Construction Of SrtA Gene Deletion Mutant Strains Of Listeria Monocytogenes And Research On Some Biological Characteristics

AbstractListeria monocytogenes（LM） is well known as one of the four most immportant foodborne pathogens, pose a great threat to the health of human and various livestock and poultry. LM can cross the intestinal barrier, the materno-fetalbarrier, the blood brain barrier（BBB） of animal and human, cause gastroenteritis, meningoencephalitis, sepsis and sbortion some clinical symptoms, especially for young female and pregnancy. LM have a variety of virulence-associated proteins that closely involved in pathogenicity on the cell wall. The one type of surface proteins comprise a COOH-terminal cell wall sorting signal, consisting of a conserved LPXTG motif that can be cleaved by sortase and anchoring peptidoglycan to present on the cell surface. LM90SB2 was isolated from the brain of illed sheep and serotype is 4b. This study have analysis of LM90SB2 srt A gene sequence and ligase to plasmid p ET32 a for prokaryotic expression, construct the LM90SB2 srt A gene deletion strain（LM90SB2-△srt A） by homologous recombination system and analyzed the biological characteristics compared to LM90SB2, such as growth characteristics, ability of infection to cells and BALB/c mice. This establishe the foundation for exploring the Srt A separation of protein spectrum, looking for new virulence proteins.It plays an important role in research on pathogenesis and screening for drug target.1. Cloning and Prokaryotic Expression of LM90SB2 srt A gene:In order to investigate the specificity of srt A genes of LM90SB2, and the expression of srt A gene in plasmid p ET32 a. The srt A gene was amplified from LM90SB2 by PCR, analyzed sequence and cloned to the plasmid p ET32 a. The recombinant expression plasmid p ET32a-srt A transformed into E.coli BL21（DE3）, induced with isopropyl-β-D-thiogalactopyranosid（IPTG）, and the predicted fusion protein was detected by SDS-PAGE and Western blotting. These results showed that the nucleotide homology were 100% when compared with LMF2365（serovar 4b,NC₀02973）, Srt A were effectively expressed in E.coli BL21（DE3）, the recombinant protein had molecular weight approximately 47 k D via analysis of SDS-PAGE and Western blotting.2. Construction and identification of LM90SB2 srt A gene deletion strain（LM90SB2-△srt A）: Gene deletion is the most common method to study genic functions. LM90SB2-△srt A deletion mutant strain was obtained by homologous recombination, laid a basis on study the function of LM90SB2 srt A gene. The primers were designed using Primer 5.0 software, the srt A gene upstream and downstream homologous sequences was amplified by PCR, then were used in splice overlap extension PCR（SOE-PCR） to obtain the fusion fragment of srt A deletion gene（Δsrt A）, p MD19-T-Δsrt A was constructed by inserting Δsrt A fragment into p MD19-T vector, and sequencing. The obtained fragments Δsrt A were cloned via the Pst I and Bam HI into the p KSV7 giving rise to plasmids p KSV7-Δsrt A. The recombinant plasmid p KSV7-Δsrt A was transferred into wild type LM90SB2 by electrophoretic transfer. The positive clones were screened in BHI-chloramphenicol agar plate, serially passaged in BHI-chloramphenicol broth at 41℃ for homologous recombination, then were confirmed by two paired primers（srt AF1 and srt AR2, srt AF and srt AR） PCR to identify mutants with correct in-frame deletions. Finally, the deletion srt A strain was continually passaged in BHI broth at 30℃ until stability of LM90SB2 with deletion srt A strain was selected. The LM90SB2-Δsrt A had no change by PCR testing after cultured in BHI at 37°C passage 20, and indicated it have good genetic stability.3. Comparative analysis the partial biological characteristics of LM90SB2-Δsrt A: The srt A gene have impact on virulence of LM90SB2 throught analyzed the biological characteristics of LM90SB2-Δsrt A compared to LM90SB2, such as growth characteristics, hemolytic activity, ability of biofilm formation and bacteria infection to cells（MBMEC, HBMEC, RAW264.7 and SIEC） and BALB/c mice. The results showed that: the srt A gene deletion has no significant effect on the LM90SB2 biochemical and acid and alkali resistant characteristics, and was slightly effective to hemolytic activity, ability of biofilm formation. LM90SB2 have different infection efficiency of adhesion, invasion and intracellular proliferation on different cell, the most efficient of RAW264.7, the brain cells efficiency is relatively low. The adhesive and invasive capacity of LM90SB2-Δsrt A to HBMEC, RAW264.7, and SIEC demonstrated a significant reduction compared to the parental strain（P<0.05）; the intracellular proliferations of deletion strain were not significantly varied on different cells except the number of bacteria decreased. The pathogenicity test showed that LD50 of mice was increased 1.63 logs with the deletion strain to that with the parental strain; In addition, overwhelming post-infection 4 days contained a reduced burden of bacteria in liver, spleen, and brain compared to LM90SB2 infection group. This study suggests that deleted srt A gene involved in reducing bacteria adhesion, invasion and intracellular proliferation on cells, further attenuating the ability of pathogenicity in mice after infection.