Effects Of CGA-N12 And CGA-N9 On The Membrane Structure Of Candida Tropicalis Cells

Fungal resistance has always been a difficult problem in the treatment of mycosis.The multi-target mechanism of antimicrobial peptides may be the best choice to solve the problem of fungal resistance.CGA-N12 and CGA-N9 are anti-Candida active peptides derived from N-terminal of chromogranin A(CGA).CGA-N12 is composed of amino acids 65-76 at N-terminal and CGA-N9 is composed of amino acids 47-55 at N-terminal.Previous studies showed that CGA-N12 and CGA-N9 could induce apoptosis of Candida cells without destroying the integrity of cell membrane,but the effect on cell membrane structure was not clear.The purpose of this paper is to explore the changes of cell membrane structure of Candida under the action of CGA-N12 and CGA-N9,enrich the theoretical system of antimicrobial peptide membrane activity mechanism,and provide theoretical basis for the development of CGA-N12 and CGA-N9 as new antifungal drugs.In this paper,the effects of CGA-N12 and CGA-N9 on the membrane potential,membrane fluidity,membrane surface structure,potassium ion leakage,ergosterol content in the plasma membrane and the pore size of liposome channel were studied by using fluorescence probe,AFM,ICP-OES and UV spectrophotometer.Furthermore,the changes of CGA-N12 and CGA-N9 in different concentrations on membrane integrity and the induced formation of channels were detected by the leakage of the fluorescent dyes calcein and CF.The results showed that CGA-N12 induced the formation of membrane channel with diameter less than 1 nm in the cell membrane of Candida tropicalis,which resulted in leakage of potassium ion,chloride ion and proton,dissipation of membrane potential,increased membrane fluidity without destroying membrane integrity,but did not affect ergosterol synthesis.CGA-N9 had a similar effect.The relationship between the concentration of antimicrobial and the membrane integrity was studied.It was found that when the concentration of the two antimicrobial peptides increased to 8 ×MIC,the pore diameter larger than 1 nm was induced on the membrane,and the membrane integrity was destroyed at 16 ×MIC.In conclusion,the antimicrobial peptides CGA-N12 and CGA-N9 have a concentration-dependent damage to cell membranes.Under the condition of low concentration,they don't damage the integrity of the cell membrane,but can induce the formation of small pores in the cell membrane,causing ion leakage and destroying the polarity of the cell membrane;as the concentration of antimicrobial peptides increase,the pore size induced on the cell membrane increase,until the integrity of the cell membrane are destroyed.