Proc. Natl. Acad. Sci. USA Vol. 93, pp. 7327-7331, July 1996 Microbiology Dimorphism and haploid fruiting in Cryptococcus neoformans: Association with the a-mating type (monokaryotic/dikaryotic/clamp connection/hyphae) BRIAN L. WICKEs, MARfA E. MAYORGA, URSULA EDMAN, AND JEFFREY C. EDMAN Department of Laboratory Medicine, University of California, San Francisco, CA 94143 Communicated by Ira Herskowitz, University of California, San Francisco, CA, March 8, 1996 (received for review October 7, 1995) ABSTRACT Cryptococcus neoformans is a major opportu- tory, desiccated yeast cells display very poor viability, an nistic fungal pathogen in AIDS and other immunosuppressed observation confirmed for environmental isolates as well (11). patients. We have shown that wild-type haploid C. neoformans Therefore, the desiccated yeast cell is an unlikely cause of can develop an extensive hyphal phase under appropriate cryptococcosis. On the other hand, C. neoformans basidio- conditions. Hyphae produced under these conditions are spores are 1.8 x 2.0 ,um in size, present on aerial hyphae, easily monokaryotic, possess unfused clamp connections, and de- aerosolized, infectious, and much more resistant to desiccation velop basidia with viable basidiospores. The ability to undergo than yeast cells (12). These characteristics would appear to this transition is determined by the presence of the a-mating favor the basidiospore over the yeast cell as the infectious type locus and is independent of serotype. The association of form. the hyphal phase with the a-mating type may explain the The ability of hyphae to form fruiting body-like structures in preponderance of this mating type in the environment and the the vegetative phase is called monokaryotic, homokaryotic, or nature of the infectious propagule of C. neoformans. haploid fruiting (13). Although haploid fruiting is common in the higher basidiomycetes (13-15), it has not been character- Since the discovery of the sexual or perfect state of the human ized in C. neoformans, and association with mating type has fungal pathogen Cryptococcus neoformans by Kwon-Chung (1, never been described. Recent observations made in this lab- 2), it has been considered a bipolar heterothallic basidiomycete oratory and a reassessment of past studies (5, 10, 16) allow us with two mating types, a and a (MA Ta and MA Ta). The to propose a new hypothesis for the biased mating type ratios imperfect state grows as a budding yeast. Under suitable and the nature of the infectious propagule of C. neoformans. environmental conditions, fusion between the two mating We show here that a strains of C. neoformans can undergo a types occurs and generates the perfect state, Filobasidiella true dimorphic transition from a haploid yeast phase to a neoformans. The perfect state is characterized by transient hyphal phase from which vegetative growth can continue dikaryotic hyphae with typical basidiomycetous clamp connec- indefinitely. The hyphal phase is induced by nitrogen starva- tions. Basidia are formed at the hyphal termini wherein tion on a solid surface. Basidia bearing viable basidiospores are karyogamy and meiosis occur. Continuous post-meiotic rep- also produced, all of which are a in mating type. The ability of lication and basidiosporogenesis result in four long chains of a but not a cells to form basidiospores under these conditions basidiospores on the basidial surface (2). Upon germination, provides an attractive explanation for both the mating type bias the haploid spores regenerate the imperfect yeast state. and the nature of the infectious propagule of C. neoformans. The various components of the life cycle of C. neoformans have been well characterized by both genetic and ultrastruc- tural studies (2, 3). Genetic analysis of the individual products MATERIALS AND METHODS of meiosis (basidiospores) has demonstrated 1:1 segregation of Strains. The strains used in this study are listed in Table 1. mating types in laboratory crosses. However, clinical and Auxotrophic strains were derived from JEC20 (MATa) and environmental isolates are predominantly of the a-mating JEC21 (MA Ta). JEC20 and JEC21 are a well- characterized, type, ranging from 40:1 (a:a) for environmental isolates to 30:1 haploid, congenic pair of prototrophic laboratory strains that for clinical isolates (4-7). These results suggest that clinical presumably differ only at the mating type locus (17). and environmental isolates do not represent sexually- Media. Media components obtained from Difco were Bacto- reproducing populations of C. neoformans. It has also been agar, Noble agar, yeast nitrogen base (YNB) without amino shown that a cells are more virulent than a cells (8). This acids, YNB without amino acids and without ammonium increased virulence could explain the preponderance of the sulfate (YNB-AS), yeast extract, and peptone. Other compo- a-mating type in clinical samples. There is, however, no nents including amino acids, agarose, carbohydrates, nucleo- adequate explanation for the overrepresentation of a in en- sides, and salts were obtained from Sigma. Yeast extract/ vironmental samples. peptone/glucose (YPG) contained 1% yeast extract, 2% pep- Infection with C. neoformans, like many other systemic tone, and 2% glucose. Synthetic dextrose (SD) contained 6.7 fungal pathogens, begins with inhalation and is first established g/liter YNB, 20 g/liter glucose, and appropriate amino acid or in the lungs. Effective penetration to the lung parenchyma nucleoside supplements. V8 juice medium contained 5% V8 requires particles <2.0 ,um in diameter (9). However, yeast juice, 0.5 g/literKH2PO4, and 4% agar adjusted to pH 7.0 with cells are 4-8.0 ,um in diameter or larger, if one includes the KOH. Filament agar contained 6.7 g/liter YNB-AS, 0.5% polysaccharide capsule. It has been demonstrated that aero- glucose, and 4% Bacto-agar at pH 5.0. Unless otherwise solized particles <2.0 ,um can cause cryptococcosis in exper- specified, solid media contained 2% Bacto-agar. Manipula- imental animals (10). This observation was attributed to yeast tions of media conditions were carried out by preparing cells that had become desiccated and, as a result, were small concentrated stocks of the individual components and diluting enough to penetrate into the alveoli. However, in the labora- to the appropriate final concentrations. The publication costs of this article were defrayed in part by page charge Abbreviations: YNB, yeast nitrogen base; YNB-AS, YNB without payment. This article must therefore be hereby marked "advertisement" in amino acids and ammonium sulfate; YPG, yeast extract/peptone/ accordance with 18 U.S.C. §1734 solely to indicate this fact. glucose; DAPI, 4',6-diamidino-2-phenylindole. 7327 Downloaded by guest on September 30, 2021 7328 Microbiology: Wickes et al. Proc. Natl. Acad. Sci. USA 93 (1996) Table 1. Strains used in this study JEC20, JEC21, JEC50, and JEC30 were used to assess linkage Strain Serotype Genotype of haploid fruiting to mating type. Strain JEC21 was used for mating type determination of basidiospores produced by hap- JEC20 D MATa loid fruiting. JEC21 D MATa JEC30 D MATa lysI JEC50 D MATa ade2 RESULTS JEC229 D MATa Discovery of the Haploid Hyphal Phase of C. neoformans. JEC230 D MATa The recognition of hyphae production from haploid yeast cells NIH332 D MATa was made during the course of a series of transformations NIH424 D MATa conditions for gene disruption in C. NIH530 D MATa intended to optimize NIH264 D MATa neoformans. A number of transformants from a disruption NIH430 D MATa experiment were patched onto YNB-AS medium. After 72-96 NIH291 A MATa hr it was found that the majority of the patches had visibly NIH371 A MATa rough edges that were unlike the typical smooth margins of NIH201 A MATa yeast colonies. Repatching to fresh YNB-AS again yielded this NIH288 A MATa morphology. Microscopic investigation revealed that the rough H99 A MATa appearence was due to the presence of abundant hyphae. The NIH112 B MATa isolates were confirmed to be C. neoformans by melanin NIH189 B MATa production on bird seed agar, lack of growth on lactose, and NIH444 B MATa the presence of urease. When the hyphae-forming isolates NIH34 C MATa were patched onto YNB, hyphae were not observed, only NIH401 C MATa typical yeast-like colonies. YNB-AS was then tested to see if it NIH strains are courtesy of K. J. Kwon-Chung (Laboratory of would support hyphal growth in untransformed strains. Hy- Clinical Investigation/National Institute of Allergy and Infectious phae were indeed produced by these strains demonstrating that Diseases Bethesda, MD). JEC strains are from the collection of J.C.E. this characteristic was not a transformation-mediated phenom- H99 is courtesy of J. Perfect (Duke University Medical Center, enon. These observations suggested that hyphae production in Durham, NC) haploid strains of C. neoformans was an innate characteristic Optimization of Hyphal Induction. A number of different that could be influenced by growth conditions. variables, both physical and nutritional, were tested for their The Hyphal Phase in C. neoformans. Fig. 1 shows C. neo- effect on hyphal induction. The physical parameters included formans growing vegetatively as hyphae. The hyphal phase is agar concentration, incubation temperature, moisture content, induced on filament agar and is characterized by extensive solid or liquid growth substrate, pH, and to a limited extent, branched hyphae that grow on and into the agar. Occasional oxygen tension. Nutritional factors included agar type, nitro- aerial hyphae can also be observed. Numerous blastospores gen concentration and source, carbohydrate concentration and can be seen that bud directly off the sides of each hypha. source, and seed culture conditions. Basidia with four chains of viable basidiospores are produced, Microscopy. Hyphal development and basidiospore produc- although the frequency is much lower than in a standard tion from haploid yeast cells were followed by patching ap- MATa-MATa cross and, on average, only 5-10 spores per proximately 1 X 107 cells of JEC21 onto filament agar plates chain were observed.