The Effect Of Quorum Sensing System For Growth Competitiveness On Shigella Flexneri

Shigella is a major pathogen of bacillary dysentery, The global annual incidence can reach 16 million, of which more than 95% of deaths occurring in developing countries. The incidence of bacillary dysentery in our country is more than 4 million every year, second to hepatitis and tuberculosis only Shigella spp. primarily spread through the fecal-oral route, about 10-100 CFU of Shigella can cause the disease. It can invade epithelial cells of intestinal tract, leading to abscesses, ulcers and strong inflammatory response.There is an increasing awareness of the importance of microbial social interactions. Although bacteria are traditionally considered as unicellular organisms, they can show complex coordinated multicellular behaviors, such as biofilm formation, antibiotic production, and secretion of virulence factors. Some of these behaviors require a large number of cooperating bacteria to be effective, that is, high cell density. Quorum sensing(QS) is a key communication system that coordinates cooperative behaviors in bacteria of high cell density.Researches showeds that, in the genomic level, Shigella and E. coli have a high degree of homology and similarity, but they have significant differences in phenotype and pathogenicity. By comparing the genome sequences of E. coli and Shigella, we found: that they both contain quorum sensing system operon genes, but there are some differences between these two strains.Compared with the full quorum sensing system genes in E. coli, Shigella spp. lacks some genes of quorum sensing systems, such as lsr K, lsr B, lsr F.In this study, the 301/p Pro-lsr BFK strain was constructed by PCR, digestion and ligation, and 301/p ET28a-lsr KG strain was constructed by Golden Gate method. The growth curves of 301/p Pro-lsr BFK strain and the 301/p ET28a-lsr KG strain was described and compared. Colony counting experiments showed that the QS system recovery strain had growth advantage over the wild-type strain when they were mixed and cultured. Further, the whole-cell protein samples of 301/p Pro-lsr BFK and 301/p ET28a-lsr KG were prepared for two-dimensional electrophoresis. Differentially expressed proteins in two-dimensional electrophoresis gels were identified by MALDI-TOF–TOF-MS analysis, and the results were carefully analyzed. The results can be summarized as follows:1. 301/p Pro-lsr BFK strain and 301/p ET28a-lsr KG strain were successfully constructed.2. We detected AI-2 in S. flexneri 2a strain 301 through the reporter bacteria Vibrio harveyi BB170, indicating that S. flexneri can produce active AI-2.3. There is no difference in carbohydrate metabolism between the 301 wild strain and 301/p Pro-lsr BFK, 301/p ET28a-lsr KG. However, bacterial density in stable of the 301/p Pro-lsr BFK, 301/p ET28a-lsr KG strains are slightly better than 301.4. By mixed and cultured 301/p Pro-lsr BFK,301/p ET28a-lsr KG and 301 wild strain, colony counting experiments showed that 301/p Pro-lsr BFK strain had no growth advantage over the wild-type strain, but the 301/p ET28a-lsr KG strain had growth advantage over the wild-type strain when they were mixed and cultured.5. The protein expressions were of 301 and 301/p Pro-lsr BFK, 301/p ET28a-lsr KG were analyzed through comparative protemics methods. Comparison 301/p Pro-lsr BFK and 301, showeds that there no difference in protein profils; While there were some signifinant different between 301/p ET28a-lsr KG and 301, These differentially expressed proteins include: Lsr B, components of the ATP-binding cassette transporter; Gro EL, related to the synthesis of proteins; Metabolism-related enzymes, icd A encodes isocitrate dehydrogenase, sdh A encodes succinate dehydrogenase, flavoprotein subunit, fab I encodes enoyl-[acyl-carrier-protein] reductase(NADH), nuo I encodes NADH dehydrogenase I chain I.