The Protective Effect And Multiple Mechanisms Of Immunomodulatory Peptide CATHPbl Against Drug-resistant S.Aureus Infections

The emergence of multiple drug-resistant Staphylococcus aureus has been of great concern in clinical settings,due to its wide area outbreak,high infection rate and high mortality.Recently,methicillin-resistant S.aureus(MRSA)infection proportion gradually increased across the world,and has become one of the three most difficult infectious diseases.Another critical issue with S.aureus is reduced susceptibility to vancomycin,such as vancomycin-intermediate S.aureus(VISA)and vancomycin-resistant S.aureus(VRSA).This situation has hastened an aggressive search for new paradigms or novel antimicrobial agents for anti?infective therapy,especially for multidrug-resistant S.aureus infections therapy.Recently,host-directed immunomodulatory therapies,has been proved to be a promising therapeutic strategy in prevention and treatment of infections.Immunomodulation offers certain advantages.By targeting the host rather than the pathogen,immunomodulation largely avoids selective pressure for the evolution of microbial resistance.Furthermore,the nonspecific nature of innate immune defenses suggests that their modulation affords broad-spectrum protection against a range of microbial pathogens,including the drug-resistant bacteria.Several innate defence regulator peptides have been tested in clinical trials to treat bacterial infections.Cathelicidins are an important immune regulatory factor in vertebrate.In addition to the potent bactericidal activity,cathelicidin has multifunctional roles in host immune responses,including directly and indirectly promotion of immune cells recruitment and differentiation,modulation of pro-and anti-inflammatory responses,promotion of angiogenesis and promotion of wound-healing,etc.This work aims to evaluate the in vivo anti-infection effects and multiple mechanisms of CATHPb1 an immunomodulatory peptide identified from P.bivittatu with potent in vitro antimicrobial and immunoregulatory activities.The main results obtained by this work were summarized below:(1)Six full-length novel natural occurring cathelicidins(CATHPbl-6)were firstly identified and characterized from P.bivittatu by RACE-PCR.The six deduced mature peptides were then screened for antimicrobial,anti-biofilm and immunomodulatory activities.CATHPbl,2,4 exhibited potent broad-spectrum antimicrobial activities against most of the tested microorganisms,especially for some drug-resistant strains.CATHPbl-4 possessed strong ability to prevent the formation of bacterial biofilms and eradicate the performed biofilms.CATHPb1,2,4,6 showed potent immunoregulatory effects in vitro.Among them,CATHPbl exhibited the best in vitro pharmacological and toxicological profile,and therefore was selected for further in vivo study.(2)We show that CATHPb1 has protective effect on a broad range of in vivo infection mice models,especially on MRSAAVRSA infection models.Either given before or after infection,CATHPb1 significantly improved the survival rate of infected mice,decreased the bacterial loads in mice peritoneal lavage and spleen,and showed a stronger protection than CPZ/SBT(20 mg/kg)in vivo even much smaller dosage of 5 mg/kg.Meanwhile,the protection of CATHPbl is also effective by both local and systemic administration.Given its neglectable toxicities such as mast cell degranulation,cytotoxicity and hemolysis,and was more resistant to protease degradation in serum,CATHPb1 has great potential to better fulfill its anti-infection therapeutic promise.(3)Studies were undertaken to uncover the mechanism by which CATHPb1 enhanced the resolution of infection.The potent in vitro antimicrobial activities of CATHPbl were achieved by inducing the cellular disruption of bacterial cells,by its amphipathic a-helix conformation.Acting through MAPK and NF-?B signaling pathways,CATHPb1 can also exactly balance or control the attendant inflammatory response of VRSA infection.In addition,the in vivo data implicated that macrophages/monocytes and neutrophils are key effector cells in CATHPbl's protection,and the protective effect of CATHPb1 against MRSA/VRSA infection might mainly achieved by modulating the trafficking and functions of neutrophils.(4)Further research on the bactericidal function of neutrophils indicated that,CATHPbl significantly promoted the respiratory burst of neutrophils,and neutrophils generated more ROS than macrophages by CATHPbl stimulation.In addition to elevating the direct killing effect of neutrophils,CATHPb1 could significantly facilitate the neutrophil extracellular traps(NETs)formation to entrap pathogen,while stabilizing the NETs structure against bacterial nuclease degradation.