Effect Of FlaA Protein On Virulence And Biofilm Formation Of Listeria Monocytogenes LM90SB₂

Listeria monocytogenes?LM?is a kind of intracellular parasitic gram-positive pathogen that can cause meningitis,miscarriage,septicemia and other diseases in animals and humans,and the mortality rate of this kind of bacteria can reach as high as 20%to 30%,which is a great danger to humans and animal husbandry.So far,there are several studies have been found that numerous virulence factors are related to the virulence of bacteria.Flagella can not only control the direction of bacteria's movement,but also play an important role in the process of bacteria's adhesion to the cells,invasion into the cells and then reproduction in the cells.Bacterial flagellin subunits were formed by fla A genes and they are one of the determinants of LM virulence.LM90SB₂ was isolated from the brain of a sheep with neurological symptoms.However,the effect of flaA gene on the virulence of LM90SB₂ and the formation of biofilm?BF?was unclear.The LM90SB₂-?flaA was constructed by homologous recombination technology in order to analyzed the influence of flaA gene on LD₅₀0 of the mice,the adhesion and invasion into cells,and the formation of BF.The main contents and results of the research are as follows:1.Construction and stability inheritance of fla A gene-deleted strain of LM90SB₂.In this study,the fusion band of?fla A was gained by using the splicing by overlap?SOE-PCR?method.After?flaA ligated with p MD19-T,the recombinant plasmid p KSV7-?fla A was constructed by double enzyme and DNA ligase reaction.The recombinant plasmid pKSV7-?flaA was transformed into freshly prepared LM90SB₂ cells and then recombined under the pressure of chloramphenicol and temperature.After 30 generations of gene recombination,double exchange gene deleted strains were constructed.Those deletion strains were stilled subcultured for generations without chloramphenicol at 30?,LM90SB₂-?flaA are not resistant of chloramphenicol and had good genetic stability.2.The effect of Fla A on LM90SB₂ motility and virulence on mice(LD₅₀)The motility of the bacteria were measured by semi-solid agar.The number of bacteria in mice internal organs were calculated by bacterial colony counting method and LD₅₀0 of mice were calculated by improved Curtis method.The colony diameter of LM90SB₂-?flaA was decreased from 1.38 cm to 0.50 cm,and the motor ability decreased by 63.77%.The bacterial load of LM90SB₂-?flaA in the liver of the infected mice was decreased compared with the wild strain?p<0.05?,the load in the spleen and kidney was lower but have no significant difference.The LD₅₀0 of LM90SB₂-?fla A?1.62×10⁷ cfu?was 3.8 times lower than the wild strain?4.27×10⁶ cfu?.The results showed that FlaA of the LM90SB₂ was related to movement and virulence.3.Effect of LM90SB₂-?fla A on adhesion and invasion of MBMEC and RAW264.7 cells.MBMEC and RAW264.7 cells were cultured as monolayers and then inoculated with LM90SB₂ and LM90SB₂-?flaA at the ratio of 5:1 or 10:1.After inoculated bacteria into MBMEC and RAW264.7 cells 1or 2 hours was lysed,and then viable bacteria were quantified by plating aliquots of serial dilutions on BHI agar.The result shown that the numbers of LM90SB₂-?flaA adherence to MBMEC cells were less than that of LM90SB₂,and the difference is significant?p<0.05?.The numbers of LM90SB₂-?flaA invasive to MBMEC cells were less than that of LM90SB₂,but with no significant difference?p>0.05?.The numbers of LM90SB₂-?flaA adherence and invasive to RAW264.7 cells were less than that of LM90SB₂,and the difference is significant?p<0.05?.Experiments have shown that the fla A gene can reduced the LM90SB₂'s ability to adhesion and invasion of cells.4.The influence of FlaA on the formation of LM90SB₂'s biofilmIn this study,LM90SB₂ and LM90SB₂-?flaA were cultured respectively in 96-well cell culture plates.After 2,6,10,12,24,and 48 hours,dye the cell culture plates with crystal violet respectively at every time point and then observe the forming of BF under a inverted fluorescence microscope.Ethanol-acetone mixture was used to dissolve BF.The amount of BF was measured quantitatively by measuring OD₅₉₅.Bacteria were cultured in 6-well cell culture plates with cover slips,and then stained them with PI or ConA,and finally placed them under the confocal laser scanning microscopy to observe the forming of BF.The results showed that under the microscope,LM90SB₂-?flaA are similar to LM90SB₂ in forming BF.They both formed a network structure within 10 hours,but the LM90SB₂-?flaA structure was loose and the compactness was poor.In OD₅₉₅,the BF formation rule of LM90SB₂-?flaA was approximately the same as that of the wild strain at 37?,and about 10 hours,they both entered into a stable period.But compared with wild strain,the amount of BF formed by LM90SB₂-?flaA declined at each time point in the process,and the difference is significant?p<0.05?.At 25?,the formation rule of BF of LM90SB₂-?flaA was roughly the same as LM90SB₂,but the amount of BF formed by LM90SB₂-?flaA decreased remarkably at each time point in the formation process,and the difference is extremely significant?p<0.01?.Through the confocal laser scanning microscopy,we found that the distribution of nucleic acids and polysaccharides in the BF structure of LM90SB₂-?flaA was loose,sparse,and scattered as compared to the wild strain.Experiments showed FlaA protein played an important role in the forming of BF in LM90SB₂.LM90SB₂-?flaA was successfully constructed by homologous recombination in this study.flaA related to the LM90SB₂'s movement and virulence on mice(LD₅₀).The motility,ability of adhesion and invasion to cells and the BF formation of the LM90SB₂-?flaA were sianificantly decreased compared with the wildtype strain LM90SB₂.This study confirmed that FlaA could affect LM90SB₂'s virulence and BF formation.