Mechanism Of Action Of An Antimicrobial Peptide CGA-N9 Against Candida Tropicalis

CGA-N9 is a peptide derived from the N-terminus of human chromogranin A comprising amino acids 47 to 55.In the present study,the antimicrobial spectrum,internalization mechanism and intracellular mechanism of action were investigated.Minimum inhibitory concentration?MIC?assays showed that CGA-N9 is a broad-spectrum antimicrobial peptide against Candida tropicalis,Candida parapsilosis,Candida glabrata,Candida krusei,Cryptococcus neoformans,Bacillus subtilis,and Listeria monocytogenes,in which Candida tropicalis is the most sensitive strain with a minimum fungicide concentration 3.9?g/mL.Hemolysis and mammalian cytotoxicity study showed that CGA-N9 has high safety in human red blood cells?HRBCs?and mouse brain microvascular endothelial cells?bEnd.3?.According to the results of flow cytometry,transmission electron microscopy?TEM?and confocal microscopy,CGA-N9 was initially localized to the cell membrane and subsequently internalized into the cytosol;the peptide accumulated in cells without destroying the integrity of the cell membrane.An investigation of the cellular internalization mechanism revealed that most CGA-N9 molecules entered the yeast cells,even at 4°C and in the presence of sodium azide?NaN₃?,both of which block all energy-dependent transport mechanisms.In addition,peptide internalization was affected by the endocytic inhibitors 5-?N-ethyl-N-isopropyl?-amiloride?EIPA?,cytochalasin D?CyD?and heparin;chlorpromazine?CPZ?also had some effect on CGA-N9 internalization.Similar results were obtained in MIC assays,whereby the anticandidal activity of CGA-N9was blocked to different degrees in the presence of EIPA,CyD,heparin or CPZ.Therefore,most CGA-N9 passes through the C.tropicalis cell membrane via direct cell penetration,whereas the remainder enters through macropinocytosis and sulfate proteoglycan-mediated endocytosis,with a slight contribution from clathrin-mediated endocytosis.The research on intracellular mechanism of action against Candida tropicalis showed that CGA-N9 damaged the calcium homeostasis,induced an over-generation of intracellular reactive oxygen species and dissipation in mitochondrial membrane potential,in which the former plays key roles in the initiation of apoptosis and the latter is a sign of the cell apoptosis.Accordingly,we assessed the apoptosis features of Candida tropicalis cells after treatment with CGA-N9 and found the following:leakage of cytochrome c into the cytosol;and apoptotic phenotypes,such as chromatin condensation and DNA degradation.In conclusion,CGA-N9 exerted antifungal effect by inducing apoptosis of C.tropicalis.The interaction of CGA-N9 and DNA in vitro showed that CGA-N9 bound with DNA in a non-insertion mode without damaging DNA structure.In summary,CGA-N9 is a highly efficient and non-toxic antimicrobial peptide.It passes through cell membrane by cell penetration and endocytosis without destroying the integrity of the cell membrane.CGA-N9 exerts its antifungal activity through apoptosis and reactive oxygen species accumulation in C.tropicalis.