EXI'IlItIMEN1AL MYCOLOGY 12, 284-2HH (lYHH) 6083 *

BRIEF NOTE

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

JULIUS PETERSI.~ AND TIMOTHY D. LEATHERS'

DcparfltU"11 of Microbiolof.:Y alld Molecular G('lIf.'fk.~. Culij"(l/'flia CO/lq.:l' (~r Aft

Accepted for publication December 6. 19B7

PETERS, J.. AND LEATHERS. T. D. 1988. Phenotypic drug adaptiltion in Mucor 1'(1C('r1//lSIf5: Con· stilulively adapted and nonadaptive mutants. Experimelllt1J A-fyrolo~y. 12, 284-2HK. WilLi·type Mucor racemoSliS acquires phenotypic resistance to cycioheximiLic after a characteristic lug peri~ od. Adapted culturcs are cross·resislant to the unrelalcd drugs trichndcrmin and amphotericin B. Mutants were isolated as either constitutively resistant (COR mut;.mtsl or nonlldaptive (NAD mutants) to cycloheximide. Mutant COR 2A was constitutively resislant 10 cycloheximide <.llone. and <.ldapted normally to trichodermin and amphotericin B. Mulant COR JA was cunstitutivdy resistant to both cycloheximide and lrichodermin. and had partially induced resi"tance 10 ampho· tericin B. Mutant NAD 67 was also pleiolropic. as it was unable 10 adilpt to any llf lhcse drugs. Phenotypic drug adaptation in M. racellfOSIiS thus appears to involve bOlh general and l.1rug·"pecific components. 1" 191\11 AC:;ldtmic: Prts~. Inc:. INDEX DESCRIPTORS: drug resistance: phenotypic adaptation: Mucor ran'/IIo.Hls: cycloheximide: trichmlt:rmin: amphotericin B; antibiotics; fungicides.

Phenotypic adaptation to antifungal coordinately involves the entire fungal pop­ agents has been described for a wide vari­ ulation (Sypherd el al., 1979). For similar ety of fungi. including the phytopathogens reasons. M. rac£'lIlosus promises to serve Sclerolillia fruclicola (Grover and Moore. as an advantageous system for the study of 1981) and Usti/a'iO lIla)'dis (Esposito and phenotypic adaptation. High-level drug re­ Holiday, 1964). and the human pathogens sistance was found to be inducible in a sin­ Cryptococcus lIeo!orlllGIIS (Bodenhoff. gle step. and coordinately involved the en­ 1968) and Clllldida albicalls (Notario £'1 al., tire fungal population. M. mcelllO.HlS devel­ 1982). Recently, drug adaptation was de­ oped phenolypic resistance to drugs (,I' scribed in the dimorphic fungus Mucor dissimilar struclures and modes of action. raC£'IIlOS/lS (Leathers and Sypherd. 1985). specifically cycloheximide. lrithodermin. !vI. rUCl'11IOSUS has long serveu as un atlrac­ and amphotericin ll. Furlhermore. adapta­ tive model for the study of fungal dimor­ tion to a single drug induced cross­ phism; morphogenesis in Ihis species is resistance to unrelated drugs. Patterns of unique in that it is easily manipulated and cross-resistance suggested that a general mechanism might function in phenotypic

I To whom correspondence should \1c addressed. resistance. for example a nonspecific ex­ ~ Current address: Clinical Microhiology Laboralo­ port system. Such a resistance mechanism ry. Children's Hospital or Los Angclcs. University of could have serious implicalions for the ag­ Southern Californiu School of Medicine. Los Angeles. ricultural and clinical control of fungi. CA Y0054. :\ Current adtlre"s: Northern Regional Research More recently. an inducible trichodermin Cenler. U.S. Deparlment of Agriculture. Pcmill. IL dCloxificalion mechanism was clucicJalcd in 61604. M. rIICl'II/O.\·/I.\' (Fonzi and Sypherd. 1986).

2K4 0147·5Y7.1/HH ~).OO ('Uf'yrijll11 1- I!JIII( hr A,;;"kllliL: 1'1<"\, 1m: All nl:hl\ III ft'f'I\'lh'l'lwll in 1m) h'llI} Il'\I'rn'd DRUG AOAllTATION MUTANTS OF M, rlln'IT/I1.HU 285

The apparent spedlicity of this mechanism tively resistant>. A eomplementary non­ (dea<.:etylation of tri<.:hodermin hy an es­ udaplive class wus also sought (NAD mu­ terase) has raised questions <.:on<.:erning the lants. for nonadaptive to drugs>. plausibility of a general me<.:hanism of drug Nitrosoquanidine-mutagenized sporan­ adaptation. An important prediction of a giospores of M. mall/oSlls were subjected general resistan<.:e model is that drug­ to one of two regimes. A simple direct se­ adaptive mutants would be pleiotropic. i.e.. lection wus possible for constitutive (COR) altered in responses to more than one drug. mutants. Mutugenized spores were germi-. We des<.:ribe here the tirst isolation of drug­ nated on solid medium unaerobically in the adaptive mutants of M. J'ace/llUSIiS. presence of ZOO fJ.g/ml cycloheximide. Un­ M. J'ace/ll(lSl/S (synonym M. {IiSillil/iclis. treated spores produced colonies under strain ATCC 1216B. NRRl 3631) served as these conditions only after a lag period of the wild type. Details of culture mainte­ 100 to 150 h. C"lonies that urose earlier nan<.:e and grow(h have been described (Pe­ than 100 h were wnsidered putative COR ters and Sypherd. In8>. Growth medium mut'lnts. These were purilied and scored on for both liquid cultures and plates was 0.5% cycloheximide plates alongside wild-type peptone. 0.05% yeast nitrogen base. and controls. 2.0% glucose. A more complicated procedure involving Anaerobic liquid cultures for adaptation counterselection wus required to obtain cy­ growth curves were sparged with 100% CO2 cloheximide-sensitive (NAD) mutants. Mu­ at 0.5 vol!culture vol/min. and were rotary tagenized spores were inoculated into liq­ shaken at 100 rmp and at 25"C. Culture uid cultures spurged with nitrogen (0.5 vol! growth was followed with a Klett­ culture vol/min) in the presence of 100 Summerson colorimeter with a green tilter fJ.g/ml cycloheximide. Under these condi­ (540. nm). Anaerobic incubations of solid tions. untreated spores coordinately medium cultures were in CO2-enriched formed hyphal "germlings" following an BBl Gas-Pak chumbers (Becton­ adaptive lag period of approximately 24 h. Dickinson and Co.. Cockeysville.. MD>. After 48 h of incubation. mutugenized cul­ Anaerobic conditions resulted in the yeast­ tures were enriched for ungerminated like morphology of M. mce/ll()SlIs. While spores by tiltration through Miraelo·th. Fil­ this morphology has obvious manipulative trates were subjected to further counterse­ advantages. the mycelial form also under­ lection against germlings by the freeze­ goes phenotypic uduptation. as previously thaw method of Peters and Sypherd (1978). described (leathers und Sypherd. 1985). (U ngerminated spores ure more resistant to Peptone and yeast nitrogen base were freeze-thawing.) Following three rounds of from Difco luboratories. Detroit. Michi­ germination. enrichment. and counterselec­ gan. Cycloheximide wus from Sigma Chem­ tion. viuble spores were selected by pluting ical Co.. St. louis. Missouri. Amphoteri<.:in on drug-free anuerohic solid medium. to B was from E. R. Squibb and Sons. Prince­ form yeastlikc colonies. After single-colony ton. New Jersey. Other chemicals were re­ purilication on drug-free plates. putative agent grade. mutants were scored on replica pi utes con­ Isolation ofmlltlints deft!('til,.'e in drug mi· taining ZOO fJ.g/ml cycloheximide. aplaliol/. Two classes of mutunts were Quantittah'e linalysis of llduptatilJ11 and sought. Since phenotypic aduptation nor­ cToss·,-esistlince in mutlints. Drug gr:owrh mally occurs only after a characteristic in­ response curves were determined for Ii'quid duction lag period. mutants were selected cultures grown in the yeastlike form. Fig­ for uninduced (constitutive) resistance to ures IA through 10 compure wild-type M. cycloheximide (COR mutants. for constitu- mcemoSlls with three representative mu- E:RRATA

Panels of Fig. 1 are out of order, and consequently misidentified in figure legends and in accompanying text.

Corrected Legend:

Legend to Fig. 1: Adaptive response of tl. racemosus strains to drugs. Early exponential cultures were split at time 0 and challenged with drugs. (A) Cycloheximide constitutively resistant mutant COR 3A; (B) Cycloheximide constitutively resistant mutant COR 2A; (C) Wild-type strain; (D) cycloheximide nonadaptive mutant NAD 67.

Corrected Text:

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10 20 30 40 50 60 70 ao '0 HOURS FIG. I. Atlaplive response of M. rt/cemo.ms strains to drugs. Early ex.ponential cultures were split at time 0 am.I challenged with drugs. fA) WilJAype strain: (8) cycloheximide constitutively resistant mutanl COR 3A: tel cycloheximide con~thutively resistant mutant eRG 2A: IDl cyclohe:ximide non~ adaptive mutant NAD 67. Culture control. no drugs ulhled \el; cycloheximitle. 100 J.Lglml (A): lri~ chouermin. 5.0 ",glml (lIl: amphotericin B. 0.4 ",glml tal.

tants. Early exponential cultures of each As previously described (Leathers and strain were split at time 0 am! challenged Sypherd, (985), wild-type M, racemosliS with cycloheximide (100 flog/mil. trichoder­ adapted to initially inhibitory concentra­ min (5.0 flog/mi, or amphotcricin B (0.'1 tions of drugs after a lag period charactcr­ flog/mi). istic for each drug at a particular conccn- DRUG ADAPTATION MUTANTS OF M. nll'l'1I/1I.\/1.\ 287 tration (Fig. IA). Auapteu growth rates latcd as llllnauaptive to cycloheximiue. As were also characteristic for eaeh urug. but shown in Fig. ID. NAD 67 also failed to not strongly uepenuent on initial urug con­ recover from either trichodermin or ampho­ centrations. tericin B after more than 140 h. at which As shown in Fig. 1B, mutant strain COR time cultures were found to he sterile. The 3A grew without lag in medium containing pleiotropic nature of NAD 67 further sup­ 100 J.Lg/ml cycloheximide. Moreover. COR ports the conclusion that dissimilar drugs 3A showed early resistance to all three test share at least some common steps in phe­ drugs. The adaptive lag period for tricho­ notypic adaptation. uermin was abolished. while the lag for am­ In addition to pleiotropic defects in drug photericin B was half that characteristic of adaptation. strain NAD 67 showed other wild type. The generation times of COR 3A unselected characteristics. The generation in the presence of drugs were similar to time of NAD 67 in drug-free medium was those of adapted wild-type M. raCe1ll0SIIS. about four times that of the wild type. Fur­ Since COR 3A was selected for early resis­ ther. the mutant was defective in morpho­ tance only to cycloheximide, its pleiotropic genesis. as it grew yeastlike in air as well as nature argues for common components in under CO2 , Cells were also atypically large resistance to dissimilar drugs. Since resis­ and vacuolated. tance to amphotericin B required an adap­ Spontaneous revertants of NAD 67 were tive lag period (although reduced from wild selected on solid medium containing cyclo­ type) amphotericin B-specific components heximide (200 fJ.g!ml). Revertants arose at 3 are also suggested. These conclusions are the surprisingly high rate of 2.8 x 10- • consistent with the incomplete reciprocity These revertants also regained the ability to of cross-resistance to amphotericin B pre­ adapt to trichodermin. NAD 67 thus ap­ viously described (Leathers and Sypheru, pears to carry an unstable mutation in an· 1985). adaptation component common to dissimi­ In contrast. constitutively resistant mu­ lar drugs. Morphology defects of NAD 67 tant COR 2A showed early resistance only appear to be unrelated to drug adaptation, to cycloheximide (Fig. IC). Like COR 3A, since revertants remained yeastlike in air. COR 2A grew without lag upon introduc­ The behavior of drug adaptive mutants tion into cycloheximide medium. with a argues that both general and urug-specific doubling time typical of wild-type M. race­ components occur in the phenotypic adap­ IIl(}SIIS adapted to the drug (about 20 h). tation mechanism of M. racemoslis. These However. adaptation to both trichodermin results are consistent with palterns of drug and amphotericin B was characteristic of cross-resistance (Leathers and Sypherd. the wild-type strain. While it is possible IY85J. and also with the recent elucidation that the cycloheximide resistance of this of a trichodermin-specific detoxification mutant was unrelated to the adaptation (Fonzi and Sypherd, 1986). Common com­ mechanism. for example involving muta­ ponents of drug adaptation might well be tion of ribosomal proteins (Stocklein and limited to regulatory elements. A global Piepersberg. 1980), the doubling time of "alarmone" signal may prove to be the ba­ COR 2A in the presence of cycloheximide sis of a general stress response. was similar to that of normally adapted wild-type cultures. The behavior of COR REFERENCES 2A thus suggests that cycloheximide­ BODF:NHOFF, J. 196R. Develnpmenl of "trains of Cry'!'­ specific components also occur in pheno­ tOCOCe/1S ll('f~/;mlwn.fj resistant 10 Wr"Hl.itin. ampho­ typic adaptation. tericin B, lrichomycin and polymyxin B. ACTa M. raCPIllOSIiS mutant NAD 67 was iso- Potlwl. Aficrnhiol. Snmd. 73: 572-5X2. ' 2BB PETERS AND LEATHERS •

ESPOSITo. R. E.• ,\1'00 HOLIDAY. R. IlJM. The dfcct NUTARIO, V,. GAI.E. E. r.. KERRIOUE, 0 .. AND W"y. of 5·l1uoroueoxyurillint: on genetic replication and MAN. F. 19M2. Phenotypic resistance to amph(lteri· mitotic crossing over in synchronized cultures of do B in Candida albiculls: Relationship to glucnn U.,'tiillgO mClydis. Genetics 50: 1009-1017. metabolism. J. Gen. I\ticmhial. 128: 761-777. FONZI. W. F .. AND SYPHERD. P. S. (9X6. Trichoder· PETERS. J.. AND SYPHERD, P. S. 197M. Enrichment of min esterase activity and trichOlh:rmin resistance in mutants of Mucor rll('('n10.HI.'i by t1ilTerential freeze· Mucor ran!ITIOSIlS. Afltimic:mb. A/:eflr.s ChemOfher. killing. J. GI!1I. Microhial. 105: 77~1. 29: 57(}"575. STOCKlEIN, W .• AND PIEI>ERSnF.RG. W. IlJHO. Allered GIlOVER, A. K .. AND MOORE. J. D. {I)!H. Adaptation ribosomal protein L29 in a cyc!oheximide·re"istant of Sdt'rOlillj{/ /runlcella and Sc:/aorillia laxu to strain of Su('cJwrol1l,\'t't!.'i c~'re\'isiul!. Curro Gt!lIt't. I: highl:r conc~ntr;'ltions of fungicides. Phy{oputllO/o}(\' 177-IX3. 51: 399-101. SYPIIERD, P. S.. ORLOWSKI, M.. ,\NO PErERS, J. 11.)79. LEATHERS. T. D.. AND SYI'IiERD. P. S. It)M5. Induc­ Models of fungal dimorphism: ConlHlI of dimor­ ible phenOl ypic mulli·urug rcsistunce in '\/I/cor ran'· phism in '\/I/cor ran'nlH.Hl.L In '\/icrohiology 1979 mosliS. Alllimicrph. Agt.'tlls Cht'lIwrher. 27: H92­ 10. St:hlessinger. Ed,). pp. 224-227. Amer. Soc. Mi· H96. crobiol.. Washington D.C.