| Listeria monocytogenes（LM）is a food-borne pathogen that widely exist in nature environment and infects people and animals through contaminated food.It is an intracellular pathogen bacterial that can cross the blood brain barrier,the intestinal barrier and the placental barrier and cause some clinical symptoms,such as diarrhea,meningoencephalitis,sepsis,neonatal hemolysis,hepatitis,abortion and so on.The morbidity and mortality of Listeriosis are higher.There are a variety of proteins on the surface of LM cell wall,among them there is one type of surface proteins that have a conserved LPXTG（Leu-Pro-X-Thr-Gly）motif in COOH-terminal and were covalently bounded to peptidoglycan by sortase A and present on the cell surface to play an important role in the virulence of LM.According to the whole genome sequence of LM EGD-e reference strain,41LPXTG motif proteins were predicted,among which the functions of Lmo0159 and Lmo0160 were unknown.In this study,LM90SB2 strain isolated from Listeriosis sheep brain was used as parent strain.The genes of lmo0159 and lmo0160 of LM90SB2 were cloned and bioinformaticsanalyzed.ThedeletionstrainsofLM90SB2-（35）lmo0159and LM90SB2-（35）lmo0160 were constructured by homologous recombination technology.The differences in environmental tolerance,biofilm formation ability,and cell-level and mouse-level virulence between deletion strains and parent strain were detected to explore the function of lmo0159 and lmo0160 genes,which provides the basis to further clarify the pathogenesis of LM.The main research contents and results are as follows:1.Cloning and sequence analysis of lmo0159 and lmo0160 genes:In order to understand the characteristics of the lmo0159 and lmo0160 genes encoding proteins of LM90SB2 strain.The lmo0159 and lmo0160 genes were amplified by PCR,connected to pMD19-T vector respectively,sequenced and analyed.The results showed that the sequence length of lmo0159gene was 2 070 bp,encoding 617 amino acids;the sequence length of lmo0160 gene was 1708 bp,encoding 475 amino acids;the nucleotide sequence homology of lmo0159 and lmo0160 gene of LM90SB2 strain were more than 99%with different source strains of 4b serotype.According to the deduced amino acid sequence,bioinformatics software analysis showed that Lmo0159 protein was a kind of acidity,stable and hydrophilic protein and Lmo0160 protein was a hydrophobic protein,both Lmo0159 and Lmo0160 proteins included collagen binding domain and collagen-binding surface protein Cna-like,B-type domain（Cna B）.The cloning and analysis of lmo0159 and lmo0160 genes of LM90SB2 strain provide a theoretical foundation for further functional study of lmo0159 and lmo0160 genes.2.Construction and identification of LM90SB2-（35）lmo0159 and LM90SB2-（35）lmo0160 deletion strain:In order to research the function of lmo0159 and lmo0160 genes of LM90SB2.In this study,the upstream and downstream sequence of lmo0159 and lmo0160genes were amplified by PCR respectively,homologous arm fragments of（35）lmo0159 and（35）lmo0160 were fused by SOE-PCR.The pMD19-T-（35）lmo0159,pMD19-T-（35）lmo0160,pKSV7-（35）lmo0159 and pKSV7-（35）lmo0160 were constructed.The pKSV7-（35）lmo0159 and pKSV7-（35）lmo0160 were transferred into LM90SB2 strain by electrotransfermation.The positive clones were subcultured in BHI-chloramphenicol（10μg/mL）broth at 42℃for homologous recombination,LM90SB2-（35）lmo0159 and LM90SB2-（35）lmo0160 deletion strains were confirmed by PCR and were subcultured in BHI broth at 37℃for 30 passages to detect the genetic stability by PCR.The results showed LM90SB2-（35）lmo0159 and LM90SB2-（35）lmo0160 deletion strains were successfully constructed and genetically stable.3.Research on environmental adaptation and biofilm formation capacity of LM90SB2-（35）lmo0159 and LM90SB2-（35）lmo0160 deletion strains:To investigate the effect of lmo0159 and lmo0160 genes deletion on LM environmental adaptability.The growth of bacteria in the different temperature,pH,NaCl concentrations,alcohol concentrations and the biofilm formation capacity were determined.The results showed that the growth rate of LM90SB2-（35）lmo0159 and LM90SB2-（35）lmo0160 were reduce compared with LM90SB2 at37℃and 42℃;The growth rate of LM90SB2-（35）lmo0159 and LM90SB2-（35）lmo0160 were lower than that of LM90SB2 in the BHI medium containing 0.5%and 2.5%NaCl,3%and3.5%alcohol.The crosslinking density of the biofilm of LM90SB2-（35）lmo0159 and LM90SB2-（35）lmo0160 were lower than that of LM90SB2 by microscopic examination.The quantitative analysis of biofilm showed that the OD57070 nm difference were greater with the extension of time,expecially at 48h,difference were extremely significant（P?0.001）.The results indicated that the deletion of lmo0159 gene and lmo0160 gene dereased the adaptability of stresses environment and biofilm formation capacity of LM90SB2.4.Virulence of LM90SB2-（35）lmo0159 and LM90SB2-（35）lmo0160 deletion strains:To investigate the effect of lmo0159 and lmo0160 genes deletion on LM virulence.Different types of cells that MBMEC,RAW264.7,SIEC and HBMEC were used as the cell model,the adhesion,invasion and intracellular growth ability of parent strain and deletion strains were detected,respectively.The LD50,the amount of bacteria in the liver,spleen and brain and the survival time of mice were measured after mice were injected intraperitoneally with parent strain and deletion strains.The results showed that compared with LM90SB2,the adhesion rate,invasion rate of the LM90SB2-（35）lmo0159 to SIEC and RAW264.7 were all decreased,and difference were extremely significant（P<0.01）;the adhesion rate,invasion rate ability of the LM90SB2-（35）lmo0160 to RAW264.7 were decreased,and difference were significant（P<0.05）and were extremely significant（P<0.01）,respectively;The intercellular growth ability of deletion strains to MBMEC,SIEC,and HBMEC were all decreased;The survival time of mice infected LM90SB2-（35）lmo0159 and LM90SB2-（35）lmo0160 strains were prolonged significantly than that of LM90SB2 strain（P<0.05）.The LD500 of the LM90SB2-（35）lmo0159and LM90SB2-（35）lmo0160 strains were higher 100.97.97 and 100.86.86 than that of LM90SB2 strain;The bacterial loading quantity of the LM90SB2-（35）lmo0159 and LM90SB2-（35）lmo0160 strains in the liver and spleen were all lower than that of LM90SB2 strain.This study suggested that after the lmo0159 and lmo0160 genes were deleted,the virulence of LM90SB2 strain was decreased. |
