| Listeria monocytogenes(Lm)is well known as one of the four most important foodborne pathogens,it can cross the intestinal barrier,the materno-fetalbarrier,the blood brain barrier of animal and human,and cause some clinical symptoms,such as meningoencephalitis,gastroenteritis and sepsis.There are a variety of virulence-associated proteins that closely involved in pathogenicity on the Lm cell wall.Among them,there are a class of proteins consisting of conserved LPXTG motif on the carboxyl terminal which can be cleaved by sortase A(SrtA),covalently anchored to peptidoglycan,presented on the surface of the bacteria.LM90SB2 was isolated from the brain of illed sheep and serotype is4 b.The differential cell wall proteins of LM90SB2 and SrtA deletion strain were screened by proteomic techniques,potential function of differential proteins were predicted by bioinformatics.The LM90SB2 cell wall protein Lmo2714 gene deletion strain(LM90SB2-?lmo2714)was.constructed by homologous recombination system and analyzed the biological characteristics compared to LM90SB2,such as ability of infection to cells and BALB/c mice,environmental adapt ability.The research will provide the foundation for the exploration of new cell wall surface proteins and pathogenic mechanism.1.Hydrolysis conditions optimization of Mutanolysin on Listeria monocytogenes: In order to improve the cell wall surface protein efficiency of LB90SB2,the proteins were obtained by enzymatic hydrolysis and TCA-acetone precipitation method.The concentration and hydrolysis time of mutanolysin were optimized.20?g/mL and 60?g/mL mutanolysin were used to hydrolyze LM90SB2 at 37? or 37? and 4 ? united action for 30min-4h,OD600 and CFU were measured before and after hydrolysis to judge membrane integrity and protoplast formation rate.Under optimal concentration of the mutanolysin,concentration of cell wall proteins were measured.The results showed that the protoplast formation rate increased from 0 to 91.00% with hydrolysis time from 30 min to 4h,proteins concentration was increased from 0.272mg/mL to 1.735mg/mL.?OD600 was change from 1.86% to14.50% with hydrolysis time from 30 min to 4h,there was no significant difference by analysis of statistics.91% of protoplast formation rate of LM90SB2 hydrolyzed by 60?g/mL mutanolysin at 37? for 4h was higher than 78% of protoplast formation rate hydrolyzed by20ug/m L mutanolysin at the same condition.So,60?g/mL mutanolysin at 37? for 4h was the best hydrolysis condition for LM90SB2.This study provides basis for the research on cell wall surface proteins.2.Screening of the differential cell wall proteins between LM90SB2 and LM90SB2-?srtA: To further explore cell wall surface proteins spectrum anchored by srtA,the Label-free quantitative methods combined with LC-MS/MS were used to screen the differential proteins,the potential function of differential proteins were predicted by bioinformatics analysis.The results showed that 697 protein groups and 3367 peptides were detected by Label-free quantitative method combined with LC-MS/MS,but there were only36 differential proteins,in which 20 proteins were detected in LM90SB2 and absent in LM90SB2-?srtA;7 proteins were detected in LM90SB2-?srtA and absent in LM90SB2;7proteins content in LM90SB2 was higher than in LM90SB2-?srtA(P<0.05);2 proteins content in LM90SB2 were less than in LM90SB2-?srt A(P<0.05).Bioinformatics analysis showed that the 36 differential proteins were related to the factors such as the internalin,the cell wall anchoring proteins.The identification of srtA sorting proteins have laid foundation for the exploration of new virulence factors and drug targets.3.Construction and identification of LM90SB2 lmo2714 gene deletion strain(LM90SB2-?lmo2714): In order to study the function of LM90SB2 Lmo2714,LM90SB2-?lmo2714 deletion mutant was constructed by homologous recombination method.First,the upstream and downstream homology arm of lmo2714 gene open reading frame were amplified by SOE-PCR,and then connected with pMD19-T vector to construct pMD19-T-?lmo2714,which was sequenced.Connected with the shuttle plasmid pKSV7 to construct pKSV7-?lmo2714,the pKSV7-?lmo2714 was transferred into the competent cell of LM90SB2 by electrophoretic transfer.The positive clones were screened in BHI-chloramphenicol agar plate,serially cultured in BHI-chloramphenicol broth at 42? for homologous recombination.LM90SB2-?lmo2714 deletion mutant were confirmed by PCR with special primers(lmo2714-L1 and lmo2714-L4,lmo2714-dele1 and lmo2714-dele2)and was continually passaged in BHI broth at 30? until pKSV7-?lmo2714 was not detected.The results showed that the bands size of deletion strain were 1033 bp and the parent strain were1981 bp observed by using special primers.After 20 passages,there was no recurrence occurred.This indicated that the LM90SB2-?lmo2714 deletion strain was successfully constructed and had a good genetic stability.4.Comparative analysis the partial biological characteristics of LM90SB2-?lmo2714: In order to explore the function of Lmo2714,biofilm formation ability,biochemical characteristics,adaptability to environment,pathogenicity to mice and adhesion,invasion,proliferation on MBMEC,SIEC,RAW264.7 and HBMEC cells were detected.The results showed that there were no significant differences in the acid and alkali adaptability and biofilm formation ability,partial difference in biochemical characteristics between LM90SB2 and LM90SB2-?lmo2714.But the LD50 of LM90SB2-? lmo2714 strain was 1.34 higher than that of the LM90SB2;The numbers of LM90SB2-?lmo2714 in the mice's liver?spleen and brain were less than that of LM90SB2 at 72 h after infection((P<0.01));the adhesion rate of LM90SB2-?lmo2714 to MBMEC was less and to SIEC was higher than that of LM90SB2(P<0.05);the invasion rate of LM90SB2-?lmo2714 to MBMEC and RAW264.7 were less than that of LM90SB2(P<0.05).The growth numbers of LM90SB2-?lmo2714 in cells were less than that of LM90SB2,but the difference was not significant.This study suggested that Lmo2714 partly involved in attenuating the ability of pathogenicity in mice. |
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