白色念珠菌与新生隐球菌对抗菌肽KK-20的敏感性差异研究
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摘要
抗菌肽KK-20是由实验室自主设计合成的,其对白色念珠菌的MIC值为128μg/L,而对新生隐球菌的MIC值为8μg/m L。此研究旨在探讨白色念珠菌和新生隐球菌对抗菌肽KK-20敏感性差异的机理。通过NPN摄取实验和PI染色实验发现抗菌肽KK-20对白色念珠菌ATCC10231细胞膜的破坏作用比新生隐球菌ATCC4906低。通过TMA-DPH标记的荧光偏振实验发现白色念珠菌的细胞膜流动性显著低于新生隐球菌。对提取的真菌细胞膜麦角固醇和脂肪酸进行分析,发现白色念珠菌的细胞膜麦角固醇含量和脂肪酸饱和度高于新生隐球菌。研究认为:麦角固醇、饱和脂肪酸和不饱和脂肪酸含量是影响真菌细胞膜对抗菌肽KK-20入膜和摄取的重要因素,从而导致两者对抗菌肽KK-20敏感性的差异,指出了真菌对抗菌肽抗性可能的机制。
The antibacterial peptide KK-20 is independently designed by the author's laboratory. The MIC values of peptide KK-20 against C. albicans ATCC10231 and C. neoformans ATCC4906 were 128 μg/m L and 8 μg/m L,respectively. The aim of this study was to investigate the mechanisms underlying the differences in the sensitivity of fungi to antibacterial peptide KK-20,such as C. albicans and C. neoformans. The results from NPN uptake and PI staining showed that the membrane destruction of antibacterial peptide KK-20 against C. albicans ATCC10231 was significantly lower than that of C. neoformans ATCC4906. Fluorescence polarization experiments through TMA-DPH labeling demonstrated that the cell membrane fluidity of C. albicans was significantly lower than that of C. neoformans. Ergosterol and fatty acids,which were from fungal cell membrane,were extracted and analyzed. Studies indicated that the cell membrane ergosterol content and fatty acid saturation of C. albicans ATCC10231 were higher than those of C. neoformans ATCC4906. In conclusion,ergosterol,saturated and unsaturated fatty acid content play a major role in the effect on the uptake and membrane penetration of antibacterial peptide KK-20,resulting in different sensitivities of KK-20 to these fungus,which indicates possible ways for these fungus against antibacterial peptide.
The antibacterial peptide KK-20 is independently designed by the author's laboratory. The MIC values of peptide KK-20 against C. albicans ATCC10231 and C. neoformans ATCC4906 were 128 μg/m L and 8 μg/m L,respectively. The aim of this study was to investigate the mechanisms underlying the differences in the sensitivity of fungi to antibacterial peptide KK-20,such as C. albicans and C. neoformans. The results from NPN uptake and PI staining showed that the membrane destruction of antibacterial peptide KK-20 against C. albicans ATCC10231 was significantly lower than that of C. neoformans ATCC4906. Fluorescence polarization experiments through TMA-DPH labeling demonstrated that the cell membrane fluidity of C. albicans was significantly lower than that of C. neoformans. Ergosterol and fatty acids,which were from fungal cell membrane,were extracted and analyzed. Studies indicated that the cell membrane ergosterol content and fatty acid saturation of C. albicans ATCC10231 were higher than those of C. neoformans ATCC4906. In conclusion,ergosterol,saturated and unsaturated fatty acid content play a major role in the effect on the uptake and membrane penetration of antibacterial peptide KK-20,resulting in different sensitivities of KK-20 to these fungus,which indicates possible ways for these fungus against antibacterial peptide.
引文
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[3]Kumariya R,Sood SK,Rajput YS,et al.Increased membrane surface positive charge and altered membrane fluidity leads to cationic antimicrobial peptide resistance in Enterococcus faecalis[J].Biochimica et Biophysica Acta(BBA)-Biomembranes,2015,1848(6):1367-1375.
[4]Abe F,Hiraki T.Mechanistic role of ergosterol in membrane rigidity and cycloheximide resistance in Saccharomyces cerevisiae[J].Biochimica et Biophysica Acta(BBA)-Biomembrances,2009,1788(3):743-752.
[5]Branco J,Ola M,Silva RM,et al.Impact of ERG3 mutations and expression of ergosterol genes controlled by UPC2 and NDT80 in Candida parapsilosis azole resistance[J].Clin Microbiol Infect,2017,23(8):575e1-575.e8.
[6]Ells R,Kock JL,Van Wyk PW,et al.Arachidonic acid increases antifungal susceptibility of Candida albicans and Candida dubliniensis[J].J Antimicrobial Chemoth,2009,63(1):124-128.
[7]Gropp K,Schild L,Schindler S,et al.The yeast Candida albicans evades human complement attack by secretion of aspartic proteases[J].Molec Immunol,2009,47(2-3):465-475.
[8]Bieberich E.Sphingolipids and lipid rafts:Novel concepts and methods of analysis[J].Chem Phy Lipids,2018,216:114-131.
[9]Scott MG,Dullaghan E,Mookherjee N,et al.An anti-infective peptide that selectively modulates the innate immune response[J].Nature Biotechnol,2007,25(4):465-472.
[10]Scott MG,Davidson DJ,Gold MR,et al.The human antimicrobial peptide LL-37 is a multifunctional modulator of innate immune responses[J].J Immunol,2002,169(7):3883-3891.
[11]Hancock RE.Peptide antibiotics[J].Lancet,1997,349(9049):418-422.
[12]Chavakis T,Cines DB,Rhee JS,et al.Regulation of neovascularization by human neutrophil peptides(alpha-defensins):a link between inflammation and angiogenesis[J].Faseb J Official Public Feder American Societies Experim Biol(FASEB),2004,18(11):1306-8.
[13]Lavaisse,Lucía M,Hollmann A,et al.Zeta potential changes of Saccharomyces cerevisiae during fermentative and respiratory cycles[J].Colloids Surfaces B:Biointerfaces,2019,174:63-69.
[14]Yu C,Wei S,Han X,et al.Effective inhibition of Cbf-14 against Cryptococcus neoformans infection in mice and its related antiinflammatory activity[J].Fungal Genetics&Biol(Fg&B),2018,110:38-47.
[15]张鹰,曾新安,温其标,等.荧光偏振法研究脉冲电场对酿酒酵母细胞膜流动性影响[J].光谱学与光谱分析,2008,28(1):156-160.
[16]Zhao X R,Lin Q,Brookes PC.Does soil ergosterol concentration provide a reliable estimate of soil fungal biomass[J].Soil Biol Biochem,2005,37(2):311-317.
[17]Blaga AC,Ciobanu C,CasCaval D,et al.Enhancement of ergosterol production by,Saccharomyces cerevisiae in batch and fedbatch fermentation processes using n-dodecane as oxygen-vector[J].Biochem Engin J,2018,131:70-76.
[18]Esther GF,Gudrun K,Rabea MW,et al.Membrane microdomain disassembly inhibits MRSA antibiotic resistance[J].Cell,2017,171(6):1354-1367.
[19]Yang H,Tong J,Lee CW,et al.Structural mechanism of ergosterol regulation by fungal sterol transcription factor Upc2[J].Nature Communic,2015,6:6129.
[20]韩旭,霍贵成,李春,等.气相色谱法测定乳酸乳球菌脂肪酸[J].东北农业大学学报,2009,40(9):92-96.
[21]Hugo A,Albertyn J,Van Rensburg WJ,et al.Polyunsaturated fatty acids cause apoptosis in C.albicans and C.dubliniensis biofilms[J].Biochimica et Biophysica Acta,General Subjects,2012,1820(10):1463-1468.
[22]刘帅,王爱武,李美艳,等.脂肪酸甲酯化方法的研究进展[J].中国药房,2014,25(37):3535-3537.
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