失株及回补株,鉴定其基本属性；(5)黏附和侵袭实验敏感细胞株的筛选,体外细胞模型的建立；(6)体外比较野生株C02011、virB9基因缺失株及回补株对人结肠癌Lovo细胞的黏附和侵袭水平；(7)腹泻动物模型的建立和初步实验；(8)明确virB9基因是否影响奇异变形杆菌对小鼠的致病能力。(9)特异基因的荧光PCR诊断方法的建立与菌株筛查。方法与结果：一、致腹泻奇异变形杆菌病原学、分子生物学特征1、选择6株奇异变形杆菌作为实验研究菌株,包括2株标准菌株：ATCC29906和HI4320;2株食物中毒分离株和2株健康人群和外环境拭子分离株。2、分析比较了不同来源的奇异变形杆菌的病原学特征和分子特征,不同来源的奇异变形杆菌生化特征基本相同,且均具有常见的毒力基因ureC、rsmA、 hpmA和zapA。说明这些基因没有特异性,不能区分致病菌株和非致病菌株。3、利用PFGE方法对奇异变形杆菌分子分型,同一起食物中毒分离株(病人标本和剩余食品分离株)的PFGE图谱一致,说明可能是由奇异变形杆菌引起的食物中毒。4、发现奇异变形杆菌中分别存在着1-3个不同大小的质粒。最大的质粒为10100bp左右,在所有菌株中普遍存在。而C05028和C02034中都存在着一个2683bp的小质粒,有四个开放读码框,其中645bp的qnrD基因为喹诺酮耐药基因,与耐药表型一致,可见该质粒与菌株的耐药性相关。5、环境分离菌株C02034携带的质粒最多(3个),其耐药性也最强,对喹诺酮类、磺胺类、氨基糖苷类和p-内酰胺类青霉素等均为耐药,由此推论其携带的另2个质粒也可能编码耐药基因,有待进一步验证。6、研究表明,凭借传统的生化分析及常见的毒力基因鉴定不能区分菌株的来源,无法确定是食物中毒菌株还是健康人群自身携带(或外环境中存在)的菌株；但PFGE和质粒分析可区分菌株来源。本研究还发现了奇异变形杆菌菌株中普遍存在的质粒,为奇异变形杆菌耐药及致病性研究提供了理论依据。二、奇异变形杆菌全基因组测序和序列比对1、为了进一步明确致腹泻菌株中的特异基因,收集了2株典型的致腹泻菌株(C05028和C02011),与1株健康人分离株(B02005)及1株外环境对照株(C02034)一起进行全基因组测序。2、通过序列比对分析,发现了仅存在于食物中毒株而非致病菌株中不存在的特异基因有7个,其功能预测结果显示这些基因有些编码溶血素,有些编码DNA核苷酸转移酶或糖基转移酶,有些是与真核细胞结合相关的转录调控原件,具体的功能有待进一步研究。这些基因为本研究首次发现,未见相关文献报道。3、在食物中毒分离株C02011中还发现了编码Ⅳ型分泌系统(T4SS)的特异基因,在奇异变形杆菌中目前尚无相关文献报道,是本课题组在食物中毒株中的首次发现。三、C02011中的Ⅳ型分泌系统(T4SS)基本特性分析针对C02011中的Ⅳ型分泌系统(T4SS)的11个基因virB1-virBll,通过同源比对、蛋白质功能域、三维结构预测、进化树、点阵图、基因结构预测、基因岛结构分析等,研究了整体的水平基因转移事件的进化关系。奇异变形杆菌C02011的T4SS主要的成分是：裂解糖基转移酶(VirB1),能量转移(VirB3-4, VirB11),融合通道(VirB2, VirB5-VirB10,脂蛋白),以及细胞黏附(VirB3-4)。结论：T4SS所有的11个基因在蛋白质和核苷酸水平上与肺炎克雷伯菌PMK1和大肠埃希菌ECOR31的T4SS基因高度相似,但与8株已测序的奇异变形杆菌菌株的核苷酸不存在同源。因此可以推断：该基因岛是近期从与其密切相关的肠杆菌科微生物大肠埃希菌和肺炎克雷伯菌中水平转移而来的。而这个事件至少发生在奇异变形杆菌C02011从其他奇异变形杆菌菌株分化之后。四、奇异变形杆菌C02011株virB9基因敲除及特性分析为了研究virB基因在奇异变形杆菌致病中的相关功能,选择编码分泌系统融合通道的virB9作为研究对象,构建virB9基因敲除株Pmi/AvirB9。利用基因同源重组的原理,通过自杀质粒PCVD442及中间宿主SM10λpir,成功构建了缺失virB9基因的突变株。并构建携带有virB9基因的Pmi/△virB9回补株,为下一步研究打下基础。在营养丰富条件下,C02011及Pmil△virB菌体的菌落形成和生长能力、生化反应并无显著差异。因而突变株适用于研究该基因对感染相关毒力的影响。五、黏附和侵袭实验敏感细胞株的筛选,体外细胞模型的建立选用人结肠癌细胞(Lovo)与人结肠腺癌细胞(Caco-2)进行体外研究,分别比对奇异变形杆菌C02011(食物中毒病人呕吐物分离株)和B02005(健康人粪便分离株)对细胞的侵袭性与黏附性,选择确定合适的细胞实验模型。结果显示,食物中毒病人呕吐物分离株C02011对Lovo、Caco-2细胞具有很强的黏附性,健康人粪便分离株B02005对Lovo、 Caco-2细胞黏附性弱,两菌株间的黏附率差异具有统计学意义(P0.01)。侵袭性实验也显示了类似的结果,两菌株对Lovo和Caco-2细胞的侵袭率差异具有统计学意义(P0.01)。本研究证实分离自食物中毒伴腹泻症状病人的呕吐物中的奇异变形杆菌C02011具有很强的黏附力和侵袭力,在引起人和动物肠道疾病中发挥重要的作用。人结肠癌细胞(Lovo)具有更高的敏感性,适宜于研究奇异变形杆菌对其的黏附和侵袭能力。六、virB9缺失降低了奇异变形杆菌C02011对Lovo细胞的黏附和侵袭能力体外模型采用Lovo单层细胞,与野生株C02011的黏附率相比,Pmil△virB9黏附Lovo单层细胞能力显著降低,二者黏附率差异具有统计学意义(P0.001)。庆大霉素保护实验结果表明,突变株在侵袭水平上也表现出明显的降低,二者侵袭率差异具有统计学意义(P0.001)。Pmil△virB9转化入回补质粒后,突变株黏附能力和侵袭能力基本得到恢复。七、腹泻动物模型的建立和初步实验为了证明部分奇异变形杆菌的确能够引起腹泻并具有不同的毒力,并建立合适的动物模型,分别采用6株不同来源的奇异变形杆菌感染小鼠,观察粪便性状及小鼠死亡情况。在经口灌胃实验中,所有菌株均未引起小鼠死亡,但食物中毒分离株引起小鼠粪便形状改变。腹腔注射实验中,7小时后,从腹泻病人呕吐物中分离的奇异变形杆菌作用的小鼠全部死亡,且小鼠的粪便稀软,小鼠的大肠切片染色结果也证实了病理改变。而从健康人群粪便及外环境分离的奇异变形杆菌作用的小鼠未死亡,且小鼠形态活力都正常。本研究成功建立了奇异变形杆菌的小鼠腹泻模型,为后续毒力基因的功能研究提供了较成熟的实验动物模型。本研究证明了奇异变形杆菌食物中毒分离株的毒力比健康人群及外环境分离株的毒力强,能引起小鼠腹泻甚至死亡,造成大肠的病理学改变。八、virB9缺失降低了奇异变形杆菌C02011对小鼠的致病能力为研究确认virB9基因的功能,在已建立好的小鼠动物模型上进行体内试验,分别采用C02011野生株、virB9基因敲除株及回补株感染小鼠,观察感染动物后的腹泻症状及病理改变,鉴别比较三者的致病能力差异。实验结果证明：C02011野生株和回补株的毒力比virB9基因敲除株的毒力强,能引起小鼠腹泻,导致肠组织含水量增多,与生理盐水对照株相比有统计学差异(t=2.7764,P=0.0193),并能造成大肠的病理学改变。virB9基因敲除株未能引起小鼠腹泻,肠组织含水量与生理盐水对照株无统计学差异(t=0.2979,P=0.7783)。说明virB9缺失降低了奇异变形杆菌C02011对小鼠的致病能力。但大肠的病理切片显示仍有一定的炎性反应,说明virB9基因不是该菌株的唯一的毒力基因,缺失virB9的敲除株仍具备一定的侵袭小鼠肠组织的毒力。有待进一步实验证实。九、致腹泻奇异变形杆菌荧光PCR检测方法的建立本研究在新发现的特异的毒力因子基础上,通过PCR筛查选取了4个在食物中毒菌株中普遍存在的毒力基因作为靶基因,采用相同标签辅助-无引物二聚体(Hand, Homo-Tag Assisted Non-Dimer)系统与改良分子信标荧光探针,建立1管同时检测奇异变形杆菌4种毒力基因的多重实时荧光PCR方法。所建立的针对致腹泻奇异变形杆菌的多重荧光PCR方法可广泛用于食物中毒或食品污染物的快速检测,区分正常人群携带株(外环境污染菌株)和致腹泻菌株,简化和完善现有的国标诊断方法,节省人力物力,满足食物中毒快速准确的诊断和传染来源追踪,减少误判和漏检,保障食品安全和人体健康。统计学分析：数据均表示为均数±标准差.两组间的比较采用t检验,多重比较采用单因素方差分析。动物实验阳性率的比较采用Pearson卡方检验。P0.05认为差异有统计学意义,检验水准为a=0.05。数据分析采用SPSS 13.0软件。结论：引起腹泻的奇异变形杆菌与健康人群及外环境分离的奇异变形杆菌经过全基因组测序和比对分析,发现了仅存在于致腹泻菌株中的一些特异的毒力基因,尤其是发现了存在于腹泻伴呕吐的食物中毒病人分离株C02011中存在着Ⅳ型分泌系统(T4SS)。该T4SS所有的11个基因(virB1-11)在蛋白质和核苷酸水平上与肺炎克雷伯菌PMK1和大肠埃希菌ECOR31的T4SS高度相似,但与8株已测序的奇异变形杆菌菌株的核苷酸不存在同源。因此推断：该基因岛是近期从与其密切相关的肠杆菌科微生物大肠埃希菌和肺炎克雷伯菌中水平转移而来的。而这个事件至少发生在奇异变形杆菌C02011从其他奇异变形杆菌菌株分化之后。食物中毒病人呕吐物分离株C02011对Lovo、Caco-2细胞均具有很强的黏附性和侵袭性,健康人粪便分离株B02005对Lovo、 Caco-2细胞黏附性和侵袭性弱,两菌株间的黏附率和侵袭率差异具有统计学意义(P0.01)。人结肠癌细胞(Lovo)具有更高的敏感性,适宜于研究奇异变形杆菌对其的黏附和侵袭能力。通过基因敲除构建的virB9基因缺失株对Lovo细胞的黏附和侵袭力下降,对小鼠的致病性降低,差异具有统计学意义(P0.01)。因此推论T4SS系统是奇异变形杆菌C02011的特异毒力因子,该毒力岛在与真核生物的黏附和侵袭作用中发挥重要作用,可能是导致人体产生腹泻和呕吐的决定性因子。构成T4SS系统的其他几个组成基因的功能将是我们后续研究中需要探索的问题。
[Abstract]:Proteus mirabilis is a common pathogenic bacterium that can cause urinary system infection. However, the relationship between the known virulence gene and diarrhea is not clear. The research on how to cause diarrhea is not conclusive. At present, the research on Proteus mirabilis mainly focuses on the strains causing urinary system infection, and the study of drug resistance is more important. Many, however, the research on the related mechanism of diarrhoea caused by Proteus mirabilis, few papers included in Pubmed. In some food poisoning events, we separated from the patient's diarrhea specimen and the remaining food to the Proteus mirabilis and the other pathogenic bacteria. The same batch isolates have the same pulse field gel electrophoresis (PFGE) type. It is suggested that there may be other specific virulence factors in the genome of Proteus mirabilis that caused diarrhoea in the genome of a healthy population and the external environment, which does not cause the above symptoms in mice, and thus suggests that there are other specific virulence factors in the genome of Proteus mirabilis, which causes diarrhea, which is the cause of food. The main reasons for the toxicosis. Around the above clues, this article mainly carried out the following research: (1) pathogenic and molecular biological characteristics analysis of Bacillus proteus diarrhoea; (2) complete genome sequencing analysis and comparison of the specific genes only existed in diarrhoea strains; (3) new type of genotypes found in diarrhoea strain C02011 Analysis of the basic characteristics of T4SS gene virBl-11 coding gene; (4) construction of virB9 gene deletion strain and remedial strain of Proteus mirabilis C02011, identification of its basic properties; (5) screening of sensitive cell lines of adhesion and invasion experiments, establishment of cell model in vitro; (6) comparison of wild strain C02011, virB9 gene deletion and recharge in vitro The adhesion and invasion level of the Lovo cells to human colon cancer cells; (7) the establishment and preliminary experiment of the animal model of diarrhea; (8) to determine whether the virB9 gene affects the pathogenic ability of the Proteus mirabilis to mice. (9) the establishment of the specific gene fluorescence PCR diagnosis method and the strain screening. Method and results: 1. Cause the pathogen of Bacillus proteus diarrhoea Study and molecular biological characteristics 1, 6 strains of Proteus mirabilis were selected as experimental strains, including 2 strains of standard strains, ATCC29906 and HI4320, 2 strains of food poisoning and 2 healthy populations and external environmental swab isolates,.2. The pathogenic and molecular characteristics of different sources were analyzed and compared. The biochemical characteristics of Bacillus proteus are basically the same, and they all have common virulence genes ureC, rsmA, hpmA and zapA. show that these genes are not specific and can not distinguish between the pathogenic bacteria and the non pathogenic strain.3. The PFGE method is used to classify the molecules of Proteus mirabilis and the PFG in the same food (the patient specimen and the remaining food isolate). The E map was consistent with the food poisoning.4 caused by Proteus mirabilis. It was found that there were 1-3 different sizes of plasmids in Proteus mirabilis. The largest plasmid was around 10100bp and was common in all strains. There was a small 2683bp plasmid in C05028 and C02034, with four open reading frames, of which 645 The qnrD gene of BP is the quinolone resistant gene, which is the same as that of the resistant phenotype. It can be seen that the plasmid is related to the resistance of the strain.5, and the environment isolate C02034 carries the most plasmids (3) and its resistance is the strongest. It is resistant to quinolones, sulfonamides, aminoglycosides and p- lactam penicillin, and thus deduces the other 2 substances carried by the strain. It is also possible to encode drug resistant genes and further verify.6. Studies have shown that traditional biochemical analysis and common virulence gene identification can not distinguish the source of the strain. It is impossible to determine the strain of food poisoning or the strain of the healthy population itself (or in the external environment); but PFGE and plasmid analysis can distinguish the source of the strain. The research also found the ubiquitous plasmids in Proteus mirabilis strain, which provided a theoretical basis for the study of resistance and pathogenicity of Proteus mirabilis. Two, the whole genome sequencing and sequence alignment of Proteus mirabilis were 1. In order to further clarify the specific genes of diarrhoea strains, 2 typical strains of diarrhoea (C05028 and C02011) were collected. The whole genome sequencing.2 was carried out with 1 healthy human isolates (B02005) and 1 external environmental control strains (C02034). Through sequence alignment analysis, 7 specific genes were found that existed only in food poisoning and non pathogenic strains. Their functional prediction results showed that some of these genes encode hemolysin and some encoded DNA nucleosides. Acid transferase or glycosyltransferase, some of which are transcriptional regulatory originals associated with eukaryotic cells, and specific functions need to be further studied. These genes were first discovered in this study and have not been reported in the relevant literature.3. The specific genes encoding the type IV secretory system (T4SS) in the food poisoning isolates C02011 are also found in the strange deformable rod. For the first time, there is no related literature in the bacteria. Three, three, the basic characteristics of type IV secretory system (T4SS) in C02011 are analyzed for the 11 gene virB1-virBll of type IV secretory system (T4SS) in C02011, through homologous comparison, egg white matter domain, three-dimensional structure prediction, evolutionary tree, lattice map, gene Structural prediction, gene island structure analysis, etc., studied the evolutionary relationships of the overall horizontal gene transfer events. The main components of T4SS of Proteus C02011 are: VirB1, VirB3-4, VirB11, VirB2, VirB5-VirB10, lipoprotein, and cell adhesion (VirB3-4). Conclusion: T4SS all The 11 genes are highly similar to the T4SS gene of Klebsiella pneumoniae PMK1 and Escherichia coli ECOR31 at protein and nucleotide levels, but have no homologous with the nucleotide of 8 strains of Proteus mirabilis. Therefore, it is inferred that this gene island is a recent Enterobacteriaceae microorganism Escherichia coli which is closely related to it. And the horizontal transfer in Klebsiella pneumoniae. This event occurred at least after the differentiation of Bacillus Proteus C02011 from other strains of Proteus mirabilis. Four, virB9 gene knockout and characterization of Proteus mirabilis C02011 strain in order to study the related functions of the virB gene in the pathogenicity of Proteus mirabilis, and select the coded secretory line VirB9 as the research object, the virB9 gene knockout strain Pmi/AvirB9. was constructed by using the principle of homologous recombination of gene and by suicide plasmid PCVD442 and the intermediate host SM10 lambda PIR, the mutant virB9 gene deletion mutant was constructed successfully, and the Pmi/ Delta virB9 recharge strain with virB9 gene was constructed to lay the foundation for the next step of the study. Under nutrient enrichment conditions, the colony formation and growth ability of C02011 and Pmil Delta virB have no significant difference in biochemical reaction. Therefore, the mutant strain is suitable for studying the effect of the gene on infection related virulence. Five, screening of sensitive cell lines in adhesion and invasion experiments, the establishment of human colon cancer cells (Lovo) and human colon gland in vitro cell model establishment Cancer cells (Caco-2) were studied in vitro to determine the cell invasiveness and adhesion of Proteus mirabilis C02011 (food intoxication patient) and B02005 (healthy human feces isolate). The results showed that the vomited isolate C02011 of the patients with food poisoning was used to Lovo, Caco-2 cells. The adhesion of healthy human fecal isolates B02005 to Lovo and Caco-2 cells was weak, and the adhesion rate difference between two strains was statistically significant (P0.01). The invasive experiment also showed similar results, and the difference of invasion rate between two strains and Lovo and Caco-2 cells was statistically significant (P0.01). This study confirmed that it was isolated from the food. C02011 has strong adhesion and invasiveness in the vomit of patients with diarrhea and diarrhea. It plays an important role in human and animal intestinal diseases. Human colon cancer cell (Lovo) has higher sensitivity. It is suitable for the study of the adhesion and invasion ability of Proteus mirabilis. Six, the loss of virB9 is reduced. The adhesion and invasion ability of Bacillus Proteus C02011 to Lovo cells in vitro model used Lovo monolayer cells. Compared with the adhesion rate of wild strain C02011, the ability of Pmil Delta virB9 to adhere to Lovo monolayer was significantly decreased, and the difference of adhesion rate between the two groups was statistically significant (P0.001). It also showed a significant decrease. The difference of invasion rate between the two was statistically significant (P0.001).Pmil Delta virB9 was converted into the supplementation plasmid, and the adhesion and invasiveness of the mutant strain were basically recovered. Seven. The establishment and preliminary experiment of the animal model of diarrhoea in order to prove that some proteus mirabilis can cause diarrhea and have different effects. The virulence and the establishment of a suitable animal model were established to infect mice with 6 different sources of Proteus mirabilis, respectively, to observe the fecal traits and the death of mice. In the oral gavage experiment, all the strains did not cause the death of the mice, but the food poisoning isolated strain caused the shape change of the feces in the mice. In the intraperitoneal injection experiment, after 7 hours, diarrhea from diarrhea. All the mice that were isolated from the vomit of the patient's vomit were all dead, and the mice's feces were thin and soft. The coloring results of the mice were also confirmed by the pathological changes. The mice that were isolated from the feces and external environment of the healthy population did not die, and the morphologic activity of the mice was normal. The study was successfully established. The mice diarrhea model of Proteus mirabilis has provided a mature experimental animal model for the follow-up of the function of the subsequent virulence genes. This study proved that the virulence of the isolated strains of Proteus mirabilis food poisoning is stronger than that of the healthy and external environmental isolates, which can cause abdominal diarrhea and even death in mice, and cause the pathological changes of the large intestine. Eight VirB9 deletion reduced the pathogenicity of Proteus mirabilis C02011 to mice and confirmed the function of virB9 gene. In the established mice animal model, the body test was carried out in vivo, with C02011 wild strain, virB9 gene knockout strain and remedial strain infected mice respectively. The diarrhea symptoms and pathological changes of the infected animals were observed and the identification ratio was compared. The experimental results showed that the virulence of the C02011 wild strain and the remedial plant was stronger than that of the virB9 gene knockout strain, causing diarrhea in mice and increasing the water content of the intestinal tissue, compared with the saline control strain (t=2.7764, P=0.0193), and could cause the pathological changes of the large intestine to change the.VirB9 gene knockout. The strain did not cause diarrhea in mice, and there was no statistical difference between the water content of intestinal tissue and normal saline (t=0.2979, P=0.7783). It indicated that the deletion of virB9 reduced the pathogenicity of Proteus mirabilis C02011 to mice. But the pathological section of the large intestine showed that there was still a certain inflammatory response, and that the virB9 gene was not the only virulence gene of the strain. The virB9 knockout strains still have a certain toxicity to the intestinal tissue of mice. Nine, the establishment of a fluorescence PCR detection method for Bacillus proteus diarrhoea, based on the newly discovered specific virulence factors, 4 virulence genes commonly found in the food poisoning strains were selected by PCR screening. For the target gene, the multiplex real-time fluorescent PCR method for simultaneous detection of 4 virulence genes of Proteus mirabilis with the same label assisted Hand (Hand, Homo-Tag Assisted Non-Dimer) system and the modified molecular beacon fluorescent probe was established. The multiplex fluorescence PCR method for the Bacillus proteus caused by diarrhoea was widely used. The rapid detection of food poisoning or food contaminants can be used to distinguish normal population and diarrhea strains, to simplify and improve the existing national standard diagnosis methods, to save manpower and material resources, to meet the rapid and accurate diagnosis of food poisoning and to trace the source of infection, to reduce misjudgement and leakage, and to ensure food safety and human health. Statistical analysis: the data were all expressed as mean + standard deviation. T test was used for comparison between the two groups, and single factor variance was used for multiple comparisons.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378

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