Mitochondrial alterations and autofluorescent conversion of Candida albicans induced by histatins.
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.
Diaz
Giacomo
G
Dipartimenti di Citomorfologia, Università di Cagliari, 09042 Monserrato (Cagliari), Italy. gdiaz@unica.it
Polonelli
Luciano
L
Conti
Stefania
S
Messana
Irene
I
Cabras
Tiziana
T
Putzolu
Martina
M
Falchi
Angela Maria
AM
Fadda
Maria Elisabetta
ME
Cosentino
Sofia
S
Isola
Raffaella
R
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002176:Candida albicans / Q000166:cytology / Q000187:drug effects* / Q000254:growth & development; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D011506:Proteins / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D012235:Rhodamines; D000099053:Histidine-Rich Glycoprotein
D000900:Anti-Bacterial Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011506:Proteins; D017382:Reactive Oxygen Species; D012235:Rhodamines; D000099053:Histidine-Rich Glycoprotein; C401833:tetramethylrhodamine methyl ester; C007740:fluorexon