Discovery of the effects of the hemiprotonic phenanthroline-phenanthroline+ against Trichophyton rubrum by inducing fungal apoptosis

Description

Trichophyton rubrum (T. rubrum) is the commonest causative agent of dermatophytosis worldwide. Development of anti-fungal drugs will contribute to treating the disease. Here we suggest a hemiprotonic compound phenanthroline- phenanthroline+ (ph-ph+), is active in inhibiting the growth and reproduction of T. rubrum, and the values of MIC and MFC were 2 g/mL and 8 g/mL, respectively. In vitro onychomycosis model, ph-ph+ killed T. rubrum by inducing apoptosis, evaluated by TEM and Annexin V-FITC/PI staining. Transcriptomic analysis and biochemical assay showed that ph-ph+ elevated iron ion content in T. rubrum cells and reduced GSH antioxidant system level, leading to increase in the contents of ROS and MDA. Therefore, the anti-fungal mechanism of ph-ph+ would be associated with iron ion-induced cell apoptosis, which is different from other known anti-fungal drugs. Further, ph-ph+ was prepared into gel for application in guinea pigs with dermatophytosis caused by T. rubrum. The results showed that the ph-ph+ gel eliminated the fungus in the animals, without skin irritation and other adverse reactions. The study would not only provide potential compound to treat dermatophytosis, but also suggests that iron ion-induced cell apoptosis might be a new approach to kill fungi.

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T. rubrum (No. 340195) was purchased from General Microbiological Culture Collection Center (Beijing, China). The fungus was cultured in SDM at 28C. The MIC and MFC values of ph-ph+ were examined by microtiter plate method followed by the protocol of CLSI. The cultured T. rubrum was diluted with media, and then the fungal suspension was spread on the plate for colony culture. After colonies grew and the number of colonies was counted, the colony forming units (CFUs)/mL was calculated by the formula: CFUs/mL = (the number of colonies/mL) × dilution ratio. T. rubrum (106 CFUs/mL) in the logarithmic growth phase was placed in microtiter plate, and then serial two-fold diluted ph-ph+ (0.25  64 μg/mL) were respectively added into the microplate. After incubation for 48 h in a thermostatic shaker (Shanghai Shanzhi Instrument Co. Ltd., China), the absorbance was measured at 600 nm by using a spectrophotometer (BioTek Instruments, Inc, USA) to determine the effect of ph-ph+ on the cells. Further, the MIC and MFC were evaluated by the Petri dish assay. Briefly, T. rubrum was respectively cultured in the center of the Sabouraud dextrose agar media containing ph-ph+ with the concentration of 2 16 g/mL. Then the diameters of the inhibition zone were recorded.

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