Dual Anti-candidal Characteristics Of Antifunal Peptide CGA-N12

The antimicrobial peptide CGA-N12?NH2-ALQGAKERAHQQ-COOH?is an antifungal peptide derived from chromogranin A?CGA?from the 65th to the 76th residue of the N-terminus of chromagranin A.Our previous research showed that CGA-N12 has anti-candidal activity,but its mechanism of action is not yet clear.Rhodamine-123 was used to detect the effect of antimicrobial peptide CGA-N12on the accumulation of intracellular reactive oxygen species.It was found that CGA-N12 could induce the accumulation of intracellular reactive oxygen species?ROS?.ROS could induce oxidative stress and reduce mitochondrial membrane potential,which play key roles in inducing apoptosis.Therefore,we investigated the apoptosis of Candida tropicalis cells induced by CGA-N12.The results revealed that CGA-N12 could depolarize cell membrane of Candida tropicalis,disspate mitochondrial membrane potential,induce cytochrome c?Cyt c?leakage frome mitochondria into plasm,promote cell and mitochondrial uptake of Ca²⁺,cysteine-dependent of Candida metacaspase was increased,and the apoptosis of Candida tropicalis was characterized by nuclear condensation and chromosomal DNA fragmentation.Therefore,CGA-N12 exerted anti-Candida activity by inducing apoptosis.Our previous research found that CGA-N12 did not destroy Candida cell membrane integrity,but could depolarize cell membrane.To detect the effect of CGA-N12 on cell membrane,we prepared liposomes containing different ion-selective fluorescent dyes.The characteristics of membrane channels induced by CGA-N12 were detected by fluorescent quenching post-treatment of CGA-N12.Membrane im-permeable halogen anion selective fluorescent dye Lucigenin was used to detect the condition of hydrated Cl^-???0.66 nm?channels;Carboxyl fluorescein?CF????1nm?was used as the membrane impermeable ion dye.the membrane-im-permeablepH-sensitivefluorescentdyeHPTS?8-hydroxypyrene-1,3,6-trisulfonic acid,trisodium salt?was used to detect the transmembrane condition of H₃O⁺channel.The results showed that CGA-N12 may induce non-ion selective channels in membrane with less than 1nm diameter.In summary,cell memdrane permeability of C.troplcalis increased post-treatment of CGA-N12 and induced the leakage of ions which resulted in the dissipation of membrane potential.CGA-N12 internalized without disturbing the cell membrane integrity and functioned on mitochondrial membrane.Followed by mitochondrial membrane deplorization,under the combind effect of decresase of cell membrane potential,the destroy of calcium hemeostasrs and mitochondrial membrane depolarization,ROS accumulation,the increase of mitochondrial permeability.Cyt.c release frome mitochondria into cytoplasm,and matecaspase was activated.All the factors promoted the process of C.tropicalis apoptosis.