Ntifungal Mechanism of MMGPFigure 10. MMGP1 induced DNA Title Loaded From File damage in C. albicans. (a) Confocal micrographs of TUNEL stained C. albicans cells treated with MMGP1 for 0, 6, 12, and 24 h (20 x magnification). The images are overlay of TUNEL (green fluorescence), Hoechst 33342 (blue) and bright field micrographs of C. albicans cells (b) Flow cytometry analysis of TUNEL stained MMGP1-treated C. albicans cells at different time intervals (0, 6, 12, and 24 h). The cells treated with H2O2 was used a TUNEL positive cells.doi: 10.1371/journal.pone.0069316.gAntifungal Mechanism of MMGPthe peptide NA condensate [23]. Similarly, in this study, proteinase K was used to hydrolyse the peptide and the bound plasmid DNA was detected on the gel, which confirms the formation of peptide NA complexes. The peptide formed highly stable complexes with DNA, which is resistant to high ionic conditions (i.e., 0.3 M of NaCl) (data not shown) and nuclease activity. In addition, the peptide inhibited the transcription processes both under in vitro and in vivo conditions, and therefore was found to interfere with cellular transcription process once inside the fungal cell. This finding is consistent with the earlier report on the antimicrobial peptide, indolicidin, which induces cytotoxicity in the target cells by binding to DNA and inhibition of macromolecular synthesis [15]. The MMGP1 is also capable of inducing endogenous production of ROS in C. albicans, which could be attributed to its binding with DNA, thereby inhibiting the transcription process within the cells. The ROS production by MMGP1 triggers a cascade of events like protein carbonylation, lipid peroxidation, mitochondrial membrane depolarization and DNA fragmentation. An earlier report suggested that the production of reactive species at subtoxic concentrations regulate cell differentiation, proliferation, signal transduction and ion transport whereas excessive accumulation of ROS within the cells can cause damage to DNA, proteins and lipids, which lead to disorganization, dysfunction and damage of membranes and proteins. Specifically, the oxidation of lipids may cause impairment of membrane function, decreased fluidity, increased permeability to ions and potentially membrane rupture [24?6]. To examine the relationship between endogenous production of ROS and antifungal mechanism, we investigated the secondary effect of ROS production in C. albicans by MMGP1. The results suggested that the hyper-induction of ROS causes oxidation of proteins, lipids and induces DNA damage. Title Loaded From File Furthermore, the growth of the MMGP1-treated C. albicans cells recovered after supplementation with glutathione, suggesting that the endogenous production of ROS could be the major mechanism for antifungal action of MMGP1 in C. albicans.Mitochondrial dysfunction due to depolarization of mitochondrial membrane is a marked characteristic of ROSmediated cytotoxicity. Interestingly, the results of mitochondrialmediated experiments suggested that the MMGP1 induced disruption of mitochondrial membrane potential in C. albicans by depolarization of inner mitochondrial membrane. This finding is consistent with the earlier report on the antimicrobial peptide, papiliocin, which induces cytotoxicity in C. albicans by disruption of mitochondrial integrity and consequent cell death [5]. Certain other antifungal peptides such as – and defensin, magainin, cathelicidin and histatin are known to disrupt the mitochondrial membrane potential and induce cytoto.Ntifungal Mechanism of MMGPFigure 10. MMGP1 induced DNA damage in C. albicans. (a) Confocal micrographs of TUNEL stained C. albicans cells treated with MMGP1 for 0, 6, 12, and 24 h (20 x magnification). The images are overlay of TUNEL (green fluorescence), Hoechst 33342 (blue) and bright field micrographs of C. albicans cells (b) Flow cytometry analysis of TUNEL stained MMGP1-treated C. albicans cells at different time intervals (0, 6, 12, and 24 h). The cells treated with H2O2 was used a TUNEL positive cells.doi: 10.1371/journal.pone.0069316.gAntifungal Mechanism of MMGPthe peptide NA condensate [23]. Similarly, in this study, proteinase K was used to hydrolyse the peptide and the bound plasmid DNA was detected on the gel, which confirms the formation of peptide NA complexes. The peptide formed highly stable complexes with DNA, which is resistant to high ionic conditions (i.e., 0.3 M of NaCl) (data not shown) and nuclease activity. In addition, the peptide inhibited the transcription processes both under in vitro and in vivo conditions, and therefore was found to interfere with cellular transcription process once inside the fungal cell. This finding is consistent with the earlier report on the antimicrobial peptide, indolicidin, which induces cytotoxicity in the target cells by binding to DNA and inhibition of macromolecular synthesis [15]. The MMGP1 is also capable of inducing endogenous production of ROS in C. albicans, which could be attributed to its binding with DNA, thereby inhibiting the transcription process within the cells. The ROS production by MMGP1 triggers a cascade of events like protein carbonylation, lipid peroxidation, mitochondrial membrane depolarization and DNA fragmentation. An earlier report suggested that the production of reactive species at subtoxic concentrations regulate cell differentiation, proliferation, signal transduction and ion transport whereas excessive accumulation of ROS within the cells can cause damage to DNA, proteins and lipids, which lead to disorganization, dysfunction and damage of membranes and proteins. Specifically, the oxidation of lipids may cause impairment of membrane function, decreased fluidity, increased permeability to ions and potentially membrane rupture [24?6]. To examine the relationship between endogenous production of ROS and antifungal mechanism, we investigated the secondary effect of ROS production in C. albicans by MMGP1. The results suggested that the hyper-induction of ROS causes oxidation of proteins, lipids and induces DNA damage. Furthermore, the growth of the MMGP1-treated C. albicans cells recovered after supplementation with glutathione, suggesting that the endogenous production of ROS could be the major mechanism for antifungal action of MMGP1 in C. albicans.Mitochondrial dysfunction due to depolarization of mitochondrial membrane is a marked characteristic of ROSmediated cytotoxicity. Interestingly, the results of mitochondrialmediated experiments suggested that the MMGP1 induced disruption of mitochondrial membrane potential in C. albicans by depolarization of inner mitochondrial membrane. This finding is consistent with the earlier report on the antimicrobial peptide, papiliocin, which induces cytotoxicity in C. albicans by disruption of mitochondrial integrity and consequent cell death [5]. Certain other antifungal peptides such as – and defensin, magainin, cathelicidin and histatin are known to disrupt the mitochondrial membrane potential and induce cytoto.

Ntifungal Mechanism of MMGPFigure 10. MMGP1 induced DNA damage in C. albicans.

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