Effects of Microtubule and Actin Inhibitors on <b><i>Cryptococcus neoformans</i></b> Examined by Scanning and Transmission Electron Microscopy

Abstract

Background:Cryptococcus neoformans is one of the most important human fungal pathogens. Its cells contain rich microtubules required for nuclear division and rich F-actin cytoskeletons for cell division. Disruption of microtubules by a microtubule inhibitor should block nuclear division, and disruption of F-actin by an actin inhibitor should block cell division. We investigated the effects of microtubule and actin inhibitors to find out whether the cytoskeletons of C. neoformans can become a new anti-fungal target for the inhibition of cell division, when examined at the ultrastructural level. Methods: Cells treated with the microtubule inhibitors vincristine (VIN) and methyl benzimidazole-2-ylcarbamate (BCM) and the actin inhibitor latrunculin A (LA), in yeast extract peptone dextrose medium, were examined by scanning (SEM) and transmission electron microscopy (TEM), and the cell number was counted using a Bürker chamber. Results: After 2 days of inhibition with VIN, BCM or LA, the cells did not divide, but later, resistant, proliferating cells appeared in all samples. With combined microtubule and actin inhibitors (VIN + LA or BCM + LA), cells did not divide during 6 or even 14 days, and no resistant cells originated. TEM showed that the inhibited cells were without cytoplasm and were dead; only empty cell walls persisted with reduced capsules, shown on SEM. Conclusion: Combined microtubule and actin inhibitors (VIN + LA or BCM + LA), have lethal effects on C. neoformans cells and no resistant cells originate.

Microtubules and Actin Cytoskeleton of Cryptococcus neoformans as Targets for Anticancer Agents to Potentiate a Novel Approach for New Antifungals

“…neoformans , IFM 41464 (CUH 34, 48-9943, 881, skin, serotype A), from the Medical Mycology Research Centre, Chiba University, Japan [16] was used. Media and cell cultivation were identical as described [9,10]. …”

“…Their effects on the F-actin cytoskeleton (AC) and the cell division of C. neoformans were investigated by fluorescence and phase-contrast microscopy in the search for new antifungal agents for the inhibition of cell division. Previously, C. neoformans was studied by electron microscopy [3], and the effects of some of these inhibitors were monitored [9,10]. Electron microscopy has also been used for examining inhibition in other yeasts [11,12,13,14,15].…”

The Effects of the F-Actin Inhibitor Latrunculin A on the Pathogenic Yeast <b><i>Cryptococcus neoformans</i></b>

“…To obtain an exponential culture, cells cultivated in YEPD medium [1% (w/v) yeast extract, 1% (w/v) peptone and 1% (w/v) dextrose] on a shaker overnight at 23°C (about 16 h) were diluted to 5 × 10 5 to 1 × 10 6 cells ml -1 by 1% YEPD and used for the application of LA [7,8]. …”

“…10 m M stock solution was prepared by dissolving 100 μg of LA (Molecular Probes, USA) in 25 μl of DMSO, kept at −20°C and then added to cells in 1% YEPD to a final concentration of 100 μ M that contained 1% DMSO [7,8,10,11]; 500-μl volumes of cultures in test tubes were shaken in the dark in a water bath. Samples for EM were taken before the LA treatment, i.e.…”

“…Recently, we studied the effects of microtubules and actin inhibitors on C. neoformans to examine whether its cytoskeleton can become a new antifungal target for the inhibition of cell division. The actin inhibitor LA was the most efficient: the actin structures [7] and cytoplasm disappeared and the inhibited cells died, with only their cell walls persisting [8]. Here, we investigated the early effects of LA on C. neoformans cells using freeze-substitution (FS) and transmission electron microscopy (EM) to identify how and why inhibited cells die.…”