本文選題：弗勞氏枸櫞酸桿菌 + 腹瀉�。� 參考：《中國疾病預(yù)防控制中心》2009年博士論文

【摘要】： 本文探索了弗勞氏枸櫞酸桿菌作為腸道病原菌的可能性。根據(jù)我們實(shí)驗(yàn)室完成的弗勞氏枸櫞酸桿菌CF72菌株的基因組序列和多位點(diǎn)序列分析(MLST)基因選擇的原則,我們選擇了7個(gè)基因,建立了弗勞氏枸櫞酸桿菌的MLST方法。根據(jù)該方法,可把36株菌分成12個(gè)序列型(ST型)。使用獲得的7個(gè)管家基因序列的種系發(fā)生關(guān)系分析發(fā)現(xiàn),弗勞氏枸櫞酸桿菌可分為A、B兩大類,A類僅包括ST9,含1株菌;B類包括其余的11個(gè)ST型。B類又可以聚為兩類。B1類包括2個(gè)ST型2株菌:ST8、ST12。B2類包括9個(gè)ST型33株菌。其中ST1、ST3、ST4親緣關(guān)系比較近,ST2、ST5親緣關(guān)系比較近。這5個(gè)型別包括29株菌,屬于優(yōu)勢(shì)ST型。從菌株的來源來看,有7個(gè)ST型僅包括人源菌株,1個(gè)ST型僅包括動(dòng)物源菌株,4個(gè)ST型,如ST1、ST2、ST3、ST5,包括動(dòng)物源和人源菌株。 根據(jù)我們實(shí)驗(yàn)室完成的基因組分析,我們發(fā)現(xiàn)弗勞氏枸櫞酸桿菌CF72菌株有49個(gè)基因組島。根據(jù)功能分為10類:其中代謝島17個(gè),轉(zhuǎn)運(yùn)島7個(gè),噬菌體相關(guān)島7個(gè),DNA/RNA組裝島5個(gè),細(xì)胞結(jié)構(gòu)島3個(gè),DNA重組島1個(gè),功能不明的1個(gè),其他1個(gè),耐藥性島1個(gè),毒力島6個(gè)。在檢測(cè)的36株菌中,只有6株菌所有檢測(cè)的49個(gè)基因島全部陽性,都屬于ST2型。噬菌體轉(zhuǎn)導(dǎo)可能是病原菌致病性進(jìn)化的重要機(jī)理之一。ST1、ST2、ST3、ST4序列型的弗勞氏枸櫞酸桿菌的噬菌體相關(guān)島的分布,也和其他ST型有明顯差異。GI22噬菌體相關(guān)島可能和生物膜形成有關(guān),在36株菌株中19株檢測(cè)陽性,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檢測(cè)為陽性,這6株菌屬于ST2型。GI36有7株菌檢測(cè)陽性,包括ST2型6株菌和ST1型1株菌。噬菌體島的分布提示,ST1、ST2、ST3、ST4、ST8的噬菌體轉(zhuǎn)導(dǎo)比較活躍,其中ST1、ST3、ST4等以GI22為主,ST2也以其他幾個(gè)噬菌體島為主。噬菌體島的分布具有ST型分布特征,主要分布在上述可能和致病性相關(guān)的ST型中。在6個(gè)毒力島中,在36株菌中都能檢測(cè)到的毒力島有3個(gè):包括編碼鐵轉(zhuǎn)運(yùn)系統(tǒng)的GI5、編碼Tol-Pal內(nèi)膜蛋白GI7、編碼Curli菌毛蛋白GI9。編碼侵襲相關(guān)菌毛蛋白GI6島,可以在35株菌中都檢測(cè)到。唯一沒有完整檢測(cè)到GI6毒力島的菌株是CF5,屬于ST9。編碼O抗原的毒力島GI24,在29株菌能夠檢測(cè)到。具有GI24島的菌株分別是為ST1、ST2、ST3、ST4、ST5。這些ST型均屬于優(yōu)勢(shì)ST型。編碼亞碲酸鉀抗性GI44島,能夠在8株菌中檢測(cè)到,其中有6株菌屬于ST2,其余兩株分別屬于ST6和ST7。 Hela細(xì)胞粘附實(shí)驗(yàn)發(fā)現(xiàn),36株菌中有34株具有甘露糖抗性粘附,其中粘附力強(qiáng)的菌株3株,分別屬于ST1、ST3、ST12;中等粘附18株,16株屬于ST1、ST2、ST3、ST5。結(jié)果提示,以ST9為代表的A類可能是致病性最弱的,它不具有粘附力,毒力島(GI6、GI24、GI44)檢測(cè)也為陰性。Hela細(xì)胞細(xì)胞毒性試驗(yàn)發(fā)現(xiàn),在36株菌中,CF74在作用10小時(shí),具有較強(qiáng)的細(xì)胞毒性作用。除了細(xì)胞毒性作用外,CF74菌株的粘附力也比較強(qiáng),屬于ST12型。 簡言之,我們發(fā)展了一種弗勞氏枸櫞酸桿菌的MLST分析方法。使用這種方法,可將36株菌分為12個(gè)ST型。其中ST1、ST2、ST3、ST4、ST5屬于優(yōu)勢(shì)型。這些ST型弗勞氏枸櫞酸桿菌在基因組島分布、毒力島分布、噬菌體島分布等方面,和其他ST型菌株相比,有明顯差異。 病原性細(xì)菌的一個(gè)最基本的特征就是入侵宿主,并在宿主體內(nèi)找到適合自己生存的小環(huán)境。經(jīng)典的EPEC(Enteropathogenic Escherichia coli腸致病性大腸桿菌)和EHEC(Enterohemorrhagic Escherichia coli腸出血性大腸桿菌)入侵宿主的第一步是在感染粘膜細(xì)胞表面形成一種經(jīng)典的病理損傷過程,稱為粘附抹平效應(yīng)(Attaching and Effacing lesions A/E)。該損傷在其致病過程中起了關(guān)鍵作用。其特點(diǎn)是:細(xì)菌緊密粘附粘膜表面,感染細(xì)胞微絨毛的丟失,細(xì)菌粘附處形成一個(gè)致密的肌動(dòng)蛋白墊。 已有的研究的發(fā)現(xiàn):該效應(yīng)是由LEE(Locus of enterocyte effacement)毒力島所編碼的三型分泌系統(tǒng)(TypeⅢsecretion systerm TTSS)產(chǎn)生的。盡管EPEC和EHEC都具有LEE系統(tǒng),但是它們的作用機(jī)制卻有很大的差別,分別是通過Nck或TccP(Tir cytoskeleton coupling protein)蛋白兩種不同的途徑來激活細(xì)胞N-WASP蛋白,從而引起細(xì)胞肌動(dòng)蛋白的聚集。因此,多年來,在體外細(xì)胞培養(yǎng)感染模型中,使用熒光染料標(biāo)記細(xì)胞肌動(dòng)蛋白聚集(Fluorescent actin stainingFAS),一直作為研究A/E損傷的模型和判斷EPEC和EHEC菌株是否具有致病力的標(biāo)準(zhǔn)。 在本研究中,我們發(fā)現(xiàn)了一大類具有代表性的EPEC O125:H6菌株,這類菌株既不能利用Nck途徑也不能利用TccP途徑,根據(jù)前述的研究結(jié)論,這類菌株將不能引起肌動(dòng)蛋白的聚集,因此在活體外器官組織培養(yǎng)感染模型中也不能引起A/E損傷。但本研究發(fā)現(xiàn):該類菌株在體外細(xì)胞培養(yǎng)感染模型中能引起微弱的肌動(dòng)蛋白的聚集,而且,在體外人腸器官組織培養(yǎng)感染模型中,EPEC O125:H6還能夠造成經(jīng)典的A/E損傷。是否存在其他途徑引起A/E損傷還是A/E損傷不需要肌動(dòng)蛋白的聚集?進(jìn)一步的研究發(fā)現(xiàn):EPEC O125:H6表達(dá)外源TccP后,可以在體外的細(xì)胞模型上引起有效的肌動(dòng)蛋白聚集,在體外人腸器官組織感染模型中粘附能力也明顯增強(qiáng),在細(xì)菌粘附處還能夠檢測(cè)到N-WASP蛋白。 上述研究結(jié)果顯示:在自然界里存在著一類不依賴于Nck或TccP途徑來粘附腸道上皮產(chǎn)生A/E損傷的大腸桿;在體外細(xì)胞培養(yǎng)感染模型上引起肌動(dòng)蛋白聚集現(xiàn)象,不應(yīng)看作是EPEC和EHEC毒力的標(biāo)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.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R378

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