Engineering Of A Listeria Phage Lysin With Improved Bactericidal Activity And Its Application In Listeria Detection

Listeria monocytogenes is a foodborne gram-positive pathogen capable of spreading from a single cell to neighboring cells.The listeria infection could cause meningitis,bacteremia or septicemia.Gentamicin and kanamycin could be used to inhibit the growth of L.monocytogenes.However,in recent years there has been an upsurge of antibiotic resistance due to the abuse of antibiotics.Therefore,there are needs to find alternative antimicrobials.Bacteriophage lysins or phage encoded peptidoglycan hydrolases,have been considered as promising alternatives to traditional antibiotics.The natural listeria lysins are specific and have a low possibility to induce bacterial resistance.However,the lytic activities of these lysins are relatively low.In this project,we created a chimeric listera lysin Ply511-N2 with improved activity by fusing a cell penetrating peptide Tat to the N-terminus of lysin Ply511.Ply511-N2 has a high active against planktonic L.monocytogenes,with reductions of 1-3 logs in viable cell number after treatment of the bacteria with 100?g/mL of Ply511-N2 for 60 minutes in PBS,but not active against intracellular L.monocytogenes.Enzymatic characterization indicated that Ply511-N2 maintains good activity within pH range of 6.0-9.0 at temperatures from 4 to 45?.Ply511-N2 showed similiar host range and specificity to its parental lysin Ply511 against L.monocytogenes,but no activity against Streptococcus agalactiae,Staphylococcus aureus,and Streptococcus mutans.MTT assay showed that CHO-K1 cells maintained more than 90%viability after exposure to 250?g/ml of either Ply511-N2 or Ply51l.Further,we developed a rapid detection method for L.monocytogenes by combining the high lytic activity of Ply511-N2 with the high sensitivity of luciferase based ATP detection.Obvious increase of ATP signal could be detected in Ply511-N2-treated L.monocytogenes cells but not Ply511-N2-treated S.aureus cells.The detection limit for L.monocytogenes was 10~4-10~5 CFU/mL.The method could be also easily performed without expensive instruments by non-technicians.Therefore,this method is promising for practical use after further improvement.In summary,this research developed a new chimeric lysin with high acitivty agaist Listeria.Based on this lysin,rapid detection of Listeria is feasible and sensitive.