Construction And Partial Biological Characteristics Of Listeria Monocytogenes ΔInlAB,ΔInlABC Mutant Strains

Listeria monocytogenes( LM) can cause signifieant foodborne zoonosis. It can cause meningitis, septicaemia, gastroenteritis of humans and animals and its mortality approaching 30%. It exists generally in natural environment and food proeessing environment and resists the aeid and salt, which can breed under low temperature.LM which as intracellular parasitic bacteria can go through the host’s intestinal barrier, the blood-brain barrier and the maternal placental barrier and it can survive and reproduce in non-phagocytic cells and phagocytic cells(such as epithelial cells, liver cells and fibroblasts, etc.). These are a direct relationship with LM internalization protein. Inl A and Inl B are unique to LM in Internalin protein family, which located the cell surface and play an important role in adhesion, invasion of host cells during its invasion of cell-mediated and internalized into phagosomes. Inl C is unique secreted protein in internalin family. There are not the receptor combining with Inl C In eukaryotic cells surface. but, expression level of Inl C is high in the late stage of bacterial infection. Affection of these important virulence factor to the growth, metabolism and LM virulence regulation are poorly understood on currently. In this study, serotype 4b isolated LM681 from food and clinical isolated LM90 from sheep encephalitis are as parent strain. LM681△Inl A△Inl B, LM681△Inl A△Inl B△Inl C and LM90△Inl A△Inl B strains are constructed basis on LM681△Inl A and LM90△Inl A strains Analyze the role of Inl A, Inl B, Inl C virulence genes by comparison between the parent and mutant strains and various mutants in the biological characteristics of pathogenic and in vitro differences, we will provide conditions and lay the foundation for further research of Inl A, Inl B, Inl C. The main contents and results are as follows:1. Construction and identification of the LM681△Inl AB, LM681△Inl ABC and LM90△Inl AB mutant strain:The homology arms of both ends of Inl B and Inl C gene were amplified from template which LM681 and LM90 strain genome. Construction of LM681△Inl AB, LM90△Inl AB, △LM681 Inl ABC mutants is successful.2. Influence of △Inl A, △Inl B, △Inl C on virulent characteristics of LM: Parent strain LM681 and mutant strains LM681△Inl A, LM681△Inl B, LM681△Inl AB, LM681 △Inl ABC were cultured under the same conditions: i) mice by Intraperitoneal injection of bacteria solution, mortality statistics determinate LD50 after rearing 10d; ii) mice after intraperitoneal inoculum, 24 h, 48 h, 72 h each period mice sterile removal of the liver, spleen, brain tissue, CFU count, Statistics contained in the amount of bacteria of the organizations. iii) each strain were streaked onto 7% sheep blood BHI plate medium and cultured 24 h at 37 ℃, observed the hemolysis of strains.Test Results: The virulence of LM681△Inl ABC decreased 1000 times which compared with the parental strain LM681, the virulence of △LM681 Inl AB decreased 100 times, virulence of △LM681 Inl A and LM681△Inl B decreased 10 times; The amount of bacterial load of each mutant srains reduced in the liver, spleen and brain tissue; The parent strain LM681 and various mutants were all β-hemolytic in the 7% sheep blood BHI medium. The results showed : the deletion of Inl B, Inl C and Inl A genes can decrease the pathogenicity of LM. The deletion of Inl B genes decreased the LM load in the liver and brain tissues, the absence of Inl A decreased the proliferation of LM in the spleen, Inl C can mediate the proliferation of LM collaborate with Inl A and Inl B in the liver, spleen and brain tissues, the deletion of Inl B, Inl C and Inl A genes did not affect the hemolysis of LM.3. The effect of △Inl A, △Inl B, △Inl C deletion mutant strains to the biological characteristics of LM in vitro.We measured the change of growth concentration of the parent strain LM681 and the mutant strains LM681△Inl A, LM681△Inl B, LM681△Inl AB, LM681△Inl ABC at ℃37 in different p H value BHI culture medium, compared the effection of Inl A, Inl B and Inl C virulence genes on the growth and acid tolerance of LM in vitro. we compared the effection of Inl A, Inl B and Inl C virulence genes to the LM metabolism by biochemical reaction experiments, compared the effection of Inl A, Inl B and Inl C virulence genes to the LM drug sensitivity by measuring drug resistance to 15 kinds of commonly used sensitive drugs in clinic. The results show: the deletion of Inl A, Inl B and ln C virulence genes decreaseded the growth ability of LM in vitro, but did not affect the acid and alkali resistance, metabolism of LM, and did not change the sensitivity of LM to the drug.All of the above results show that: the deletion of Inl A, Inl B and Inl C gene decreased the virulence of LM, The internalized function of LM is regulated by the complex network structure of the interaction between different internalization. The results provided an important reference for the further studying the role of the internalization in the pathogenesis of LM.