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Updated: Aug 17, 2026
Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis
Published on: March 19, 2019
Mitochondrial alterations and autofluorescent conversion of Candida albicans induced by histatins
Giacomo Diaz1, Luciano Polonelli, Stefania Conti
1Dipartimenti di Citomorfologia, Università di Cagliari, 09042 Monserrato (Cagliari), Italy. gdiaz@unica.it
Abstract:
The mechanism of the candidacidal activity of histatins 3 and 5 (Hst) is still a matter of debate. Previous studies have indicated that Hst induce cell permeabilization, generation of reactive oxygen species (ROS) by mitochondria, inhibition of the respiratory chain, and energy-dependent cytotoxic release of ATP. On the other hand, the multiplicity of effects and the apparent contrast between experimental data continue to render the mechanism of Hst-induced killing of C. albicans unclear. In this investigation, using fluorescent probes (the potential-sensitive mitochondrial probe tetramethylrhodamine methyl ester perchlorate, TMRM; the ROS-sensitive probe dihydrofluorescein diacetate, DHF; the membrane-impermeant probe, calcein) and autofluorescence data we observed that Hst induce ROS generation by mitochondria undergoing a high energy swelling condition, accompanied by oxidation of cytosolic NAD(P)H and mitochondrial flavoproteins. ROS generation and swelling, attributable to an inhibition of the respiratory chain and to impairment of the K/H-exchanger, were followed by mitochondrial depolarization. Mitochondrial changes were accompanied by massive calcein influx, indicative of cell permeabilization, and prominent alterations of the cell size, shape, and optical density. The loss of proliferative activity was correlated, on a single cell basis, to the acquisition of a lipofuscin-like autofluorescence.
Insights
Histatins 3 and 5 (Hst) kill Candida albicans by inducing mitochondrial dysfunction, including swelling and reactive oxygen species (ROS) generation. This leads to cell permeabilization and loss of proliferation.
Area of Science:
- Antimicrobial Peptides
- Molecular Biology
- Cellular Physiology
Background:
- Histatins 3 and 5 (Hst) are known to possess candidacidal activity against Candida albicans.
- Previous studies suggest Hst induce cell permeabilization, mitochondrial reactive oxygen species (ROS) generation, and ATP release, but the exact mechanism remains unclear.
- Conflicting experimental data necessitate a deeper investigation into the precise molecular events underlying Hst-mediated fungal killing.
Purpose of the Study:
- To elucidate the detailed mechanism of candidacidal activity of histatins 3 and 5 (Hst) on Candida albicans.
- To investigate the role of mitochondrial function, ROS generation, and cell integrity in Hst-induced cell death.
- To correlate single-cell events with the loss of proliferative activity.
Main Methods:
- Utilized fluorescent probes including tetramethylrhodamine methyl ester perchlorate (TMRM), dihydrofluorescein diacetate (DHF), and calcein.
- Employed autofluorescence measurements to assess cellular changes.
- Analyzed mitochondrial swelling, membrane potential, ROS production, cytosolic NAD(P)H and flavoprotein oxidation, and cell permeabilization.
Main Results:
- Histatins induced ROS generation in mitochondria experiencing high-energy swelling, coupled with cytosolic NAD(P)H and flavoprotein oxidation.
- Mitochondrial depolarization followed ROS generation and swelling, which were linked to respiratory chain inhibition and K/H-exchanger impairment.
- Observed massive calcein influx, indicating cell permeabilization, significant alterations in cell morphology, and a correlation between lipofuscin-like autofluorescence and loss of proliferation.
Conclusions:
- Histatins trigger a cascade of mitochondrial events including swelling, ROS production, and depolarization, leading to cell permeabilization and death in Candida albicans.
- The findings clarify the mechanism of Hst-induced candidacidal activity, highlighting the critical role of mitochondrial dysfunction.
- The study establishes a link between specific cellular changes, such as lipofuscin-like autofluorescence, and the loss of fungal proliferative capacity.
