Identification Of RpoE Gene For Regulation Of Salmonella Biofilm Formation And Biological Characteristics Of RpoE Deletion Mutants

Salmonella is a common zoonosis pathogen, which can infect both human and animals. Its infection causes enormous economic losses to animal husbandry and food processing industry. Salmonella can form biofilm, which can increase the ability of bacteria resistance to adverse conditions. The bacteria in biofilm phynotype are not to be removed through sterilization disinfectant and antibiotics easily, which can cause bacterial persistent infection and recurrent infection. The biofilm formation is mostly regulated by rpoS, while there are also some rpoS gene-independent Salmonella biofilm formation strains. In this study, biofilm forming ability of 120 Salmonella strains was detected by crystal violet assay. The rpoS gene-dependent or-independent strains for biofilm formation were determined by catalase test. The Real-time PCR method was established to compare the expression of six different Sigma factors during the period of biofilm formation. Further, rpoE mutants were constructed using the Red recombination system, the biofilm-formation ability of mutants was tested and their pathogenicity was determined. This study may help to elucidate the regulatory mechanism of salmonella biofilm formation.1 Identification of Sigma factors for regulation of biofilm formation in Salmonella strainsThe biofilm-forming ability of 120 salmonella strains was detected by crystal violet assay. The Congo red/Brilliant blue plate and calcofluor plate were used to detect the components of biofilm. The rpoS gene-dependent or-independent strains for biofilm formation were determined by catalase test. Finally, the Real-time PCR method was established to compare the expression of six different Sigma factors during the period of biofilm formation. Results showed that there were four different phynotypes of the bacteria grow in the Congo red/Brilliant blue plate:(1) Red, dry and rough (RDAR), (2) Brown, dry and rough (BDAR), (3) Pink, dry and rough (PDAR), (4) Smooth and with (SAW). Cellulose production was assessed by comparing the fluorescence of the test strains under UV light.75% of test strains formed biofilms, in which 11 strains were tested as the rpoS-independent strains. Six recombinant standard plasmids cloned with different Sigma factors were constructed for preparation the quantitative curve by using absolute methods. Three strains with different biofilm formation characteristics were selected to compare the expression of six different Sigma factors during the period of biofilm formation. The result showed that the expression of rpoE gene was the highest during 4 h and 24 h post-incubation in the rpoS-independent strains, indicating that the rpoE gene was one of biofilm formation regulation genes in Salmonella rpoS-independent strains.2 Construction of deletion mutants of Sigma factors related with biofilm formation in Salmonella strainsTwo rpoS-independent strains, Salmonella Pullourm S6702 and Salmonella Typhimurium S016, and one rpo-S-dependent strain, Salmonella Typhimurium S025, were selected for construction of deletion mutants with deficiency of genes rpoS or rpoE using the Red recombination system. Six deletion mutants were confirmed by PCR and RT-PCR amplification, combined with gene complementation. Growth curve revealed that the growth rates of rpoS deletion mutants were as same as wild-type strains; rpoE deletion mutant of Salmonella Pullorum grew faster than wild-type strain, while rpoE deletion mutants of Salmonella Typhimurium grew slower than wild-type strain. A quantitative microtitre asssy with Crystal Violet staining in 96-well plate showed that in two rpoS-independent strains, ΔrpoS mutants kept their the biofilm-forming ability, while ΔrpoE mutants could not produce biofilm, when compared to wild-type strains. In rpoS-dependent strain, both ΔrpoS and ΔrpoE mutants could not form biofilm. The complementation strains ΔrpoSR and ΔrpoER recovered the biofilm formation ability. In conclusion, the rpoE gene was identified as one of regulation genes for biofilm formation in Salmonella rpoS-independent strains.3 Study on pathogenicity of Salmonella rpoE deletion mutantsThe adherence to and invasion of Hela cells, proliferation of macrophages of the wild-type strains and the rpoS or rpoE deletion mutants were determined. The results showed that there was no significant difference in adherence ratio, invasion ratio and proliferation ration between the rpoS deletion mutants and wild-type strains. The rpoE deletion mutants of Salmonella Typhimurium showed lower adherence ratio, invasion ratio and proliferation ration than the wild-type strain, while the rpoE mutants of Salmonella Pullourm showed higher adherence ratio, invasion ratio and proliferation ration than the wild-type strain. To determine the LD50,1-day-old SPF chickens were challenged orally with the wild-type strain and the deletion mutants. The LD50s of deletion mutants ΔrpoS were similar as that of the wild-type strains. The LD50 of S6102ΔrpoE was higher than that of the wild-type strain, while the LD50S of S016ΔrpoE and S025 ΔrpoE were lower than that of the wild-type strain. Bacterial distribution test in vivo revealed that the numbers of bacteria were no significant difference between the mutants and wild-type strains at 12 h postchallenge. At 48 h postchallenge, the numbers of bacteria in spleen and bile of chickens in S6702ΔrpoE group were higher than that of the wild-type strain challenge group, while the numbers of bacteria in spleen and bile of chickens in S016ΔrpoE and S025ΔrpoE groups were lower than that of the wild-type strain challenge group. In conclusion, the effect of rpoE gene on Salmonella virulence was diverse in different serotypes.