Construction And Some Biological Characteristics Of Lmo0331 Gene Deletion Mutant Strain Of Listeria Monocytogenes

Listeria monocytogenes?LM?is an important pathogenic bacteria that can cause serious listeriosis in huamans and animals,poses a great threat to the health of human and various animals.LM can cross the intestinal,blood-brain,maternofetal barriers through various virlence factors and causes sepsis,meningoencephalitis and abortion and other diseases.The mortality rate can be as high as 30%.LPXTG motif surface protein plays an important role in the LM infection.The Lmo0331 protein is a member of the LPXTG surface protein predicted from the LM genome sequence,and its function remained unknown.In this study,LM90SB2was used as bacterial material which isolated from the brain of illed sheep and serotype is 4b.Firstly,sequence analysis and prokaryotic expression of lmo0331 gene of LM90SB2 were performed.Secondly,lmo0331 gene deletion strain of LM90SB2 was constructed by using homologous recombination technology,and then,the adhesion and invasion ability to different cells,pathogenicity to mice,and environmental adaptation of LM90SB2 and LM90SB2?lmo0331 were determined,in this way the role of LM90SB2 Lmo0331 protein in the stress tolerance and virvlulence was probed.The research lays a foundation for further study of the pathogenic mechanism of LM.1.The bioinformatic analysis and prokaryotic expression of LM90SB2 lmo0331 geneIn order to clone the lmo0331 gene from the Listeria monocytogenes clinical isolate?LM90SB2?,analyze the protein domain and express it in prokaryotic system.Specific primers were designed according to the sequence of lmo0331 in GenBank.The lmo0331 gene of LM90SB2 was amplified by PCR and the amplified product was cloned into pMD19-T vector and sequenced for nucleotide sequence and protein domain analysis.The expression plasmid pET32a-0331 was transformed into E.coli BL21?DE3?competent cells.After induced by IPTG,the recombinant proteins were detected by SDS-PAGE and Western blotting.The results showed that the homologies of LM90SB2 lmo0331 gene with NTSN,F2365,LL195 and WSLC 1033 strains were 100.0%and the homologies with N2306,02-1792 and H34 strains were 99.9%.The lmo0331 gene of LM90SB2 was 1 956 bp in length and 1 857 bp in ORF,encoding a total of 618 amino acids.Protein domain prediction showed that Lmo0331 protein had NEL,LRR,IR,PulA and LPXTG domains.SDS-PAGE results showed about 88 ku recombinant protein band,and Western blotting showed that the protein could specifically bind with mouse anti-His-tag monoclonal antibody.2.Construction and identification of LM90SB2 lmo0331 gene deletion strainIn order to constructed the LM90SB2 lmo0331 gene deletion strain.The upstream and downstream homology arms of lmo0331 gene were amplified from LM90SB2.The two homology arms of lmo0331 gene were fused together to get the?lmo0331 fragment by using spliced overlap extension PCR?SOE-PCR?technique and then cloned into the suicide plasmid pKSV7.The recombinant plasmids pKSV7-?lmo0331 were electransformed into LM90SB2strains.The positive clones were screened by PCR.The positive transformants were subcultured in BHI with chloramphenicol at 41?,and LM90SB2?lmo0331 were confirmed by detection primers.LM90SB2?lmo0331 was subcultured in BHI without chloramphenicol at 30?to lost plasmid pKSV7 and identify genetic stability of LM90SB2?lmo0331.Results showed LM90SB2?lmo0331 was constructed by homologous recombination technology and the mutant had genetic stability.3.Influence of?lmo0331 on the in vitro environmental tolerance characterics of LM90SB2In order to explore the effect of lmo0331 gene on the adaptation of LM environment.The growth curve of Wild-type strain?LM90SB2?and lmo0331 gene deletion strain?LM90SB2?lmo0331?under different temperatures,pH,osmotic pressure and biofilm formation ability were detected.Results showed that the growth of LM90SB2?lmo0331 was better than that of LM90SB2 at 4??P<0.05?.Acid and alkali resistance test in vitro,the alkali resistant ability?pH=9?of LM90SB2?lmo0331 was lower than that of LM90SB2?P<0.05?.The growth of LM90SB?lmo0331 was significantly lower than LM90SB2 in BHI culture medium contain 4.5%ethanol.The growth of LM90SB?lmo0331 was significantly better than LM90SB2 in BHI culture medium contain 0.5%NaCl?P<0.05?.The above results suggested that Lmo0331 protein of Listeria monocytogenes plays an important role in alkali resistance.4.Influence of?lmo0331 on virulent characterics of LM90SB2In order to explore the effect of lmo0331 gene on the virulence of LM.The adhesion and invasion capacity to different cell lines and pathogenicity to mice of wild-type strain and mutant strain were determined.Results showed the adhesion capacities of LM90SB2?lmo0331 mutant strain to RAW264.7,MBMEC,HBMEC and SIEC cells significantly reduce comparing with LM90SB2 strain?P<0.05?.The invasion capacities of LM90SB2?lmo0331 mutant strain into RAW264.7,MBMEC cells significantly reduce?P<0.05?.Infection capacity of LM90SB2?lmo0331 to BALB/c mice decreased and the LD₅₀ was 0.97 times higher than LM90SB2.The microbial load of LM90SB2?lmo0331in liver and brain of mice were significantly less than that of LM90SB2 at different time points.The microbial load of LM90SB2?lmo0331 in spleen of mice were significantly less than that of LM90SB2 at 48h point after infection.These data indicated that surface protein Lmo0331 may be involved in LM virulence in mice.