Construction Of Salmonella Enterica Serovar Enteritidis RfaQ Mutant And Its Virulence Determination

Salmonella enterica serovar Enteritidis (S. Enteritidis, SE) is a Gram-negative facultative intracellular pathogen with a broad range of hosts. Salmonella mainly grows and replicates in the macrophages and dendritic cells (DCs) of the hosts in natural infection. A large number of experiments showed that colonizing within macrophages is essential for Salmonella to elicit systemic infection. The intracellular bacteria can infect mesenteric lymph nodes and spread to the other organs through the blood circulation. The secreted effectors can promote the macrophage apoptosis, leading the bacteria spread cell-to-cell. The lipopolysaccharide (LPS) was proved involved in the virulence of Salmonella. LPS is a highly acylated saccharolipid located on the outer leaflet of the outer membrane of Gram-negative bacteria. LPS is critical to maintaining the barrier function preventing the passive diffusion of hydrophobic solutes such as antibiotics and detergents into the bacterial cell. LPS has been considered as.an essential component for the outer membrane biogenesis and the cell viability based on former studies in the model of Gram-negative organisms Escherichia coli and Salmonella enterica.In this study, we constructed the rfaQ deletion mutant of SE by the λ-Red recombination system. Its complemented strain was introduced by the pBR322 plasmid. The virulence of the wild-type (WT) strain and the mutant has been determined by infecting macrophages in vitro and inoculating the BALB/c mice. It was demonstrated that the rfaQ gene is involved in the pathogenicity and virulence.1 The prokaryotic expression of protein RfaQ and construction and identification of rfaQ mutant of S. EnteritidisIn this study, we constructed the prokaryotic expression plasmid pET30a-rfaQ, and then transformed it into E.coli BL21 (DE3). The RfaQ protein was expressed efficiently after the IPTG induction. Expressed products were confirmed by SDS-PAGE, which proved that most of the recombinant protein was existed in the form of the inclusion body. The BALB/c mice were immunized with the purified recombinant protein for the preparation of specific antiserum against RfaQ.The rfaQ gene mutant of SE was constructed using λ-Red recombination system, denoted as C50041 ΔrfaQ. And its complementary strain was constructed subsequently, called C50041Δ rfaQ::rfaQ. The rfaQ gene of SE C50041 ΔrfaQ could not be detected at the level of transcription in the realtime-PCR assay, nor product could react to the anti-RfaQ polyclonal antibodies evaluated by Western blotting assay. Western blotting analysis of the WT and mutant strains revealed that RfaQ protein of complementary strain can be detected using RfaQ antiserum. This showed that the rfaQ has been knocked out successfully and the complementary strain could express RfaQ protein which has immunological activity. The results of biochemical identification showed that the biochemical criterion O129R of rfaQ mutant is negative in contrast to WT strain. The growth characteristic and LPS structure of rfaQ mutant were consistent with those of the parent strain C50041. The motility of rfaQ mutant was decreased compared with WT strain C50041.2 The virulence determination of S. Enteritidis WT strain and rfaQ mutantIn this study, we detected the LD50 of rfaQ mutant is 1×109 CFU, which was 1000-fold higher than that of the parent strain (1×106 CFU) in the oral infection of BALB/c mice. The murine macrophage-like cell line RAW264.7 and chicken macrophage-like cell line HD-11 were used as the cell model to investigate the traits of the mutant. The results suggested that the adherence efficiency of rfaQ mutant was significantly declined compared with C50041 in RAW264.7. However, there is no significantly difference on the invasion ability. The adherence efficiency of rfaQ mutant was consistent with that of its parent strain C50041, whereas the invasion efficiency of rfaQ mutant was significantly declined in HD-11. The results of HD-11 intracellular replication assay indicated that there was no distinct difference in the proliferation ability between WT and rfaQ mutant strain at different point-in-times. Interestingly, compared with C50041, the proliferation of rfaQ mutant was higher than WT strain at 5 h and 10 h post-infection in RAW264.7. In vitro and in vivo competition assay showed that rfaQ deletion strain is quite incompetent compared with the WT strain.