Construction, Virulence And Environmental Tolerance Ofâ–³RsbV Deletion Strain Of Listeria Monocytogenes

LM is a facultative intracellular parasitic bacteria which is Gram-positive and it is an Zoonosis pathogen with a greater hazard to animals and humans, which can cause pregnant livestock (women) miscarriage and meningitis. LM widely presents in the natural environment, such as water and soil, because it has a strong environmental adaptability to survive in a variety of complex environmental pressure of the acidic, high salt and low temperature, which is an important reason of a high incidence of listeriosis. Immunization is an effective means to prevent the disease, so it has important significancethe to research attenuated vaccine of LM.LM could go through major physiological barrier of a host(intestinal barrier, the blood-brain barrier and the placental barrier) and it could survive and reproduce in full-time and part-time phagocytic cells (such as epithelial cells, liver cells and fibroblasts, etc.) after invasion of the body. Specific virulence factors of LM were involved in parasitic and cell proliferation. LM owns many virulence genes, complex adjustment mechanism and a multi-level regulation system. Studies have shown that the virulence of LM is mainly regulated by positive adjustment factor (PrfA), response regulator factor (VirR) and environmental stress factor SigmaB. SigmaB operon encodes RsbR, RsbS, RsbT, RsbU, RsbV, RsbW, SigmaB and RsbX, a total of eight factors. SigmaB can regulate the main virulence factors of LM such as PrfA, inlA, inlB and LLO. However, the role of RsbV is not clear in the regulation of virulence and environmental tolerance of LM. So, we first constructed RsbV gene deletion strain of LM, researched on its virulence, immunogenicity and environmental tolerance, and then discussed the regulation role of RsbV in virulence and environmental tolerance of LM. All of this provides a scientific basis for the research and development of LM attenuated vaccine. The main research contents and results are as follows:1. Construction and molecular identification of LM RsbV gene deletion strain:Both the upstream homologous arm and downstream homology arm was amplified by PCR, and then the two arms was connected together to get the RsbV missing fragment by SOE-PCR. The RsbV missing gene fragmentwas was then inserted into the the shuttle vector PKSV7, to construct the recombinant shuttle plasmid pKSV7-ΔRsbV The resulting plasmid, pKSV7-ΔRsbV was electroporated into LM-XS5, and the detection primers was used to search for the positive transformants which can get two target fragments of807bp and479bp by PCR. The positive transformants were subcultured for15generations in the pressure of chloramphenicol at42℃, to get the single-exchange strain with chloramphenicol resistance. Then, the strain was continued to be passaged in the absence of chloramphenicol at42℃, to get the double-exchange strain called LM-Δ RsbV without chloramphenicol resistance, which only amplified a479bp target fragment by PCR. In order to identify whether LM-Δ RsbV had genetic stability, it was stilled subcultured for20generations without chloramphenicol at37℃2. The effects of RsbV gene detection on virulence and immunogenicity of LM:This experiment studyed the differences in virulece betwen LM-XS5and LM-ARsbV through the determination of LD50<bacterial counts of liver and spleen-, virulence genes transcription levels and macrophages RAW264.7infection experiment. The immune effect of LM-ARsbV was studyed by challenge experiments in mice. Research results showed that:LD50of LM-ARsbV was10981CFU and LD50of LM-XS5was10556CFU. so LD50of gene-deleted strain was104higher than that of the wild strain(P<0.01);The numbers of LM-Δ RsbV in the mouse’s liver and spleen were fewer than that of the wild strain at various times (P<0.05);The transcription levels of four virulence factors(inlA、LLO、PlcA and PrfA) of LM-ΔRsbV were lower than that of the wild strain (P<0.05);The survival numbers of LM-Δ RsbV in macrophages were fewer than that of LM-XS5between3-24hours (P<0.05); The protection rate of LM-Δ RsbV for the mice was90.0%. The results showed that RsbV plays an important role in the regulation of LM virulence, and LM-ΔRsbV still had a good immunogenicity which had potential to be treated as attenuated vaccine.3. The effects of RsbV gene deletion on environmental stress respone of LM:In order to detect the changes of the deletion mutant in environmental tolerance, we constructed stress environment of high temperature, low temperature, high salt, acid, surfactant and alcohol pressure. Our experiment results showed that LM-ΔRsbV had a lower tolerance to low temperature, osmotic pressure, alcohol and acidic stress compared with LM-XS5, and there was no difference in tolerance to the surfactant Triton-X-100between the two strains. This results confirmed that RsbV was needed in the stress of low temperature, osmotic pressure, alcohol force and acidic stress.