Characterization Of The WaaI And RfbA Genes Of Shigella Flexneri2A

Shigella Spp. is the pathogen causing human bacillary dysentery, and uses a type III secretion system to inject proteins into human cells, leading to a vigorous inflammatory response. Dysentery is a worldwide high incidence of intestinal infectious diseases, and more than one million people were dead in this disease each year. The main treatment for dysentery is the use of antibiotics, and resulting in more than95%strains producing the resistance to various antibiotics. Enhanced bacterial resistance caused by the abuse of antibiotics has become one of the difficulties for the treatment of bacterial infections. More recently, the first bacterial N-glycosylation pathway was found in Campylobacter jejuni. PgIB, the key enzyme of the pathway, has opened up the field of glycoengineering, particularly with respect to the development of glycoconjugate vaccines.The N-glycosylation pathway has significant similarities with lipopolysaccharide synthesis. First, oligosaccharide precursor is synthesized on a lipid carrier undecaprenol. The flippase then translocates the oligosaccharide precursor from cytoplasm side to the periplasm side, where the oligosaccharyltransfase PgIB transfers the oligosaccharide to the protein acceptor, or the oligosaccharide precursor is first polymerized into O-PSs and then transferred to lipidA-core by the O-antigen ligase WaaL. When gene rfbA what catalyzes deoxythymidine diphosphate-rhamnose was deleted, the oligosaccharide precursor could not be synthesized, and then the proteins could not be glycosylated; when the oligosaccharyltransfase PgIB and its protein substrate were cloned into the waal gene deletion mutant of Shigella flexneri2a301, glycosylated AcrA with the O-PSs of S. flexneri2a301could be obtained. In this study, we explore the potential glycoprotein in Shigella and hope to lay the foundation for the development of sugar-conjugated vaccine for Shigella.In this article, genes waal and rfbA of S. flexneri2a301were deleted to construct waal and rfbA deletion mutants by using λ-Red recombination system. Then, waal recovery mutant was constructed by using low copy plasmid. Phenotypic identification of the mutants was performed by LPS silver staining experiment. The results showed that the mutant strains could not synthesize lipopolysaccharide. No ladder was found in waal and rfbA deletion mutant. Then, growth of the two mutants, the recovery strain and wild strain at37℃were measured respectively, and some biochemical events of them were comparatively investigated. At the same time, the evaluation of virulence was performed by the sereny test of mutants and wild strain. The growth curves of the waal, rfbA mutants and the wild-type presented non-significant difference in LB medium. Compared with wild strain, the ability to utilize raffinose by waal mutant was lower. In the Sereny tests, the mutant strains could not cause corneal inflammation in guinea pigs, while wild type and the recovery strain were able to provoke strong kerotoconjunctivitis in guinea pigs. After preparation of protein samples of the wild type and mutant strains grown at37℃, comparative proteomic analyses were performed using two-dimensional electrophoresis. Analysis of comparative proteomics showed that differential expressed proteins were found in waal and rfbA mutant strains. Subsequently, the oligosaccharyltransfase PgIB and its protein substrate were cloned into the waal gene deletion mutant of S. flexneri2a301. After induction with IPTG, AcrA can be expressed, but PgIB can not be expressed and AcrA can not be glycosylation.