Abstract
The incidence of mucormycosis has increased drastically in immunocompromised patients. Also the array of targets whose inhibition results in Mucorales death is limited. Recently, researchers identified mitochondria as important regulators of detoxification and virulence mechanisms in fungi. In this context, targeting the mitochondrial respiratory chain may provide a new platform for antifungal development. We hypothesized that targeting respiratory pathways potentiates triazoles activity via apoptosis. We found that simultaneous administration of antimycin A (AA) and benzohydroxamate (BHAM), inhibitors of classical and alternative mitochondrial pathways respectively, resulted in potent activity of posaconazole (PCZ) and itraconazole (ICZ) against Rhizopus oryzae. We observed cellular changes characteristic of apoptosis in R. oryzae cells treated with PCZ or ICZ in combination with AA and BHAM. The fungicidal activity of this combination against R. oryzae was correlated with intracellular reactive oxygen species accumulation (ROS), phosphatidylserine externalization, mitochondrial membrane depolarization, and increased caspase like activity. DNA fragmentation and condensation assays also revealed apoptosis of R. oryzae cells. These apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine. Taken together, these findings suggest that the use of PCZ or ICZ in combination with AA and BHAM makes R. oryzae exquisitely sensitive to treatment with triazoles via apoptosis. This strategy may serve as a new model for the development of improved or novel antifungal agents.
Highlights
The mortality rate in immunocompromised patients with mucormycosis remains high [1,2]
We observed that R. oryzae germlings treated with PCZ or ICZ in combination with antimycin A (AA) and BHAM, exhibited increased intracellular reactive oxygen species accumulation (ROS) production (3.5 fold) than did untreated germlings and those treated with AA and BHAM alone (Figure 1A and B)
We found that the levels of cyt c in mitochondria and the cytosol in R. oryzae germlings treated with PCZ or ICZ in conjunction with AA and BHAM -induced apoptosis differed from those in untreated and AA and BHAM treated germlings
Summary
The mortality rate in immunocompromised patients with mucormycosis remains high [1,2]. 60% of all disease manifestations and 90% of all rhinocerebral cases are caused by Rhizopus oryzae [3]. The rapid growth rate and the angioinvasive nature of the disease lead to an overall mortality of 50% [3]. R. oryzae, is resistant to most antifungal agents, with the exception of the polyene amphotericin B (AMB) and the third generation azole, posaconazole (PCZ) [2]. Both AMB/PCZ target the cell membrane, the need for identification of novel cellular targets whose inhibition results in loss of viability of Mucorales species is dire
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