Cryptococcus neoformans is a major opportunistic fungal pathogen in AIDS and other immunosuppressed patients. We have shown that wild-type haploid C. neoformans can develop an extensive hyphal phase under appropriate conditions. Hyphae produced under these conditions are monokaryotic, possess unfused clamp connections, and develop basidia with viable basidiospores. The ability to undergo this transition is determined by the presence of the a-mating type locus and is independent of serotype. The association of the hyphal phase with the a-mating type may explain the preponderance of this mating type in the environment and the nature of the infectious propagule of C. neoformans.Since the discovery of the sexual or perfect state of the human fungal pathogen Cryptococcus neoformans by Kwon-Chung (1, 2), it has been considered a bipolar heterothallic basidiomycete with two mating types, a and a (MA Ta and MA Ta The ability of hyphae to form fruiting body-like structures in the vegetative phase is called monokaryotic, homokaryotic, or haploid fruiting (13). Although haploid fruiting is common in the higher basidiomycetes (13-15), it has not been characterized in C. neoformans, and association with mating type has never been described. Recent observations made in this laboratory and a reassessment of past studies (5, 10, 16) allow us to propose a new hypothesis for the biased mating type ratios and the nature of the infectious propagule of C. neoformans. We show here that a strains of C. neoformans can undergo a true dimorphic transition from a haploid yeast phase to a hyphal phase from which vegetative growth can continue indefinitely. The hyphal phase is induced by nitrogen starvation on a solid surface. Basidia bearing viable basidiospores are also produced, all of which are a in mating type. The ability of a but not a cells to form basidiospores under these conditions provides an attractive explanation for both the mating type bias and the nature of the infectious propagule of C. neoformans. MATERIALS AND METHODSStrains. The strains used in this study are listed in Table 1. Auxotrophic strains were derived from JEC20 (MA Ta) and JEC21 (MA Ta). JEC20 and JEC21 are a well-characterized, haploid, congenic pair of prototrophic laboratory strains that presumably differ only at the mating type locus (17).Media 7327The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Respirometry using modified cell culture microplates offers an increase in throughput and a decrease in biological material required for each assay. Plate based respirometers are susceptible to a range of diffusion phenomena; as O2 is consumed by the specimen, atmospheric O2 leaks into the measurement volume. Oxygen also dissolves in and diffuses passively through the polystyrene commonly used as a microplate material. Consequently the walls of such respirometer chambers are not just permeable to O2 but also store substantial amounts of gas. O2 flux between the walls and the measurement volume biases the measured oxygen consumption rate depending on the actual [O2] gradient. We describe a compartment model-based correction algorithm to deconvolute the biological oxygen consumption rate from the measured [O2]. We optimize the algorithm to work with the Seahorse XF24 extracellular flux analyzer. The correction algorithm is biologically validated using mouse cortical synaptosomes and liver mitochondria attached to XF24 V7 cell culture microplates, and by comparison to classical Clark electrode oxygraph measurements. The algorithm increases the useful range of oxygen consumption rates, the temporal resolution, and durations of measurements. The algorithm is presented in a general format and is therefore applicable to other respirometer systems.

Kirsten (Ki)-ras cDNA clones were prepared from human lung and colon carcinoma cell lines expressing an activated c-Ki-ras2 gene. DNA sequence analysis and transfection studies indicate that different point mutations at the same codon can activate the gene; that most human c-Ki-ras2 mRNA uses sequences from a fourth coding exon distinct from that of its viral counterpart; and that at least one cell line is functionally homozygous for the activated gene.

SummaryCryptococcus neoformans possesses two mating types, MAT␣ and MATa. ␣-Cells are more virulent than a-cells and are also, unlike a-cells, capable of producing extensive hyphae in the haploid phase. The molecular analysis of hyphae production in C. neoformans has resulted in the identification of a gene which displays substantial similarity to other fungal STE12 genes, including the presence of a highly conserved homeodomain. Overexpression of the C. neoformans gene resulted in poor growth, altered morphology and the presence of hyphal projections, phenotypes reported in similar studies of the Saccharomyces cerevisiae STE12 gene. Overexpression was also found to induce MF␣, a pheromone, and CNLAC1, a confirmed C. neoformans virulence gene. The C. neoformans STE12␣ gene, however, has one striking difference from other fungal STE12 genes; it is found only in ␣-cells. The existence of STE12␣ in C. neoformans suggests that this fungus has elements of a conserved MAP kinase cascade, which may be organized in a novel manner.

Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinji is a member of the Fungi, sequence analysis and chromosomal localization show that DHFR is neither physically nor genetically linked to thymidylate synthase. Expression of recombinant P. carinji DHFR in heterologous hosts provides an abundant source of the enzyme that may form a basis for the development of new therapies for this enigmatic pathogen. Studies with the recombinant enzyme show that trimethoprim is a very poor inhibitor of P. carinji DHFR and, in fact, is a more potent inhibitor of human DHFR.Pneumocystis carinii pneumonia is a leading cause of morbidity and mortality in AIDS (1, 2). Since the onset of the AIDS epidemic, the incidence of P. carinii pneumonia has risen from ==200 to >25,000 cases per year in the United States. Due to the lack of a continuous in vitro culture system and the cumbersome nature of the rat model of P. carinii pneumonia, the development of anti-P. carinji therapy has rested largely on the assumption that antiprotozoan agents were likely to be effective (3-5). Despite the fact that P. carinii has recently been shown to be a member of the Fungi (6), the two principal therapeutic modalities, trimethoprim/ sulfamethoxazole and pentamidine, were developed on this basis. Before the AIDS epidemic, these agents were sufficient for treatment of the rare cases of P. carinii pneumonia. However, in the human immunodeficiency virus-positive patient, therapy and prophylaxis with the standard anti-P. carinii agents are complicated by frequent toxic and allergic side effects (7). New compounds active against P. carinii are clearly needed.The inability to propagate P. carinii reliably in vitro and the limited quantities of P. carinii enzymes that can be purified from infected rat lungs have hindered the search for anti-P. carinii agents. Purification and characterization of the intracellular targets for such agents would enable the development of new therapies for P. carinii pneumonia. Of the known anti-P. carinii agents, the dihydrofolate reductase (DHFR;5,6,7,3) inhibitors are the most thoroughly characterized. DHFR plays a central role in the de novo synthesis of nucleic acid precursors. DHFR inhibitors (methotrexate, trimethoprim, pyrimethamine) are effective antineoplastic, antibacterial, and antiprotozoal agents. P. carinii pneumonia clearly responds to the combination of a DHFR inhibitor (trimethoprim or pyrimethamine) and a sulfonamide. Despite their obvious efficacy when used in conjunction with a sulfonamide, available information suggests that trimethoprim and pyrimethamine, are in themselves poor inhibitors of P. carinii DHFR (IC50 values of 39,600 and 2400 nM, respectively, compared with 8 and 2500 nM for Escherichia coli DHFR at similar substrate concentrations; refs. 8 and 9). Other antifolates have b...