Antibacterial Mechanism Of Mammalian Cathelicidins Against Pseudomonas Aeruginosa Infection

Pseaudomonas aenuginosa(P.aeruginosa)is an important opportunistic pathogen,which can infect many kinds of animals and leads to many diseases including mastitis,pneumonia and sepsis.It is also a nosocomial pathogen which often causes pulmonary infection.P.aeruginosa growing in a biofilm state is very difficult to treat because of the considerable degree of protection from the host immune system attack or antibiotic therapy,posing great challenge to the global public health.The ability of biofilm formation and the recent worldwide spread of antibiotic-resistant strains makes this pathogen even more difficult to treat.As a result,there is an urgent need for developing new antimicrobial drugs.Mammalian cathelicidins act as the potent microbicidal molecules for controlling bacterial infection,and are considered promising alternatives to traditional antibiotics.In this study,four mammalian cathelicidins which have ?-helix structure were synthesized,including human-derived LL-37,bovine-derived BMAP-27 and BMAP-34,murine-derived mCRAMP.The aims of this study were to compare the antibacterial and anti-biofilm activities of the four cathelicidins,to determine their immunomodulatory activity on neutrophils,and to evaluate the therapeutic efficacy of the combination of BMAP-27 and LL-37 against P.aeruginosa infection using a mouse pulmonary infection model.The results showed that all four cathelicidins exhibited strong antibacterial abilities against Escherichia coli,P.aeruginosa,Acinetobacter baumannii,Klebsiella peumoniae,Salmonella enterica,Staphylococcus aureus,Enterococcus faecium.The bactericidal activities of the four cathelicidins were more efficient against gram-negative strains than gram-positive strains.The results of PI uptake assay and TEM demonstrated that all of the four cathelicidins effectively killed bacteria via rapid induction of membrane permeabilization.The results showed that BMAP-27,mCRAMP and LL-37 effectively inhibited biofilm formation,while BMAP-34,mCRAMP and LL-37 exerted immunomodulatory functions with varying degrees of efficacy by stimulating the chemotaxis of neutrophils,inducing the production of reactive oxygen species,and facilitating the formation of neutrophil extracellular traps.BMAP-27 exhibited the most excellent bactericidal activity against diverse bacterial pathogens,but its immunomodulatory ability is weak,without the chemotactic function or ability to induce NETs at a low concentration.A mouse pulmonary infection model was used to evaluate the therapeutic efficacy of four peptides against P.aeruginosa infection.The results showed that the combination of LL-37 and BMAP-27 effectively enhanced the clearance of P.aenuginosa by reducing 1 000-fold number of bacteria,and alleviated the organ injuryin infected mice.Together,these findings highlight that identifying the appropriate synergistic combination of mammalian cathelicidins with different beneficial properties may be an effective strategy against bacterial infection,and have important guiding significance for the clinical application of new antimicrobial drugs including antimicrobial peptides.