Construction And Characterization Of Listeria Monocytogenes△hfq Mutant Strains

Gram-positive brevibacterium Listeria monocytogenes(Lm) is an important zoonotic foodborne pathogen engaging in both saprophytism and parasitism. As a facultative intracellular parasitic bacterium, Lm has relatively broad host spectrum. In addition, it has strong ability to adapt to cold, acid, alkaline and osmotic stress, therefore, it exists widely in nature. Under different environmental stress challenges, Lm can adapt, survive and display pathogenicity, which is associated with the regulatory network consisting of regulating proteins. Hfq protein is a newly discovered virulence regulatory factor of Lm, and currently it is little known about the role of Hfq in bacterial growth, metabolism and virulence regulation. The ecological distribution and virulence of different serotypes of Lm are obvious variations,we chose the serotype1/2a standard strain EGDe and serotype4b virulent isolate NTSN as the parental strains, and successfully constructed deletion strains of hfq with homologous recombination technology. Then the ability of stress resistance and virulence to mice of deletion mutant strains were evaluated and compared with their parental strains, in this way the role of Lm Hfq protein in the stress tolerance and virulence was probed.1. Construction and identification of hfq gene deleted and reverse listerial strainsBased on homologous recombination technology, we successfully constructed hfq gene mutant strains EGDe△hfq and NTSN△hfq,respectively.Firstly, homologous fragments of target gene were amplified, then spliced together with SOEing PCR. Nextly, the fusion fragments and shuttle vector pKSV7were digested with double enzymes respectively, then purified, ligated and transformed into competent cell E. coli DH5a. After identified, the recombinant plasmids were transformed by electrotransformation to competent cells of EGDe and NTSN respectively. Under the pressure of temperature and chloramphenicol, with two rounds of recombination, the mutant strains EGDeAhfq and NTSN Ahfq were obtained after identification of recombinant clones by PCR and RT-PCR. In addition, the hfq gene of NTSN and its promoter sequence were ligated into the vector pERL3and transformed into NTSN△hfq competent cells, then a recovery strain of NTSN△hfq, named NTSN△hfq-hfq was successfully constructed. The growth curve, physiological and biochemical characteristics, hemolytic activity of parent strains, deletion mutants and recovery strain were measured. The results showed that growth trends of EGDeAhfq and NTSN△hfq were in accordance with their parental strains, and there was no significant change of haemolytic activity, but EGDeAhfq had lost the a-glucosidase (AGLU) enzyme activity, and NTSN△hfq had lost the Leucine arylamidase (LeuA) activity and Lactose oxidation capability. The above results indicated that the normal growth and metabolism of hfq gene deletion strains were not obviously affected, but the specific carbon and nitrogen metabolic pathways were impacted to some extent.2. The biological characteristics of Listeria monocytogenes Ahfq mutantHfq protein is widely existed in Gram-negative bacteria and some Gram-positive bacteria, it can regulate the expression of a large number of target genes, and plays an integral role in the regulation of a sereies of process such as pathogenicity in order to adapt to the living environment both in vivo and in vitro. In this study, the phenotypic characteristics, anti-stress and bacterial virulence of Lm were studied by measurement the culture characteristics under different stress in vitro, the invasion ability on Caco-2cell line and infection experiments in murine model in vivo. The results showed that, comparing with their parental strains, the growth of EGDe△hfq and NTSN△hfq was significantly inhibited under cold temperature (p<0.05), salt medium containing10%NaCl and the medium containing4.5%ethanol. In addition, EGDeAhfq and NTSN△hfq were significantly more sensitive to1%TritonX-100stress compared to their parental strains (p<0.01, p<0.001). The ability of biofilm formation of mutant in BHI, gastric fluid medium was reduced significantly (p<0.05), similarily, the invasion rate to Caco-2cell lines was reduced. Virulence to BALB/c mice test showed the LD50of EGDeAhfq was6times higher than EGDe, moreover, the LD50of NTSN△hfq was16times higher than NTSN, heretofore, the virulence of EGDe△hfq and NTSN△hfq was significantly reduced. Interestingly, compared to EGDe△hfq, NTSN△hfq was more sensitive to cold, TritonX-100, invasion ability to Caco-2cell lines and virulence to mice was higher significantly reduced. The above results thus suggested that Hfq protein was asscociated in regulation stress response, biofilm formation and virulence. The construction and biological characteristics of Listeria monocytogenes Ahfq mutant strains afford materials for studying the function of Hfq, provide the possibilities to elucidate the mechanisms of Lm in resisting the stress and pave ways to the development of novel strategies for the prevention and control of Lm infections.