Study On Regulation Of Type Ⅵ Secretion System By The Quorum Sensing System

Salmonella is a widely distributed important zoonotic pathogen. Most of Salmonella are pathogentic to humans and animals, which can cause a variety of salmonellosis. Salmonella is divided into two species, namely the Salmonella enterica and Salmonella bongori. Salmonella enterica including 2500 serotypes is a representative pathogen that can cause salmonellosis in human and animals worldwidely. A new secretion system, termed type Ⅵ secretion system(T6SS), is discovered in gram-negative bacteria, through which virulence effectors are transported to the extracellular across plasma membrane. T6 SS is widespread, of which characterized components include an Icm F homologue, the ATPase Clp V, a regulatory FHA domain protein and the secreted Vgr G and Hcp proteins. Previous studies have shown that T6 SS can enhance adaptability of bacteria to the external environment, improve the adaptability to the host cell and affect the biofilm formation. Like the other secretion system, the transcriptional and post-transcriptional level of T6 SS are tightly regulated. The lux S gene of quorum sensing(QS) system has a significant impact on T6 SS in Vibrio cholerae. Thus, this study was carried out to determine whether QS system affects T6 SS in Salmonella. 1. Developement and application of multiplex PCR assay for detection of virulence genes in SalmonellaRapid and sensitive multiplex PCR assays were developed to detect virulence genes in Salmonella Pathogenicity Island. A total of 16 primer pairs were designed according to sequences from the Genbank, and 4 multiplex PCR assays had been established by optimizing reaction conditions,. The distribution of virulence genes, bacterial resistance to antibiotics and bacterial median lethal dose(LD50) were determined. The results showed distribution rate of sop E gene was only 15%, but distribution rates of the other 15 genes were over 90%. The results of antibiotic sensitivity test indicated that Salmonella strains show different resistance and sensitivity to common antibiotics, and antibiotic resistance of Salmonella strains has been much serious. More than 90% Salmonella strains were resistant to erythromycin, azithromycin and clindamycin, neomycin, spectinomycin, tetracycline and rifampin. Moreover, the multi-drug resistance strains have been very popular. Virulence test indicated that the Salmonella isolates were virulent to the mice.. The results indicated that virulence genes of Salmonella strains are widely distributed, Salmonella strains have a high resistance to antibiotics, and these strans have a high virulence to mice. 2. Construction and characteration of lux S gene mutant strainThe lux S gene mutant strain was constructed successfully to determine its role in the phenotypes of Salmonella. Then, the characteration of lux S gene mutant strain were determined. The results showed that growth curve, biofilm formation, virulence, adhesion and invasion ability of mutant type were not significantly different to that of wild type strain. However, the motibility capablity was significantly increased of mutant type compared to the wild type strain. 3. Analysis of the regulation of T6 SS by the Lux S proteinT6SS core components, Hcp1, Hcp2 and Clp V were cloned and expressed by the p ET system. Then, Anti-Hcp1, Hcp2 and Clp V polyclonal antibodies were raised in rabbits. Real-time PCR and western blot assays were carred out to detect transcriptional and post-transciptional level of T6 SS core components. The results showed there were no significant difference in transcriptional and post-transcriptional level of T6 SS core components between the lux S mutant strain and wild type strain. We can conclude that Lux S protein does not directly regulate T6 SS core components in Salmonella. The regulation of T6 SS in Salmonella should be studied in the furture.