Studies On Antibacterial Mechanism Of ε-Poly-L-lysine Against Pathogens In Molecular Level And Its Effect On Intestinal Microbial Diversity

ε-Poly-l-lysine (ε-PL), a natural homo-poly-amino acid, is characterized as being edible, water soluble, biodegradable, stable and low toxic, and widely used as an antibacterial agent for its broad antimicrobial activity against bacteria, yeasts, molds and others. Despite of wide using in food, the mechanism of ε-PL against pathogens at the molecular level has not been elucidated.In this study, microbial cultivation, atomic force microscopy (AFM), transmission electron microscopy (TEM), real-time quantitative polymerase chain reaction (RT-qPCR), suppression subtractive hybridization (SSH) and gene knockout technology was used to investigate the antibacterial activity of ε-PL against typical foodborne and nosocomial pathogens, in order to understand the antimicrobial mechanism in molecular level. In addition, the effect of ε-PL on the fecal microbiota of mice was analyzed by using denaturing gradient gel electrophoresis (DGGE) and RT-qPCR analysis.The whole research work is described in six chapters. In Chapter1, the type, character and antimicrobial range of food preservatives (especial from natural sources) were described. As a kind of natural food preservative, the physical and chemical characters, antimicrobial activity and mechanism, and the application range as well development tendency of ε-PL were reviewed.In Chapter2, E. coli O157:H7(CMCC44828) was chosen as a model to investigate the antimicrobial activity and mechanism of ε-PL against pathogens. The results indicated that the antibacterial effect of ε-PL increased with rise of temperature (4℃,25℃and37℃) and prolonging of the treatment time. The inhibition effect of ε-PL was stable in the range of pH5-7, but decreased in alkaline environments (when pH up to8).Verification of the destructive effect of ε-PL on cell structure and membrane integrity was performed by AFM and TEM. The surface of treated bacteria was rough and had an uneven shape. The cell membrane of cells exposed to ε-PL showed collapsed, lysed and rough membranes, a cavitated cell shape, and non-integral membranes. Results showed a positive correlation between ROS levels and ε-PL concentration in cells. RT-qPCR results in this work showed that ε-PL induced the up-regulation of bacterial oxidative stress response, elicited the SOS response, and reduced the transcription of virulence genes.In order to analyze the effects of ε-PL at molecular level and examine its effect on gene expression, C. albicans, a frequent mucosal infection and hardly handling pathogen was studied as another model of pathogen in Chapter3. Suppression subtractive hybridization (SSH) was used to identify differentially expressed genes from in responses to ε-PL. A total of10subtracted clones sharing high homologies with known genes of C. albicans were isolated under the sublethal concentrations of ε-PL. RT-qPCR was used to validate these results, which suggested that ε-PL might inhibit carbohydrates metabolic and lead to lipid metabolism disorders of C. albicans through reduced expression of glycolysis and lipid metabolism associated genes. Increased expression of arginyl tRNA synthetase ARGS, translational activator GCN1and ubiquitin protein ligase Asi3suggested the cell structures of C. albicans were destroyed during ε-PL exposure, even enhancing protein degradation.Gene absence by gene knockout technology is an effective method to verify the antibacterial mechanism of ε-PL. Salmonellae are the major foodborne pathogens in contaminated food, which uses two-component regulatory systems to regulate virulence and respond to cationic antimicrobial peptide resistance. In Chapter4, to understand the inhibition effect of ε-PL on S. enleritidis in regulatory systems(phoP and rcsF), gene of PhoP-PhoQ and RcsFCDB was knocked out. The results showed antibacterial activity of ε-PL againsted ΔphoP mutant significantly increased. The expression level of downstream genes regulated by PhoP-PhoQ increased. However, the growth rate of ΔrcsF mutant did not change when compared with the wild strain, indicating that PhoP-PhoQ but not RcsFCDB plays an important role in S. enteritidis facing the challenge of ε-PL ε-PL is an antimicrobial peptide used in many countries as a safe and natural antibacterial food preservative. However, no studies were conducted to investigate its effect on microbial diversity in the intestine. In Chapter5, the effect of ε-PL’s administration for two weeks on mice fecal microbiota was analyzed by using DGGE and RT-qPCR. Results indicated that ε-PL significantly increased the microbiota composition of Lactobacillus species. No significant variation was demonstrated in the proportion of Lactobacillus, Bacteroides, and Enterobacteriaceae when compared with the initial microbiota. The results showed that ε-PL may not significantly affect the stabilization of microbial population in the gastrointestinal tract.