Research Of Antimicrobial Peptide With Trp Bactericidal Mechanism To Drug-resistant Staphylococcus Epidermidis

Multidrug-resistant Staphylococcus epidermidis has become major pathogens that caused nosocomial infection due to the lack of appropriate antimicrobial drugs.Therefore,the development of new antimicrobial agents has become essential.Antimicrobial peptides were expected to become a new alternative to conventional antibiotic which were difficult to generate bacterial resistance and had good stability,broad-spectrum antimicrobial activity.Natural antibacterial peptide temporin-1CEb was isolated from the Chinese forest frog skin secretions.We replaced Leu and Pro in the hydrophobic surface with lysine(Lys),and obtained the modified peptide L-K6.L-K6 has a broad-spectrum antimicrobial activity and low hemolytic activity.Previous studies showed that the Trp-substitution L-K6 exhibited different antibacterial effect to sensitive bacteria and drug-resistant bacteria.The study on antibacterial mechanism demonstrated that the modified peptide could combine bacterial surface charge through electrostatic interaction and then destroy the integrity of cell membrane.In this thesis,we chose a series of L-K6 analogues in which the assigned sites were replaced by tryptophan(Trp)to explore the interaction of modified peptides with bacterial biofilms,bacterial outer membranes,membrane-related gene expressions of clinical multidrug resistant Staphylococcus epidermidis.The results indicated that I1 W showed the ability to make both biofilm decomposition or formation inhibition at the concentration of 3.12μM,and there is no significant difference to sensitive and resistant Staphylococcus epidermidis.The biofilm formation was fully inhibited by I4 W at the concentration of 6.25μM.The reason for this difference was mainly due to the MIC value of I4 W and I4 W showed stronger inhibit biofilm formation and permeate outer membrane ability to sensitive bacteria than drug-resistant bacteria.But the inhibiting biofilm formation of drug-resistant Staphylococcus epidermidis by L5 W and L11 W and these two peitides showed lower ability to decompose the biofilm of drug-resistant bacteria at high concentration.L5 W exhibited higher ability to permeate outer membrane of drug-resistant bacteria.Compared with other antimicrobial peptides,L12 W could significantly decrease the biofilm formation.Only at very high concentration,the peptides I1WL5 W and L4WL5 W can inhibit biofilm formation.I4WL5 W showed lower biofilm decomposition ability to resistant bacteria than sensitive bacteria.Real-time PCR experiments indicated that seven kinds of antimicrobial peptides could downregulate icaA and icaD gene expression after on thepeptides interacted with resistant bacteria for 24 hours,among which L12 W had almost no obvious effect and the gene expression of two icaA and icaD was not significant.The results showed that we could get different effect when the specific site of L-K6 was displaced byTrp.The structural difference may lead different antibacterial mechanisms.L-K6 analogues had multiple mode of action on drug-resistant bacteria,and was hard to make the bacteria drug resistance,and these peptides were expected to become an alternative to traditional antibiotic and new effective antimicrobial agents to treat infections caused by drug-resistant bacteria.