Construction And Some Biological Characteristics Of Lmo2193 Gene Deletion Mutant Strain Of Listeria Monocytogenes

Listeria monocytogenes?LM?is an intracellular pathogen that mainly infects individuals with lower immune function LM can cause sepsis,meningitis,and fetal infection or abortion.Oligopeptide transporter?Opp?is a type of protein that transports essential amino-aicd intracellular growth and is responsible for bacterial virulence in oligopeptide transport system.Based on the use of pan-genome technology,the lmo2193 gene was found having Opp activity in LM studies,however,the specific function is still unknown.In this study,the LM90SB2 which isolated from Xinjiang sheep with encephalitis was used.After lmo2193 gene were analyzed using bioinformatics and cloned into the prokaryotic expression vector,the gene deletion strain and the complementary strain were constructed by overlap extension PCR and homologous recombination technology.To investigate the function of lmo2193 as an Opp in LM and how does it effect on pathogenicity,the environmental adaptability,biofilm formation ability,cell adhesion invasiveness and intracellular proliferation virulence were analyzed among the deletion mutant,complementary strain and wild strain.Objective:?1?To putative the function of lmo2193 gene in LM90SB2 by bioinformatics analysis.?2?To verify the function of lm2193 by constructing the gene deletion strain and complementary strain.?3?To study the effect of lmo2193 gene deletion by detecting environmental adaptability and biofilm formation ability.?4?To study the effect of gene deletion on LM pathogenicity by virulence detection.Methods:?1?lmo2193 gene was amplificated by PCR,after cloning and sequencing,bioinformatics analysis,the expression of the protein was identified by SDS-PAGE gel electrophoresis and Western Blot technology.?2?The upstream and downstream arms were obtained using PCR amplification,the fusion fragment were amplificated by overlap extension PCR,ligated with the shuttle plasmid pKSV7 positive transformants was screened using electroporation into the competent strain of the wild strain,the gene deletion strain was obtained under double pressure subculture.The integrated plasmid pIMK2 was ligated and electroporated into the competent strain to screen the gene complementary strain;the genetic stability was examined.?3?The growth curves and the biofilm formation at different time periods were compared among the deletion strain,the wild strain and the complementary strain were compared under different environmental stress conditions.?4?Cell adhesion and invasion,intracellular proliferations were compared among the three different strains.The difference of bacterial burdens in the liver,the spleen and the brain of mice were compared and LD₅₀0 was detected after mice infected the three strains respectively.Results:?1?Bioinformatics analysis showed that lmo2193 protein was an ATP superfamily,the gene was presumed to be oppD.The product of prokaryotic expression was about 60 kDa which was consistent with expectation.?2?LM90SB2?oppD and CLM90SB2?oppD were successfully constructed,and maintained a good genetic stability.?3?LM90SB2?oppD had a slower growth rate than LM90SB2 and CLM90SB2?oppD when they were under different temperature,EtOH concentration,pH and NaCl concentration,the ability of mesh density and biofilm formation in LM90SB2?oppD were lower than LM90SB2 and CLM90SB2?oppD.?4?The cell adhesion and invasion rate decreased in LM90SB2?oppD compared with LM90SB2 and CLM90SB2?oppD.Interestingly,in SIEC cells,the amount of bacterial proliferation decreased gradually,but in RAW264.7 In MBMEC and HBMEC,the amount of bacteria increased gradually.LD₅₀ showed that the virulence in LM90SB2?oppD was 1.34 and 0.5 orders of magnitude higher than that of LM90SB2 and CLM90SB2?oppD,respectively.Bacterial burdens were decreased in LM90SB2?oppD infected mice but not in LM90SB2 and CLM90SB2?oppD.Conclusion:?1?It is presumed that the function of lmo2193 gene in LM90SB2 strain is OppD.?2?The genetically stable LM90SB2?oppD and CLM90SB2?oppD were successfully constructed.?3?The environmental adaptability and biofilm formation ability of LM90SB2 strain was decreased due to the deletion of lmo2193.?4?The deletion of the lmo2193 gene results in the decreased virulence in LM90SB2strain.