Mechanism Of Mig-14 Participating In Salmonella Enterica Serovar Typhi Resistance To Polymyxin B

Objective:mig-14 was a horizontally acquired host-induced virulence gene in Salmonella enterica serovar Typhi(S. Typhi), which plays an important role in host invasion and polymyxin B resistance. However, the mechanism remains unclear. The aim of this study is to explore the molecular mechanism of Mig-14 contributes to S. Typhi resistance to PB.Methods:(1) The wild-type strain and △mig-14 mutant strain of S. Typhi were grown in LB broth containing PB with the final concentration of 0.1μg/mL at 37℃, 250 r/min for 5 hours. The total RNA was extracted from wild-type and △mig-14 mutant of S. Typhi. Then cDNAs were synthesized by reverse transcription and labeled with cy3- or cy5-dCTP.The gene expression profiles of the wild-type strain and △mig-14 mutant of S. Typhi under the exposure of PB were investigated by a geneomic DNA microarray analysis. Quantitative RT-PCR was utilized in selective genes to determine the relative transcription of specific genes of the two different strains to confirm the microarray data.(2) The 3×Flag fusion with ompC and ompF in the WT of S. typhi and △mig-14 of S. typhi were prepared by homologous recombination mediated by suicide plasmid. The 3 × Flag tag was inserted directly before the termination codon of ompC or ompF in the wild-type of S.typhi and △ mig-14 S. typhi to construct WT-Omp C::3 × Flag,WT-Omp F::3 × Flag fusion strain and △ mig-14-Omp C::3 × Flag, △mig-14-Omp F::3×Flag fusion strain.(3) WT-Omp C::3 × Flag fusion strain, △ mig-14-Omp C::3 × Flag fusion strain, WT-Omp F::3×Flag fusion strain and △mig-14-Omp F::3×Flag fusion strain were grown in LB broth containing PB with the final concentration of 0.1 μg/mL at 37℃, 250 r/min for 5 hours. Then bacterial cells were harvested, and Western-blot analysis was used to detect the protein levels of Omp C, Omp F in the WT of S. typhi and △mig-14 of S.typhi with anti-FLAG.(4) Mutant strains of S. Typhi used in this study were prepared by homologous recombination mediated by suicide plasmid pGMB151. Two pairs of specific PCR primers were designed for upper- and down-stream of the ompC gene of S. Typhi to amplify two homologous DNA fragments and then inserted into the suicide plasmid pGMB151 BamH I site. The positive clones were then transferred into the target S. Typhi wild-type strain, △ompF mutant, △mig-14 mutant by electroporation.The recombination was visualized by PCR, and the complete recombinant strain was selected as the ompC deleted mutant strain(△ompC),△ompC/ompF, △mig14/ompC double mutant strain. The pGMB151 containing the homologous ompF deletion fragment were transferred into△mig-14 and △mig14/ompC to construct △mig14/ompC and△mig14/ompC/ompF mutant strains, which were confirmed by PCR and sequencing analysis.(5) The growth curves of these strains treated by PB were detected to analyze their survival abilities under the treatment of PB.(6) The wild-type and △mig-14 mutant strain were grown in LB containing 0.1 μg/mL PB and incubated at 37℃, 250 r/min for 2 hours,which were grown to log-phase and inoculated into a 96 well plates and cultured for 3 days at 30℃. It was then fixed with methyl alcohol and crystal violet added for 15 minutes. The wells were washed 3 times with PBS. The plates were air-dried for 2 h at room temperature and stained biomass was dissolved with 200 μl 30%(v/v) Acetic acid. The OD570 was measured to quantify biofilm formation.Results:(1) Gene expression profile analysis revealed that 768 genes showed significant differences between the △mig-14 and the WT strain in the presence of PB. Flagella associated genes and outer membrane porin proteins ompF and ompC were significantly up-regulated inΔmig-14 strain. Invasion and virulence associated genes were down-regulated in Δmig-14 strain. qRT-PCR comfirmed the results of the microarray analysis.(2) PCR and sequencing analysis showed that WT-Omp C::3×Flag fusion strain, △mig-14-Omp C::3×Flag fusion strain, WT-Omp F::3×Flag fusion strain and △ mig-14-Omp F::3 × Flag fusion strain were successfully constructed.(3) Western-blot analysis showed that compared with the WT strain,the protein levels of Omp C and Omp F in △mig-14 mutant were also elevated in the presence of PB.(4) PCR and sequencing analysis showed that mutant strains of△ompC, △ompC/ompF, △mig-14/ompC, △mig-14/ompF and△mig-14/ompC/omp F were successfully constructed.(5) The Δmig-14 strain was more sensitive to PB compared with the wild type strain. Deleting ompC or/and ompF in Δmig-14 decreased the sensitivity to PB caused by deleting mig-14 gene in S. Typhi.However, their growth was still slower than that of the wild-type strain.(6) The results of the biofilm assay showed that the biofilm levels in△mig-14 mutant strains were lower than those in the wild-type stains.Conclusions:Exposure to PB, Mig-14 can control a specific set of genes expression. Deleting mig-14 in S. Typhi resulted in the up-regulation of ompF and ompC in mRNA and protein leves under the treatment of PB.Deleting ompC or/and ompF in Δmig-14 can increase the survival ability of the bacteria, However, their growth was still slower than that of the wild-type strain. We further found that Mig-14 can promote the biofilm formation in S. Typhi. As a conclusion, Mig-14 may contribute to PB resistance by decreasing the permeability of the outer-membrane and promoting bioflim formation.