O-antigen Modification Medicated By Serotype-converting Bacteriophages In Shigella Flexneri And Enhancement Of Virulence Functions Through Glucosylation Of S. Flexneri LPS

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 â…¢ 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 transformant036â‘¥-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 （036â‘¥-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.