Construction Of InlC-deleted Strains Of Listeria Monocytogenes And Study On Its Virulence And Environmental Tolerance

The bacterial pathogen Listeria monocytogenes(LM) is a common food-borne pathogens that can cause human listeriosis:abortion in pregnant animal(women),meningitis and other diseases.The bacterium is an important Gram-positive model organism,which has a great harm to humans and animals. LM is bacillus,arranged in a V-shape or in pairs, micro-aerobic, can grow in normal medium,is able to resist alkali,salt,heat environment.The growth temperature of LM is usually 30to38 ℃,but it can grow or reproduce still at 4 ℃, the lowest growth temperature is 0.1 to 0.4 ℃. According to bacterial and flagellar antigen, pathogenic serotype are 1 / 2a, 1 / 2b, 1 / 2c, 3a, 3b, 3c, 4a, 4b,however,type 4b cause higher incidence.LM is a typical intracellular parasites,not only grow in the phagocytic cell, but also in the epithelial cells and liver cells, it can encroach the central nervous system through the damaged mucosa to the nerve endings of the tunica vaginalis. There were two clusters of virulence genes related to intracellular parasitic life cycle, called Listeria monocytogenes pathogenicity island 1(LIPI-1) and Listeria monocytogenes pathogenicity island 2(LIPI-2). LIPI-1 has six genes, both sides were prs and ldh sites. From the beginning of prs downstream, prfA, plcA, hly, mpl, actA, plcB successively,which were encoding gene of transcriptional activator.LIPI-2, also known as internalization island, means of a leucine-rich repeat protein family.It divided into two subfamilies, subfamily1 is representative of InlA and InlB,a gene encoded by InlAB. Subfamily 2 is composed of a relatively small molecular protein composition, the prototype from InlC of Listeria monocytogenes.In view of this, the study constructed LM90SB2- △ InlC gene deletion strains,carried out the virulence and environmental tolerance study on the gene deletion strain,discussed the regulatory role of InlC in virulence and environmental tolerability,provided a scientific basis for vaccines research. The main contents and results are as follows:1. Construction of Inl C-deleted strains of Listeria monocytogenes: Got the InlC gene sequence from NCBI,desigened upstream and downstream primers homologous arm for the deletion Firstly,the upstream and downstream homology arms of InlC gene were amplified from LM-90SB2,which serotype was 4b.Then, the two homology arms was connected together to get the △InlC fragment through SOE-PCR.The △InlC fragment was inserted into the suicide plasmid pKSV7, to construct the recombinant suicide vector p KSV7-△InlC.The recombinant vector was transformed by electroporation into the competent cells of the LM-90SB2.The positive transformants were subcultured for 15 generations in the pressure of chloramphenicol(10ug/ml) at 41℃, to get the single-exchange strain. Then, the strain was continued to be passaged to the 108 th generation in the absence of chloramphenicol at 41℃to get the double-exchange strain, and the detection primers was used to identify for the mutant. In order to lost plasmid and identify genetic stability of LM90SB2-△InlC, it was stilled subcultured for 30 generations without chloramphenicol at 37℃. LM90SB2-△InlC was constructed. The detection primers was used to detect for the mutant which can get only one target fragment of 1480 bp by PCR,at the same time,the mutant had genetic stability.2. The effects of LM90SB2-△InlC on virulence of LM:This experiment studyed the differences in virulece betwen LM90SB2 and LM90SB2-△InlC through the determination of LD50、bacterial counts of liver and spleen、 hemolysis test、adhesion、invasion and intracellular growth in MBMEC. Research results showed that: LD50 of LM90SB2-△InlC was 107 CFU and LD50 of LM90SB2 was 104.5 CFU, so LD50 of gene-deleted strain was 102.5higher than that of the wild strain(P<0.01); The numbers ofLM90SB2-△InlC in the mouse’s liver and spleen were fewer than that of the wild strain at various times(P<0.05); The result of hemolysis test is both were β-hemolytic. The adhesion rate of LM90SB2-△InlC was2.63 percent and the adhesion rate of LM90SB2 was3.72 percent,the difference was1.09percent(P<0.01);The invasion rate of LM90SB2-△InlC was0.033 percent and the adhesion rate of LM90SB2 was1.4percent,the rate of LM90SB2 Significantly higher than the other(P<0.01);The survival numbers of LM90SB2-△InlC in cells were fewer than that of LM90SB2 between 2-12 hours(P<0.05).The results showed that InlC has related to the virulence of LM.3. The effects of LM90SB2-△InlC on environmental stress respone of LM : In order to detect the changes of the deletion mutant in environmental tolerance, bacteria OD600 were detected in different temperature and different pH pressure. Our experiment results showed that there was no difference in tolerance to the different environment between the two strains. This results confirmed that InlC was not needed in the stress of temperature and pH stress.