| Invasive candidiasis is a life-threatening fungal infection.Candida tropicalis is a major non-albicans species that causes invasive candidiasis.Due to the scarcity of antifungal drugs and the rapid emergence of resistant strains that develop resistance to antifungal drugs,there is an urgent need to develop antifungal drugs with novel mechanisms of action.Antimicrobial peptides(AMPs)have the advantages of low drug resistance,broad spectrum activity and low cytotoxicity.AMPs are potential drugs to combat multi-drug resistant pathogens and to treat invasive candida infections.But compared with antibiotics,the lower antibacterial activity of AMPs has become one of the important factors limiting their development.Structural modification of target and transmembrane transport efficiency are two important ways to improve the activity of antimicrobial peptides.CGA-N12(ALQGAKERAHQQ)and CGA-N9(RILSILRHQ)are two non-membrane breaking specific anti-candida peptides discovered in the laboratory.To improve the anti-candida activities of the antimicrobial peptides CGA-N12 and CGA-N9,the following studies were carried out:(1)Modification of CGA-N12 structure based on the target C.tropicalis β-1,6-glucan synthetase(Ct KRE9).Based on Ct KRE9 structure,a series of CGA-N12 analogues were designed by molecular docking technique.The antimicrobial activity,hemolytic activity,and stability of the prepared antimicrobial peptide CGA-N12 and its analogues were determined.Six analogues with improved antimicrobial activity,low cytotoxicity and good stability were obtained,which showed high antimicrobial activity against C.tropicalis.Compared with fluconazole,it showed stronger inhibitory effect on the growth of refractory C.tropicalis.Among them,CGA-N12-0801 was the best analogue with the minimum antimicrobial concentration of 3.46 μg/m L,which was 29.09 times higher than that of CGA-N12.Then,the mechanism of action of CGA-N12 structure analogized was explored,and molecular docking technology was used to explore the molecular interaction between CGA-N12 and Ct KRE9.The inhibition of Ct KRE9 protease activity was analyzed by enzyme activity assay.The effect of CGA-N12 on C.tropicalis biofilm was measured by crystal violet staining method and 96-well microtitration plate method.The effects on cell wall and membrane of C.tropicalis were observed by scanning electron microscope and transmission electron microscope.The results showed that reducing the docking energy of CGA-N12 and Ct KRE9 and increasing the positive charge number of CGA-N12 could significantly improve the antimicrobial activity against C.tropicalis.CGA-N12 analogues had the ability to inhibit biofilm formation and eliminate formed biofilms.Compared with Fluconazole,they showed a stronger ability to eliminate formed biofilms.The results of electron microscope observation and Ct KRE9 protease activity inhibition test showed that CGA-N12 and its analogues killed C.tropicalis by destroying cell wall synthesis and cell membrane integrity.(2)Excavation of transmembrane transporters of CGA-N12 and CGA-N9.C.tropicalis cells were treated with CGA-N12 at different times to obtain the transcriptome of C.tropicalis.Analysis of m RNA expression patterns showed that the expression patterns of oligopeptide transporter gene Ct OPT1,Ct OPT2,Ct OPT9 and Ct OPT12 of C.tropicalis were positively correlated with the transmembrane amount of antimicrobial peptide CGA-N12.It is speculated that Ct OPT1,Ct OPT2,Ct OPT9 and Ct OPT12 assist the transmembrane transport of CGA-N12.The coding sequences of 12 OPT genes(CtOPTs)were cloned from C.tropicalis by RT-PCR.Molecular docking results showed that Ct OPT1,Ct OPT9 and Ct OPT12 contribute to the transmembrane transport of CGA-N9,and Ct OPT1 and Ct OPT12 are stronger in facilitating transmembrane transport of CGA-N9.In conclusion,the computer aided drug design technology based on target proteins is a simple and reliable method to rationally design antimicrobial peptides,which can accelerate the development of novel antimicrobial peptides and provide an ideal drug candidate for invasive candida infections.At the same time,it is very important to explore the transmembrane transport proteins and the mechanism of their assistance in the transmembrane transport of antimicrobial peptides,so as to improve the antibacterial activity of non-membrane breaking antimicrobial peptides. |
