Antimicrobial activity of natural and mutant variants of free and immobilized antimicrobial peptides against Listeria and E. coli
Over the past decade, the emergence and rapid spread of microorganisms resistant to antibiotics have an enormous impact on morbidity and mortality as well as on healthcare costs. One potential candidate of novel antimicrobial compounds is antimicrobial peptides (AMPs), which are relatively short peptides that have the ability to penetrate the cell membrane, form pores leading to cell death. This study mainly investigates the antimicrobial activity of natural and mutant variants of free and immobilized antimicrobial peptides against Listeria and E. coli from three aspects. The first part of the study compares both antimicrobial activity and cytotoxicity of native and two mutants, namely, melittin I17K (GIGAVLKVLTTGLPALKSWIKRKRQQ) with a higher charge and lower hydrophobicity and mutant G1I (IIGAVLKVLTTGLPALISWIKRKRQQ) of higher hydrophobicity. The antimicrobial activity against different strains of Listeria were investigated by bioassay, viability studies, fluorescence and transmission electron microscopy. Cytotoxicity was examined by lactate dehydrogenase (LDH) assay on mammalian Caco-2 cells. The minimum inhibitory concentration (MIC) of native, mutant I17K, mutant G1I against L. monocytogenes were 0.315±0.008, 0.814±0.006 and 0.494±0.037 μg/ml respectively, whereas the minimum bactericidal concentration (MBC) values were 3.263±0.0034, 7.412±0.017 and 5.366±0.019 μg/ml respectively. Lag time for inactivation of L. monocytogenes was observed at concentrations below 0.20 and 0.78 μg/ml for native and mutant melittin I17K respectively. The antimicrobial activity against L. monocytogenes was in the order native > G1I > I17K. Native melittin was cytotoxic to mammalian Caco-2 cells above concentration of 1 μg/ml, whereas the two mutants exhibited negligible cytotoxicity up to a concentration of 8 μg/ml. Pore formation in cell wall/membrane was observed by transmission electron microscopy. Molecular dynamics (MD) simulation of native and its mutants indicated that (i) surface native melittin and G1I exhibited higher tendency to penetrate a mimic of bacterial cell membrane and (ii) transmembrane native and I17K formed water channel in mimics of bacterial and mammalian cell membranes. Due to strong antimicrobial activity of AMPs and their superior mechanism of pore formation, foodborne pathogen can be effectively deactivated by AMPs at a certain low concentrations. It is well known that low intensity ultrasound may also help deactivate microorganisms by thinning the cell membranes as a result of regular oscillations of the bubbles produced by cavitation. The second part of the study characterized the antimicrobial activity of melittin against L. monocytogenes F4244 in the presence of low power ultrasound. Combination of low power ultrasound with melittin treatment is shown to result in a significantly greater reduction of L. monocytogenes F4244 (CFU reduction of 2.5 x 105 ) when treated with both 60 W ultrasound and 0.78 µg/ml antimicrobial peptide compared to melittin treatment alone (CFU reduction of 100) and low power ultrasound alone (CFU reduction of 104) respectively. Promising results obtained in this study indicates the possibility of application of this more economical and effective sterilization process of combination of low power ultrasound and natural antimicrobial peptide in microbial deactivation on fresh produce. The third part of the study characterized the antimicrobial activity of Cecropin P1C that was adsorbed or immobilized onto silica nanoparticles by covalent linkage using polyethylene glycol (PEG) cross-linker. Antimicrobial activity of adsorbed Cecropin P1C against E. coli O157:H7 EDL933 was identical to that in solution. The surface coverage of immobilized Cecropin P1C onto silica nanoparticles was varied from 12.3% to 83.8% and three different linker lengths, namely, (PEG)2, (PEG)6 and (PEG)20 were investigated. Immobilized Cecropin P1C lost its secondary α helical structure for (PEG)2 and (PEG)6 whereas part of the secondary structure was retained for (PEG) 20 as evidenced by circular dichroism. MIC for immobilized Cecropin P1C was found to be much higher than that for free and adsorbed Cecropin P1C and decreased with an increase in surface coverage, from 86.06 ± 1.41 µg/ml for 12.3% to 17.84 ± 0.11 µg/ml for 83.8%. MIC increased slightly from 24.38 ± 0.21 µg/ml to 37.59 ± 0.05 µg/ml when the linker length was decreased from 20 to 6. Further decrease of linker length to 2, however, resulted in a drastic decrease in antimicrobial activity as indicated by its MIC of 109.82 ± 0.21 µg/ml. MD simulation of PEGylated Cecropin P1C interacting with DOPG/DOPC (1:3) mixed membrane show that PEGylation affects the interaction between Cecropin P1C and lipid bilayer by significantly blocking the binding of peptide to the bilayer. In addition, PEGylation destabilizes the α-helical structure of Cecropin P1C, which qualitatively explains the experimental observation of a decrease in antimicrobial activity of immobilized Cecropin P1C.
Narsimhan, Purdue University.
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