【作者】 罗霞；

【导师】 徐建国；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2013， 博士

【摘要】 志贺菌（Shigella）是细菌性痢疾的主要病原菌,它通过毒性大质粒上的Ⅲ型分泌系统（type Ⅲ secretion system, T3SS）将分泌的效应蛋白分子注入到宿主细胞内,从而导致细菌对人体肠道的侵袭并引起强烈的炎症反应,引起腹泻、腹痛、发热等症状,严重的可导致死亡。志贺菌属包含痢疾志贺菌（Shigella dysenteriae, A群）、福氏志贺菌（Shigella flexneri, B群）、鲍氏志贺菌（Shigella boydii, C群）和宋内志贺菌（Shigella sonnei, D群）四个血清群,其中宋内志贺菌是发达国家的优势血清群,福氏志贺菌是发展中国家的主要致病血清群。根据O-抗原结构的差异,福氏志贺菌又可以分为众多血清型,目前报道的福氏志贺菌血清型至少有19种,包括1a,1b,1c,1d,2a,2b,3a,3b,4a,4av,4b,5a,5b, Y, Yv, X, Xv,6和7b。除血清型6外,其它血清型O-抗原具有共同的结构：一个N-乙酰葡萄糖胺（N-acetylglucosamine, GlcNAc）和三个鼠李糖（Rhamnose, Rha）构成的四糖骨架重复单元,即Y血清型结构。四糖骨架中特定糖的糖基化或/和乙酰化或/和磷酸乙醇胺化修饰决定了福氏志贺菌的型和群特异性抗原决定簇,从而形成了福氏志贺菌血清型的多样性。福氏志贺菌O-抗原的糖基化和乙酰化修饰是由血清型转换噬菌体所介导的,而磷酸乙醇胺化修饰是由质粒介导的。目前已经发现的福氏志贺菌血清型转换噬菌体有SfI,SfIC, SfII, Sf6, SfIV, SfV和SfX,除噬菌体Sf6外,其他噬菌体均携带由3个基因（gtrA,gtrB和gtr）组成的基因簇,gtr基因编码特异性葡萄糖基转移酶（Glucosyltransferase,Gtr）,介导葡萄糖基以不同的连接方式添加到O-抗原四糖重复单位的不同糖基上导致血清型的转换。福氏志贺菌在不同地区、不同时间具有优势血清型的变化,如2a血清型福氏志贺菌长期以来是包括中国在内亚洲国家的优势血清型。近年来一些新的血清型菌株出现并在一些地区造成流行,如Xv血清型菌株在中国河南2002～2006年取代2a血清型菌株成为优势血清型。我们实验室研究证实了Xv血清型是由X血清型经磷酸乙醇胺化修饰转换而来的,并分离到血清型转换噬菌体SfX:1c血清型在80年代末的孟加拉出现并在一段时期成为越南及埃及的主要流行血清型,有研究提示1c血清型菌株是由1a血清型菌株感染噬菌体SflC而来。不同血清型福氏志贺菌流行差异及流行血清型变迁的原因目前仍不清楚,除血清型转换这一作用外,血清型转换噬菌体介导的O-抗原变化是否也会导致细菌毒力的改变有待进一步研究。本研究对一株Y血清型的福氏志贺菌036进行了血清型转换研究：将036感染血清型转换噬菌体SfX,可将福氏志贺菌Y血清型转化为X血清型（菌株036X）,将新转换的福氏志贺菌Y血清型菌株036X感染血清型转换噬菌体SfI,得到一种新的血清型菌株,与Ⅰ型特异性抗血清和群7,8特异性抗血清发生凝集,我们将它命名为福氏志贺菌1d血清型（菌株-036₁d）。随后我们在中国监测的福氏志贺菌株中发现5株1d血清型菌株。进一步研究发现,受体菌株获得血清型转换噬菌体,O-抗原被修饰后,可影响细菌的毒力。细菌对HeLa细胞的侵袭方面,获得血清型转换噬菌体SfX的036_X及获得血清型转换噬菌体SfX和SfI的036₁d的侵袭能力均高于Y血清型菌株036,且036₁d与036的差异有统计学意义；豚鼠角膜实验也发现0361d所致豚鼠角膜结膜炎症程度高于036,提示O-抗原的糖基化可以影响细菌的侵袭力。用乳酸脱氢酶释放量来检测细菌对巨噬细胞的毒性发现,036₁d对小鼠巨噬细胞Raw264.7的细胞毒性明显高于036,且差异有统计学意义。Raw264.7细胞和036₁d菌株相互作用后,培养液上清的IL-1p及IL-6的释放量也稍高于036菌株。对上述3株菌全菌蛋白质组的比较分析研究发现,036₁d菌株有一个表达明显上升的蛋白Sap,该蛋白在036及036X菌株中没有表达。使用QRT-PCR分析发现,036₁d菌株的sap基因的表达水平比036及036_X菌株显著提高。据此推测sap基因具有重要功能。我们继而构建了携带sap基因表达载体的036菌株-036⑥-1菌株,研究Sap蛋白表达对细菌毒力的影响。结果发现036⑥-1菌株的sap基因的转录水平显著高于036、036_X及036₁d菌株。036⑥-1对上皮细胞的侵袭力及对巨噬细胞的毒性均高于036,但仍明显低于036₁d。志贺菌Sap蛋白与大肠杆菌Antigen43（Ag43）蛋白具有96%的高度同源性。大肠杆菌Ag43蛋白在大肠杆菌中具有介导自我聚集与促进生物膜形成的作用,通过介导细胞的聚集能起到抵抗过氧化氢物质杀死的作用。关于志贺氏菌Sap蛋白的作用有待于进一步的实验研究。本研究发现血清型转换噬菌体介导的O-抗原糖基化修饰菌株对上皮细胞的侵袭性及巨噬细胞毒性均高于O-抗原未糖基化修饰菌株,提示O-抗原糖基化修饰可以促进福氏志贺菌的毒力。更多还原

【Abstract】 Shigella is the major pathogen causing shigellosis or bacillary dysentery in developing countries. It is well known that Shigella injects effector proteins into human cells through a type Ⅲ secretion system （T3SS）, resulting in bacterial invasion and a vigorous inflammatory response. The symptoms of shigellosis includes watery diarrhea, strong abdominal cramps, fever, even death. The genus Shigella includes four species named S. dysenteriae （serogroup A）, S. flexneri （serogroup B）,S. boydii （serogroup C）, and S. sonnei （serogroup D）. Among the four Shigella species, S. sonnei is the most common serogroup found in industrialized countries and S. flexneri is the predominant species in developing countries.S. flexneri serotyping are based on structure of the O-antigen lipopolysaccharide. There are19known serotypes up to now, including1a,1b,1c,1d,2a,2b,3a,3b,4a,4av,4b,5a,5b,6, X, Xv, Yv,7b and Y. Except for serotype6, all share a common tetrasaccharide backbone of repeating units of N-acetylglucosamine-rhamnose-rhamn-ose-rhamnose. The basic O-antigen is referred to as serotype Y and the addition of glucosyl and/or O-acetyl groups and/or a phosphoethanolamine （PEtN） group to different sugars of the tetrasaccharide unit results in the presence of type-and group-specific antigenic determinants. S. flexneri O-antigen glucosylation and O-acetylation are mediated by temperate bacteriophages. The addition of phosphoethanolamine to the O-antigen is medicated by a plasmid carried gene, Ipt-O （LPS phosphoethanolamine transferase for O-antigen）. Seven different serotype-converting phages or prophages, SfI, SfIC, SfII, Sf6, SfIV, SfV and SfX, have been identified and characterized. Except for Sf6which carries a single gene oac for O-antigen acetylation, the other phages carry O-antigen glucosylation locus （gtrA, gtrB and gtrtype） responding for the addition of glucosyl molecules to sugar residue（s） on the basic O-antigen repeating unit. Some S. flexneri serotypes are more prevalent than others, with serotype2a being the most predominant serotyps in China and other Asian countries. Recently, novel and untypical serotypes had been reported in different regions of world. Serotype Xv, which was firstly emerged in Henan province of China in2001, had become the most prevalent serotype in Henan and other provinces of China during2002-2006. A PEtN group was found attached to the rhamnose of O-antigen giving rise to the MASF IV-1positive phenotype in Xv serotype and other serotypes. Phage SfX, which responding for the presence of7;8antigen, was successfully induced from serotype Xv strain2002017. Serotype lc was firstly identified in Bangladesh in the late1980s and has been the predominant serotype in Vietnam and rural Egypt in many years. Serotype1c was originated from serotype1a by acquiring a phage named SfIC, which carrying a gtrlC gene cluster mediating the glucosylation modification. Apart from serotype conversion, little is known on the contribution of serotype-converting phages to the virulence of S. flexneri.In this study, we constructed a novel serotype1d by sequentially infecting serotype Y strain036with serotype-converting phages SfX and SfI. The novel serotype1d strain agglutinated with both of the serotype1a-specific typing sera I and serotype X-specific grouping sera7;8, and differed from subserotypes1a,1b and1c.5strains with serotype1d serological feature were also identified from1650S. flexneri strains collected in Chian during a surveillance program performed by China CDC.The virulence of strains036,036_X, and036₁d were further analized. Results indicated that O-antigen glucosylation enhanced the virulence of S. flexneri. The efficiency of HeLa cells invasion infected with036X and036₁d was higher than that of036, with statistically significance found between036₁d and036（p<0.05）. In Sereny test,036₁d induced a more severe keratoconjunctivitis reaction that was indistinguishable from that induced by036. The these findings suggested that O-antigen glucosylation promotes the invasion of S. flexneri. The cytoxicity of strain036₁d and036was tested by lactate dehydrogenase （LDH） release assay. Results showed that the LDH induced by036₁d was significantly higher than that of036（p<0.05）. The expression of IL-6and IL-1β induced by036was lower than that of036₁d at12and24h time points. We also performed comparative proteome analysis on strain036,036X and036₁d, and found that a protein Sap in036₁d was higher expression than that in strain036and036X. To evaluate the function of Sap, we detected the transcription levels of sap gene by quantitative real-time PCR （QRT-PCR）. Results indicated that the transcription levels of sap gene in036₁d was higher than that in036and036_X. Futhermore, To evaluate the function of Sap, we cloned gene sap and transformed into strain036to form transformant036⑥-1. Results revelaed by QRT-PCR indicated that the transcription level of sap gene was higher in036⑦-1than that of036,036_X and036₁d. Additionally, invasion assay indicated that strains with increased expression of Sap （036⑥-1） presented enhanced cellular invasion and macrophages cytotoxicity than036. Sap in S. flexneri shares high similarity （96%, protein level） with Antigen43（Ag43） in E. coli, a protein responding for biofilm formation and protection against hydrogen peroxide killing. Based on this data, we proposed that Sap in S. flexneri may play the same role in enhancing virulence, and more research are needed. These data suggested that the glucosyl modification of O-antigen promotes virulence of target cells by altering the conformation of LPS.更多还原