S on treatment with MMGP1(Figure 9a). Figure 9b shows the NAO staining of mitochondria isolated from C. albicans treated with and without MMGP1. The intensity of NAO fluorescence diminished afterDiscussionEarlier, it was reported in our laboratory that the MMGP1 peptide induces cell death in C. albicans cells in a nondisruptive manner through energy-independent direct penetration mechanism [12]. Several Hexokinase II Inhibitor II, 3-BP antifungal peptides are translocated across cell membrane and are found inside the cell, wherein they can induce various inhibitory activities,Antifungal Mechanism of MMGPFigure 5. In vivo inhibition of transcription in C. albicans by MMGP1. (a) Confocal micrographs showing inhibition of transcription in C. albicans by MMGP1. The images are overlay of TMR-florescent azide (red), Hoechst 33342 (blue) and bright field micrographs of C. albicans cells. Intense EU staining (red fluorescence) was observed in nucleus after 2 16574785 h of treatment with MMGP1 and prolonged treatment of cells with peptide showed decrease in EU signal in the nucleus (b) Quantification of transcription inhibition in MMGP1-treated C. albicans by flow cytometry (X2-C. albicans cells showing TMR-A fluorescence i.e cells that are transcriptionally active).doi: 10.1371/HIF-2��-IN-1 chemical information journal.pone.0069316.gAntifungal Mechanism of MMGPFigure 6. MMGP1 induced ROS production in C. albicans. (a) ROS induction in C. albicans cells treated with MMGP1. 1-C. albicans cells without MMGP1 (negative control panel); 2-C. albicans cells treated with MMGP1 for 6 h (Test panel); 3-C. albicans cells treated with H2O2 for 6 h (b) Time-scale measurement of intracellular ROS in MMGP1 treated C. albicans (0.57 ) by flow cytometry. The fluorescence obtained with the cells treated with 1 mM of H2O2 serves as positive control and the cells without peptide serves as negative control.doi: 10.1371/journal.pone.0069316.gdisrupting normal cell functions primarily not linked with cell penetration [4]. In the present study, we investigated the mechanisms of antifungal action of MMGP1 in C. albicans. TheMMGP1 showed a remarkable non-specific DNA-binding property in vitro. The use of SDS or trypsin to remove the peptide allows the direct analysis of the status of bound DNA inAntifungal Mechanism of MMGPFigure 7. Effect of glutathione on viability of MMGP1-treated C. albicans cells. The cells were treated with peptide (0.57 ) in 23727046 the presence and absence of glutathione for 24 h. The cell density was measured at 600 nm for every 6 h interval. A-without peptide; B-with peptide; C, D, E-with peptide in the presence of 1, 10 and 50 mM glutathione, respectively.doi: 10.1371/journal.pone.0069316.gFigure 8. MMGP1-induced intracellular oxidation of proteins and lipids in C. albicans. (a) Time-dependent measurement of protein carbonyls in MMGP1 treated C. albicans cells by DNPH assay. (b) Time-dependent measurement of TBARS production in MMGP1 treated C. albicans cells by TBA assay.doi: 10.1371/journal.pone.0069316.gAntifungal Mechanism of MMGPFigure 9. Mitochondrial membrane depolarization in MMGP1 treated C. albicans cells. (a) Measurement of mitochondrial membrane potential in MMGP1 treated C. albicans cells by flow cytometry (b) Measurement of inner mitochondrial membrane depolarization by MMGP1 in C. albicans cells. 1-mitochondria of C. albicans cells without treatment; 3-mitochondria of C. albicans cells treated with 1 mM H2O2; 2-mitochondria of C. albicans cells treated with MMGP1 for 24 h.doi: 10.1371/journal.pone.0069316.gA.S on treatment with MMGP1(Figure 9a). Figure 9b shows the NAO staining of mitochondria isolated from C. albicans treated with and without MMGP1. The intensity of NAO fluorescence diminished afterDiscussionEarlier, it was reported in our laboratory that the MMGP1 peptide induces cell death in C. albicans cells in a nondisruptive manner through energy-independent direct penetration mechanism [12]. Several antifungal peptides are translocated across cell membrane and are found inside the cell, wherein they can induce various inhibitory activities,Antifungal Mechanism of MMGPFigure 5. In vivo inhibition of transcription in C. albicans by MMGP1. (a) Confocal micrographs showing inhibition of transcription in C. albicans by MMGP1. The images are overlay of TMR-florescent azide (red), Hoechst 33342 (blue) and bright field micrographs of C. albicans cells. Intense EU staining (red fluorescence) was observed in nucleus after 2 16574785 h of treatment with MMGP1 and prolonged treatment of cells with peptide showed decrease in EU signal in the nucleus (b) Quantification of transcription inhibition in MMGP1-treated C. albicans by flow cytometry (X2-C. albicans cells showing TMR-A fluorescence i.e cells that are transcriptionally active).doi: 10.1371/journal.pone.0069316.gAntifungal Mechanism of MMGPFigure 6. MMGP1 induced ROS production in C. albicans. (a) ROS induction in C. albicans cells treated with MMGP1. 1-C. albicans cells without MMGP1 (negative control panel); 2-C. albicans cells treated with MMGP1 for 6 h (Test panel); 3-C. albicans cells treated with H2O2 for 6 h (b) Time-scale measurement of intracellular ROS in MMGP1 treated C. albicans (0.57 ) by flow cytometry. The fluorescence obtained with the cells treated with 1 mM of H2O2 serves as positive control and the cells without peptide serves as negative control.doi: 10.1371/journal.pone.0069316.gdisrupting normal cell functions primarily not linked with cell penetration [4]. In the present study, we investigated the mechanisms of antifungal action of MMGP1 in C. albicans. TheMMGP1 showed a remarkable non-specific DNA-binding property in vitro. The use of SDS or trypsin to remove the peptide allows the direct analysis of the status of bound DNA inAntifungal Mechanism of MMGPFigure 7. Effect of glutathione on viability of MMGP1-treated C. albicans cells. The cells were treated with peptide (0.57 ) in 23727046 the presence and absence of glutathione for 24 h. The cell density was measured at 600 nm for every 6 h interval. A-without peptide; B-with peptide; C, D, E-with peptide in the presence of 1, 10 and 50 mM glutathione, respectively.doi: 10.1371/journal.pone.0069316.gFigure 8. MMGP1-induced intracellular oxidation of proteins and lipids in C. albicans. (a) Time-dependent measurement of protein carbonyls in MMGP1 treated C. albicans cells by DNPH assay. (b) Time-dependent measurement of TBARS production in MMGP1 treated C. albicans cells by TBA assay.doi: 10.1371/journal.pone.0069316.gAntifungal Mechanism of MMGPFigure 9. Mitochondrial membrane depolarization in MMGP1 treated C. albicans cells. (a) Measurement of mitochondrial membrane potential in MMGP1 treated C. albicans cells by flow cytometry (b) Measurement of inner mitochondrial membrane depolarization by MMGP1 in C. albicans cells. 1-mitochondria of C. albicans cells without treatment; 3-mitochondria of C. albicans cells treated with 1 mM H2O2; 2-mitochondria of C. albicans cells treated with MMGP1 for 24 h.doi: 10.1371/journal.pone.0069316.gA.
