Cloning,Structure And Function Analysis Of Cathelicidin Family Antimicrobial Peptides From Pelophylax Nigromaculata

Cathelicidin is an important family of antimicrobial peptides(AMPs),which plays a key role in the immune response of vertebrates against microbial infections.At present,four cathelicidin-related drugs are undergoing clinical trials,which indicates that cathelicidin have great potential to be developed as new antimicrobial drugs.Therefore,it is of great importance to identify more novel cathelicidin from vertebrates.The black-spotted frog belongs to the genus Amphibian,Anura,Ranidae,and Pelophylax,and is one of the most widely distributed amphibians in China.In the present study,two novel cathelicidin family AMPs PN-CATH1(KKCNFFCKLKKKVKSVGSRNLIGSATHHHRIYRV)and PN-CATH2(EGCNILCLLKRKVKAVKNVVKNVVKSVG)were identified from the skin of Pelophylax nigromaculata.PN-CATHs showed potent and broadspectrum antimicrobial activity against the tested bacteria,including human and aquatic pathogenic bacteria.Mechanism study showed that they kill bacteria mainly by destroying the bacterial membrane,and inducing the formation of intracellular reactive oxygen species(ROS).Besides,they exhibited strong anti-biofilm activity and are capable of eliminating persister bacteria,indicating that they have potential to combat infections caused by persister bacteria in biofilms.In addition to direct antimicrobial activity,PN-CATHs also exhibited strong anti-inflammatory activity.They could effectively inhibit the production of pro-inflammatory cytokines such as IL-6,IL-1β,and TNF-α in primary mouse peritoneal macrophages induced by lipopolysaccharide.PN-CATHs can directly bind to LPS and neutralize the toxicity,which may be partly responsible for their antiinflammatory activity.Furthermore,PN-CATHs exhibited strong in vitro free radical scavenging activity,and their gene expression in the frog skin was significantly increased after ultraviolet radiation.They may play important roles in host response to ultraviolet radiation.The cytotoxicity of PN-CATHs toward B16 F10,Ha Cat,MPMs and mouse red blood cells was also determined.They exhibited weak cytotoxicity and extremely low hemolytic activity toward the tested cells.In summary,PN-CATHs represent the first discovery of cathelicidin family AMPs with both potent anti-infective and antioxidant activities.The discovery of PN-CATHs provides possibility for the development of new anti-infective and antioxidant drugs.