EXPERIMENTAL MYCOLOGY 12,284-288 (1988)

BRIEF NOTE

Phenotypic Drug Adaptation in Mucor racemosus: Constitutively Adapted and Nonadaptive Mutants

JULIUS PETERSI,' AND TIMOTHY D. LEATHERS~

Department of Microbiology and Molecular Genetics, California College of Medicine, University of California, Irvine, California 92717

Accepted for publication December 6, 1987

PETERS, J., AND LEATHERS, T. D. 1988. Phenotypic drug adaptation in Mucor racemosus: Con- stitutively adapted and nonadaptive mutants. Experimental Mycology. 12, 284-288. Wild-type Mucor racemosus acquires phenotypic resistance to cycloheximide after a characteristic lag peri- od. Adapted cultures are cross-resistant to the unrelated drugs trichodermin and amphotericin B. Mutants were isolated as either constitutively resistant (COR mutants) or nonadaptive (NAD mutants) to cycloheximide. Mutant COR 2A was constitutively resistant to cycloheximide alone, and adapted normally to trichodermin and amphotericin B. Mutant COR 3A was constitutively resistant to both cycloheximide and trichodermin, and had partially induced resistance to ampho- tericin B. Mutant NAD 67 was also pleiotropic, as it was unable to adapt to any of these drugs. Phenotypic drug adaptation in M. racemosus thus appears to involve both general and drug-specific components. 0 1988 Academic Press, Inc.

INDEX DESCRIPTORS: drug resistance; phenotypic adaptation; Mucor racemosus; cycloheximide; trichodermin; amphotericin B; antibiotics; fungicides.

Phenotypic adaptation to antifungal agents has been described for a wide vari- ety of fungi, including the phytopathogens Sclerotinia fructicola (Grover and Moore, 1981) and Ustilaqo maydis (Esposito and Holiday, 1964), and the human pathogens Cryptococcus neoformans (Bodenhoff, 1968) and Candida albicans (Notario et al., 1982). Recently, drug adaptation was de- scribed in the dimorphic fungus Mucor racemosus (Leathers and Sypherd, 1985). M. racemosus has long served as an attrac- tive model for the study of fungal dimor- phism; morphogenesis in this species is unique in that it is easily manipulated and

’ To whom correspondence should be addressed. * Current address: Clinical Microbiology Laborato-

ry, Children’s Hospital of Los Angeles, University of Southern California School of Medicine, Los Angeles, CA 90054.

3 Current address: Northern Regional Research Center, U.S. Department of Agriculture, Peoria, IL 61604.

coordinately involves the entire fungal pop- ulation (Sypherd et al., 1979). For similar reasons, M. racemosus promises to serve as an advantageous system for the study of phenotypic adaptation. High-level drug re- sistance was found to be inducible in a sin- gle step, and coordinately involved the en- tire fungal population. M. racemosus devel- oped phenotypic resistance to drugs of dissimilar structures and modes of action, specifically cycloheximide, trichodermin, and amphotericin B. Furthermore, adapta- tion to a single drug induced cross- resistance to unrelated drugs. Patterns of cross-resistance suggested that a general mechanism might function in phenotypic resistance, for example a nonspecific ex- port system. Such a resistance mechanism could have serious implications for the ag- ricultural and clinical control of fungi.

More recently, an inducible trichodermin detoxification mechanism was elucidated in M. racemosus (Fonzi and Sypherd, 1986).

284 0147-5975188 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

DRUG ADAPTATION MUTANTS OF M. racemosux 285

The apparent specificity of this mechanism (deacetylation of trichodermin by an es- terase) has raised questions concerning the plausibihty of a general mechanism of drug adaptation, An important prediction of a general resistance model is that drug- adaptive mutants would be pleiotropic, i.e., altered in responses to more than one drug. We describe here the first isolation of drug- adaptive mutants of M. racemosus.

M. racemosus (synonym M. lusitanicus, strain ATCC 1216B, NRRL 3631) served as the wild type. Details of culture mainte- nance and growth have been described (Pe- ters and Sypherd, 1978). Growth medium for both liquid cultures and plates was 0.5% peptone, 0.05% yeast nitrogen base, and 2.0% glucose.

Anaerobic liquid cultures for adaptation growth curves were sparged with 100% CO, at 0.5 vohculture vol/min, and were rotary shaken at 100 rmp and at 25°C. Culture growth was followed with a Klett- Summerson calorimeter with a green filter (540 nm). Anaerobic incubations of solid medium cultures were in CO,-enriched BBL Gas-Pak chambers (Becton- Dickinson and Co., Cockeysville, MD). Anaerobic conditions resulted in the yeast- like morphology of M. rucemosus. While this morphology has obvious manipulative advantages, the mycelial form also under- goes phenotypic adaptation, as previously described (Leathers and Sypherd, 1985).

Peptone and yeast nitrogen base were from Difco Laboratories, Detroit, Michi- gan. Cycloheximide was from Sigma Chem- ical Co., St. Louis, Missouri. Amphotericin B was from E. R. Squibb and Sons, Prince- ton, New Jersey. Other chemicals were re- agent grade.

Isolation of mutants defective in drug ad- aptation. Two classes of mutants were sought. Since phenotypic adaptation nor- mally occurs only after a characteristic in- duction lag period, mutants were selected for uninduced (constitutive) resistance to cycloheximide (COR mutants, for constitu-

tively resistant). A complementary non- adaptive class was also sought (N tants, for nonadaptive to drugs).

Nitrosoquanidine-mutagenized spor giospores of M. racemosus were subjec to one of two regimes. A simple direct se- lection was possible for constitutive (C mutants. Mutagenized spores were ge nated on solid medium anaerobically in the presence of 200 pg/ml cycloheximide. Un- treated spores produced colonies u these conditions only after a lag peri 100 to 150 h. Colonies that arose e than 100 h were considered putative mutants. These were purified and scar cycloheximide plates alongside wild-type controls.

A more complicated procedure involving counterselection was required to obtai cloheximide-sensitive (NAD) mutants tagenized spores were inoculated into uid cultures sparged with nitrogen (0. culture vol/min) in the presence of 100 bg/ml cycloheximide. Under these condi- tions, untreated spores coordinately formed hyphal “germlings” foi~owi~~ an adaptive lag period of approximately 24 h. After 48 h of incubation, mutagenized cd- tures were enriched for u~gerrn~~at~d spores by filtration through Miraclot trates were subjected to further counterse- lection against germlings by the freeze- thaw method of Peters and Sypherd (197 (Ungerminated spores are more resistant to freeze-thawing.) Following three rounds of germination, enrichment, and counterselec- tion, viable spores were selected by plating on drug-free anaerobic solid mediums to form yeastlike colonies. After single-colony purification on drug-free plate mutants were scored on replica taining 200 pg/ml cycloheximide.

Quantitative unaiysis of a cross-resistance in mutants. response curves were determme cultures grown in the yeastlike for ures IA through 1D compare wild- racemosus with three representative mu-

286 PETERS AND LEATHERS

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FIG. 1. Adaptive response of M. racemosus strains to drugs. Early exponential cultures were split at time 0 and challenged with drugs. (A) Wild-type strain; (B) cycloheximide constitutively resistant mutant COR 3A; (C) cycloheximide constitutively resistant mutant CR0 2A; (D) cycloheximide non- adaptive mutant NAD 67. Culture control, no drags added (0); cycloheximide, 100 pg/ml (A); tri- chodermin, 5.0 pg/ml (A); amphotericin B, 0.4 pg/ml (0).

tams. Early exponential cultures of each As previously described (Leathers and strain were split at time 0 and challenged Sypherd, 1983, wild-type M. racemosus with cycloheximide (100 pg/ml), trichoder- adapted to initially inhibitory concentra- min (5.0 kg/ml, or amphotericin B (0.4 tions of drugs after a lag period character- w/ml). istic for each drug at a particular concen-

DRUG ADAPTATION MUTANTS OF 44. racemosus 287

tration (Fig. 1A). Adapted growth rates were also characteristic for each drug, but not strongly dependent on initial drug con- centrations.

As shown in Fig. lB, mutant strain COR 3A grew without lag in medium containing 100 yg/ml cycloheximide. Moreover, COR 3A showed early resistance to all three test drugs. The adaptive lag period for tricho- dermin was abolished, while the lag for am- photericin B was half that characteristic of wild type. The generation times of COR 3A in the presence of drugs were similar to those of adapted wild-type M. racemosus. Since COR 3A was selected for early resis- tance only to cycloheximide, its pleiotropic nature argues for common components in resistance to dissimilar drugs. Since resis- tance to amphotericin B required an adap- tive lag period (although reduced from wild type) amphotericin B-specific components are also suggested. These conclusions are consistent with the incomplete reciprocity of cross-resistance to amphotericin B pre- viously described (Leathers and Sypherd, 1985).

In contrast, constitutively resistant mu- tant COR 2A showed early resistance only to cycloheximide (Fig. 1C). Like COR 3A, COR 2A grew without lag upon introduc- tion into cycloheximide medium, with a doubling time typical of wild-type M. ruce- mosus adapted to the drug (about 20 h). However, adaptation to both trichodermin and amphotericin B was characteristic of the wild-type strain. While it is possible that the cycloheximide resistance of this mutant was unrelated to the adaptation mechanism, for example involving muta- tion of ribosomal proteins (Stocklein and Piepersberg, 1980), the doubling time of COR 2A in the presence of cycloheximide was similar to that of normally adapted wild-type cultures. The behavior of COR 2A thus suggests that cycloheximide- specific components also occur in pheno- typic adaptation.

M. racemosus mutant NAD 67 was iso-

lated as nonadaptive to cycloheximide. As shown in Fig. ID, NAD 67 also failed to recover from either trichodermin or ampho- tericin B after more than 140 h, at w time cultures were found to be sterile. pleiotropic nature of NAD 67 further sup- ports the conclusion that dissimilar d share at least some common steps in phe- notypic adaptation.

In addition to pleiotropic defects in dr adaptation, strain NAD 67 showed st unselected characteristics. The generation time of NAD 67 in drug-free medium was about four times that of the wild type. ther, the mutant was defective in mor genesis, as it grew yeastlike in air as well as under CO,. Cells were also aty~ical~y large and vacuolated.

Spontaneous revertants of MAD 67 were selected on solid medium containing cyclo- heximide (200 pglml). Revertants arose at the surprisingly high rate of 2.8 x 1 These revertants also regained the ability to adapt to trichodermin. NAD 67 thus ap- pears to carry an unstable mutation in an adaptation component common tcs mi- lar drugs. Morphology defects of 67 appear to be unrelated to drug adaptation, since revertants remained yeasthke in air.

The behavior of drug adaptive mutants argues that both general and drug-s~ec~~c components occur in the phenotypic adap- tation mechanism of M. racemosus. These results are consistent with patterns of drug cross-resistance (Leathers and Sy~~~rd, 19SS), and also with the recent elucidation of a trichodermin-specific detoxificat~Q~ (Fonzi and Sypherd, 1986). Common com- ponents of drug adaptation might well be limited to regulatory elements A global “alarmone” signal may prove to be the sis of a general stress response.

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288 PETERS AND LEATHERS

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