Effect of Auristatin PHE on Microtubule Integrity and Nuclear Localization in Cryptococcus neoformans

Abstract

The mechanism of action of the fungicidal peptide auristatin PHE was investigated in Cryptococcus neoformans. Since auristatin PHE causes budding arrest in C. neoformans (T. Woyke, G. R. Pettit, G. Winkelmann, and R. K. Pettit, Antimicrob. Agents Chemother. 45:3580-3584, 2001), microtubule integrity and nuclear localization in auristatin PHE-treated cells were examined. Iterative deconvolution in conjunction with an optimized C. neoformans microtubule immunolabeling procedure enabled detailed visualization of the microtubule cytoskeleton in auristatin PHE-treated C. neoformans. The effect of auristatin PHE on C. neoformans microtubule organization was compared with that of the tubulin-binding agent nocodazole. Both drugs produced complete disruption first of cytoplasmic and then of spindle microtubules in a time-and concentrationdependent manner. Sub-MICs of auristatin PHE caused complete microtubule disruption within 4.5 h, while 1.5 times the nocodazole MIC was required for the same effect. For both drugs, disruption of microtubules was accompanied by blockage of nuclear migration and of nuclear and cellular division, resulting in cells arrested in a uninucleate, large-budded stage. Nocodazole and the linear peptide auristatin PHE are remarkably different in structure and spectrum of activity, yet on the cellular level, they have similar effects.The basidiomycetous budding yeast Cryptococcus neoformans was identified as a human pathogen more than 100 years ago (5). Due to the growing number of immunocompromised patients worldwide, the number of invasive infections caused by this encapsulated yeast has been increasing dramatically (6,15,29,42). The most frequent clinical manifestation of cryptococcal infection is meningoencephalitis, which is fatal if left untreated. Existing therapies, including polyene and azole antifungals (11,29,42,49), are limited by host toxicities (42) and the emergence of drug-resistant strains (18,20).The pentapeptide auristatin PHE (dovaline-valine-dolaisoleunine-dolaproine-phenylalanine-methyl-ester) is a synthetic modification of the natural marine product dolastatin 10 (37, 38), which is currently undergoing phase I and phase II cancer clinical trials. Auristatin PHE has fungicidal activity against C. neoformans and fungicidal or fungistatic activity against various species of Trichosporon (39, 50). Auristatin PHE caused C. neoformans cells to arrest in a large-budded stage (50), suggesting that it might interfere with the microtubule dynamics in a manner similar to that of nocodazole in Saccharomyces cerevisiae (17).To investigate this possibility, we optimized cell wall digestion and microtubule labeling conditions for C. neoformans. This procedure, in conjunction with iterative deconvolution microscopy, allowed detailed visualization of the C. neoformans microtubule network. In iterative deconvolution, the focal Z plane sections are digitally captured and out-of-focus light in each section is mathematically removed and/or reduced by examination of neighboring sections (2, 43). T...

Differential Gene Expression in Auristatin PHE-Treated Cryptococcus neoformans

“…A complete C. neoformans genome annotation and the availability of cDNA microarrays would make a multi-time point study much more feasible. We demonstrated microscopically that auristatin PHE affects microtubule dynamics in C. neoformans, resulting in abnormal nuclear migration and arrested cell division (62). In C. neoformans, there are two ␤-tubulin genes, TUB1 and TUB2 (12).…”

“…This technique, as well as RT-PCR, was used due to the unavailability of C. neoformans cDNA microarrays. A portion of the cryptococcal transcriptome was analyzed in response to a single dose of auristatin PHE at a single time point, a dose and duration of exposure in which cells are viable but a fraction of the population begins to exhibit microtubule and cell division aberrations (43,61,62). A complete C. neoformans genome annotation and the availability of cDNA microarrays would make a multi-time point study much more feasible.…”

“…Total RNA was isolated from C. neoformans ATCC 90112, a cerebrospinal fluid isolate, the same strain used to examine the mechanism of action of auristatin PHE via immunofluorescence (62). Cells were treated with 1.5 times the MIC (7.89 M or 6 g/ml, as determined by broth microdilution in YM) of auristatin PHE or an equivalent volume of DMSO (control) for 90 min.…”

“…Compared to dolastatin 10, auristatin PHE had lower MICs (43,61), exhibited concentration-dependent killing between the MIC and four times the MIC (43), and demonstrated increased activity as well as prolonged postantifungal effects in the presence of human serum (43,61). Iterative deconvolution in conjunction with an optimized C. neoformans microtubule immunolabeling procedure enabled us to investigate the mechanism of action of auristatin PHE (62). Detailed visualization of the microtubule cytoskeleton in auristatin PHE-treated C. neoformans revealed complete disruption of first cytoplasmic and then spindle microtubules in a time-and concentration-dependent manner (62).…”

“…Iterative deconvolution in conjunction with an optimized C. neoformans microtubule immunolabeling procedure enabled us to investigate the mechanism of action of auristatin PHE (62). Detailed visualization of the microtubule cytoskeleton in auristatin PHE-treated C. neoformans revealed complete disruption of first cytoplasmic and then spindle microtubules in a time-and concentration-dependent manner (62). Sub-MICs of auristatin PHE caused complete microtubule disruption within 4.5 h, accompanied by blockage of nuclear migration and nuclear and cellular division, resulting in cells arrested in a uninucleate, large-budded stage (62).…”