弗劳氏枸橼酸杆菌分子分型和毒力岛分析

发布时间：2018-04-27 00:26

本文选题：弗劳氏枸橼酸杆菌 + 腹泻　；参考：《中国疾病预防控制中心》2009年博士论文

【摘要】： 本文探索了弗劳氏枸橼酸杆菌作为肠道病原菌的可能性。根据我们实验室完成的弗劳氏枸橼酸杆菌CF72菌株的基因组序列和多位点序列分析(MLST)基因选择的原则,我们选择了7个基因,建立了弗劳氏枸橼酸杆菌的MLST方法。根据该方法,可把36株菌分成12个序列型(ST型)。使用获得的7个管家基因序列的种系发生关系分析发现,弗劳氏枸橼酸杆菌可分为A、B两大类,A类仅包括ST9,含1株菌;B类包括其余的11个ST型。B类又可以聚为两类。B1类包括2个ST型2株菌:ST8、ST12。B2类包括9个ST型33株菌。其中ST1、ST3、ST4亲缘关系比较近,ST2、ST5亲缘关系比较近。这5个型别包括29株菌,属于优势ST型。从菌株的来源来看,有7个ST型仅包括人源菌株,1个ST型仅包括动物源菌株,4个ST型,如ST1、ST2、ST3、ST5,包括动物源和人源菌株。根据我们实验室完成的基因组分析,我们发现弗劳氏枸橼酸杆菌CF72菌株有49个基因组岛。根据功能分为10类:其中代谢岛17个,转运岛7个,噬菌体相关岛7个,DNA/RNA组装岛5个,细胞结构岛3个,DNA重组岛1个,功能不明的1个,其他1个,耐药性岛1个,毒力岛6个。在检测的36株菌中,只有6株菌所有检测的49个基因岛全部阳性,都属于ST2型。噬菌体转导可能是病原菌致病性进化的重要机理之一。ST1、ST2、ST3、ST4序列型的弗劳氏枸橼酸杆菌的噬菌体相关岛的分布,也和其他ST型有明显差异。GI22噬菌体相关岛可能和生物膜形成有关,在36株菌株中19株检测阳性,包括ST1(7/13株)、ST2(7/7株)、ST3(3/4株)、ST4(1/3株)、ST8(1/1株)。在36株菌中,有6株菌的GI13、GI29、GI30检测为阳性,这6株菌属于ST2型。GI36有7株菌检测阳性,包括ST2型6株菌和ST1型1株菌。噬菌体岛的分布提示,ST1、ST2、ST3、ST4、ST8的噬菌体转导比较活跃,其中ST1、ST3、ST4等以GI22为主,ST2也以其他几个噬菌体岛为主。噬菌体岛的分布具有ST型分布特征,主要分布在上述可能和致病性相关的ST型中。在6个毒力岛中,在36株菌中都能检测到的毒力岛有3个:包括编码铁转运系统的GI5、编码Tol-Pal内膜蛋白GI7、编码Curli菌毛蛋白GI9。编码侵袭相关菌毛蛋白GI6岛,可以在35株菌中都检测到。唯一没有完整检测到GI6毒力岛的菌株是CF5,属于ST9。编码O抗原的毒力岛GI24,在29株菌能够检测到。具有GI24岛的菌株分别是为ST1、ST2、ST3、ST4、ST5。这些ST型均属于优势ST型。编码亚碲酸钾抗性GI44岛,能够在8株菌中检测到,其中有6株菌属于ST2,其余两株分别属于ST6和ST7。 Hela细胞粘附实验发现,36株菌中有34株具有甘露糖抗性粘附,其中粘附力强的菌株3株,分别属于ST1、ST3、ST12;中等粘附18株,16株属于ST1、ST2、ST3、ST5。结果提示,以ST9为代表的A类可能是致病性最弱的,它不具有粘附力,毒力岛(GI6、GI24、GI44)检测也为阴性。Hela细胞细胞毒性试验发现,在36株菌中,CF74在作用10小时,具有较强的细胞毒性作用。除了细胞毒性作用外,CF74菌株的粘附力也比较强,属于ST12型。简言之,我们发展了一种弗劳氏枸橼酸杆菌的MLST分析方法。使用这种方法,可将36株菌分为12个ST型。其中ST1、ST2、ST3、ST4、ST5属于优势型。这些ST型弗劳氏枸橼酸杆菌在基因组岛分布、毒力岛分布、噬菌体岛分布等方面,和其他ST型菌株相比,有明显差异。病原性细菌的一个最基本的特征就是入侵宿主,并在宿主体内找到适合自己生存的小环境。经典的EPEC(Enteropathogenic Escherichia coli肠致病性大肠杆菌)和EHEC(Enterohemorrhagic Escherichia coli肠出血性大肠杆菌)入侵宿主的第一步是在感染粘膜细胞表面形成一种经典的病理损伤过程,称为粘附抹平效应(Attaching and Effacing lesions A/E)。该损伤在其致病过程中起了关键作用。其特点是:细菌紧密粘附粘膜表面,感染细胞微绒毛的丢失,细菌粘附处形成一个致密的肌动蛋白垫。已有的研究的发现:该效应是由LEE(Locus of enterocyte effacement)毒力岛所编码的三型分泌系统(TypeⅢsecretion systerm TTSS)产生的。尽管EPEC和EHEC都具有LEE系统,但是它们的作用机制却有很大的差别,分别是通过Nck或TccP(Tir cytoskeleton coupling protein)蛋白两种不同的途径来激活细胞N-WASP蛋白,从而引起细胞肌动蛋白的聚集。因此,多年来,在体外细胞培养感染模型中,使用荧光染料标记细胞肌动蛋白聚集(Fluorescent actin stainingFAS),一直作为研究A/E损伤的模型和判断EPEC和EHEC菌株是否具有致病力的标准。在本研究中,我们发现了一大类具有代表性的EPEC O125:H6菌株,这类菌株既不能利用Nck途径也不能利用TccP途径,根据前述的研究结论,这类菌株将不能引起肌动蛋白的聚集,因此在活体外器官组织培养感染模型中也不能引起A/E损伤。但本研究发现:该类菌株在体外细胞培养感染模型中能引起微弱的肌动蛋白的聚集,而且,在体外人肠器官组织培养感染模型中,EPEC O125:H6还能够造成经典的A/E损伤。是否存在其他途径引起A/E损伤还是A/E损伤不需要肌动蛋白的聚集?进一步的研究发现:EPEC O125:H6表达外源TccP后,可以在体外的细胞模型上引起有效的肌动蛋白聚集,在体外人肠器官组织感染模型中粘附能力也明显增强,在细菌粘附处还能够检测到N-WASP蛋白。上述研究结果显示:在自然界里存在着一类不依赖于Nck或TccP途径来粘附肠道上皮产生A/E损伤的大肠杆;在体外细胞培养感染模型上引起肌动蛋白聚集现象,不应看作是EPEC和EHEC毒力的标志。
[Abstract]:In this paper, we explored the possibility of citrate citrate as intestinal pathogenic bacteria. According to the principle of genomic sequence and multiple point sequence analysis (MLST) gene selection, we have selected 7 genes and established the MLST method of citric acid bacilli in our laboratory. According to this method, the MLST method of citric acid bacilli was established. 36 strains of bacteria were divided into 12 sequence type (ST type). Using the phylogenetic relationship of the 7 housekeeper gene sequences obtained, it was found that citric acid bacilli could be divided into A, B two, a only including ST9, and 1 strains of bacteria; the B class including the other 11 ST type.B classes can also be clustered into two classes of.B1 including 2 ST type 2 strains: ST8, ST12.B2 category includes 9 ST type 33. The relationship between ST1, ST3 and ST4 is close, and the relationship of ST2 and ST5 is close. These 5 types include 29 strains of bacteria and belong to the dominant ST type. From the source of the strain, 7 ST types include only human strains, and 1 ST types only include animal source strains, and 4 ST types, such as ST1, ST2, ST3, and humans, including animal and human strains.
According to genome analysis completed in our laboratory, we found that the CF72 strain of citric acid bacilli has 49 genome islands. According to its function, there are 10 categories: 17 metabolic islands, 7 transshipment islands, 7 phage related islands, 5 assembly islands, 3 cell structure islands, 1 DNA recombinant islands, 1 unidentified functions, 1 other, drug resistance island 1. There are 6 virulence islands. Among the 36 strains tested, only 6 strains of all 49 gene islands are all positive, all of which belong to type ST2. Phage transduction may be one of the important mechanisms of pathogenic evolution of pathogenic bacteria,.ST1, ST2, ST3, and ST4 sequence type of phage related islands of citric acid bacilli, which are also significantly different from those of other ST types,.G I22 phage related islands may be related to biofilm formation, and 19 of the 36 strains are positive, including ST1 (7/13 strain), ST2 (7/7 strain), ST3 (3/4 strain), ST4 (1/3 strain), ST8 (1/1 strain). In 36 strains, there are 6 strains of GI13, which belong to 7 bacteria detection positive, including 6 bacteria and 1 bacteria. Phage island distribution suggests that phage transduction of ST1, ST2, ST3, ST4, ST8 is more active, in which ST1, ST3, ST4, etc. are dominated by GI22, and ST2 is dominated by several other phage islands. The distribution of phage islands has a ST distribution feature, mainly in the ST type that may be associated with pathogenicity. In 6 virulence islands, 36 strains can be detected. There are 3 tested virulence islands, including the GI5 of the encoded iron transport system, the encoding of the Tol-Pal intima protein GI7, and the encoding of the Curli pili protein GI9. encoding the associated pili protein GI6 Island, which can be detected in the 35 strains. The only strain that does not fully detect the GI6 virulence Island is CF5, the virulence island of ST9. encoded O antigen is GI24, and the 29 strains can be found. The strains with GI24 Island were ST1, ST2, ST3, ST4, and ST5., all of which belonged to the dominant ST. The potassium tellurite resistant GI44 island was encoded in 8 strains, 6 of which belonged to ST2 and the other two belonged to ST6 and ST7..
Hela cell adhesion experiment found that 34 of the 36 strains had mannose resistance adhesion, of which 3 strains with strong adhesion were ST1, ST3, ST12, 18 of medium adhesion and 16 of ST1, ST2, ST3, ST5., indicating that a class of ST9, represented by ST9, might be the weakest, it did not have adhesion, and the test of virulence Island (GI6, GI24, GI44) was also The cytotoxicity test of negative.Hela cells showed that in 36 strains of bacteria, CF74 had a strong cytotoxic effect for 10 hours. In addition to cytotoxicity, the adhesion force of CF74 strain was also strong, which belonged to ST12 type.
In short, we developed a MLST analysis method for citrate virulence. Using this method, 36 strains of bacteria can be divided into 12 ST types. Among them, ST1, ST2, ST3, ST4, ST5 are dominant. The distribution of the genome Island, the distribution of virulence Island, the distribution of phage Island, the distribution of virulence Island, and the distribution of phage islands are compared with other ST type strains. Distinct differences.
One of the most basic characteristics of pathogenic bacteria is to invade the host and find a small environment for its own survival in the host. The first step of the classic EPEC (Enteropathogenic Escherichia coli enteropathogenic Escherichia coli) and EHEC (Enterohemorrhagic Escherichia coli enterohaemorrhagic Enterobacter) is to infect the mucous membrane The cell surface forms a classic pathological process, called Attaching and Effacing lesions A/E, which plays a key role in its pathogenesis. The characteristics are that bacteria adhere to the surface of the mucous membrane closely, infect the cell microvilli and form a compact actin pad at the bacterial adhesion.
The discovery that the effect is produced by the type three secretory system (Type III secretion systerm TTSS) encoded by LEE (Locus of enterocyte effacement) virulence island. Although EPEC and EHEC have LEE systems, their mechanisms are very different. ) protein is activated by two different ways to activate cell N-WASP protein and thus cause actin aggregation. Therefore, for many years, in an in vitro cell culture infection model, a fluorescent dye is used to mark actin aggregation (Fluorescent actin stainingFAS), which has been used as a model to study A/E damage and to determine whether EPEC and EHEC strains are isolated. A standard of pathogenicity.
In this study, we found a large class of representative EPEC O125:H6 strains, which neither use Nck nor TccP pathway. According to the previous research, these strains will not cause actin aggregation. Therefore, it can not cause A/E damage in the infection model of living external organ tissue culture. The study found that the strain can cause weak actin aggregation in the cell culture infection model in vitro, and the EPEC O125:H6 can also cause the classic A/E damage in the human intestinal tissue culture infection model in vitro. Whether there are other pathways that cause A/E damage and the A/E damage does not require actin aggregation? The study found that EPEC O125:H6 can induce effective actin aggregation on the cell model in vitro, and the adhesion ability of the human intestinal organ infection model in vitro is obviously enhanced, and the N-WASP protein can be detected at the bacterial adhesion.
The above results show that there is a class of coliform rods that do not rely on Nck or TccP to adhere to the intestinal epithelium to produce A/E damage, and that actin aggregation in an in vitro cell culture infection model should not be regarded as a sign of EPEC and EHEC toxicity.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378

【参考文献】