CGA-N9, an antimicrobial peptide derived from chromogranin A: direct cell penetration of and endocytosis by Candida tropicalis

CGA-N9 is a peptide derived from the N-terminus of human chromogranin A comprising amino acids 47–55. Minimum inhibitory concentration (MIC) assays showed that CGA-N9 had antimicrobial activity and exhibited time-dependent inhibition activity against Candida tropicalis, with high safety in human red blood cells (HRBCs) and mouse brain microvascular endothelial cells (bEnd.3). According to the results of transmission electron microscopy (TEM), flow cytometry and confocal microscopy, CGA-N9 accumulated in cells without destroying the integrity of the cell membrane; the peptide was initially localized to the cell membrane and subsequently internalized into the cytosol. 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 (NaN(3)), 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 the MIC assays, whereby the anticandidal activity of CGA-N9 was 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.