Formulation of a defined V8 medium for induction of sexual development of Cryptococcus neoformans

Abstract

For over 3 decades, sexual development in the human fungal pathogen Cryptococcus neoformans and other fungi has been initiated by growing compatible mating partners on V8 juice medium. Although this medium is an efficient inducer of sexual development, the mechanism by which it promotes the process is unknown. To understand how V8 juice medium induces sexual development, we attempted to purify inducing factors from V8 juice, and we carried out a complete compositional analysis of V8 juice. We discovered that no single factor is responsible for the effects of V8 juice medium. Rather, the unique composition of V8 juice medium provides the proper nutrient composition for inducing and sustaining complete sexual development. Utilizing these findings, we developed a defined V8 (DV8) medium that mimics V8 juice medium in sexual development assays. Then, using DV8 as a tool, we explored the roles that specific molecules play in enhancing sexual development. Surprisingly, we discovered that copper is a key factor, leading to an upregulation of the mating pheromone genes MFa and MFalpha, both required for the initial steps in sexual development. The utilization of DV8 to investigate the effects of copper on sexual development presented here is an example of how defining the conditions that induce sexual development will advance the study of C. neoformans.

FIG. 1.

Sexual development of C. neoformans. Large ovals represent yeast cells. Black and white circles represent a and α nuclei. (Section 1) Under appropriate environmental conditions, yeast cells of opposite mating types sense one another via pheromones and pheromone receptors. (Section 2) Yeast cells fuse, but their nuclei do not. (Section 3) After fusion, a new developmental program occurs, leading to dikaryotic, filamentous growth. (Section 4) In response to unknown signals, filamentous growth arrests, a basidium is formed, and nuclear fusion takes place. (Section 5) Meiosis and sporulation result in four chains of spores that reside on the surface of the basidium.

FIG. 2.

Sexual development activity fractionates with the soluble portion of V8 juice. Crosses between wild-type a and α strains were placed on agar media containing different HPLC fractions of V8 juice. The macroscopic view reveals white spots, which indicate robust sexual development due to filament formation, whereas dark spots indicate little or no sexual development.

FIG. 3.

DV8 medium mimics V8 juice medium. Crosses between wild-type strains were carried out with agar medium containing 5% filtered V8 juice or defined components determined from a compositional analysis. Panels show the periphery of a cross under ×200 magnification.

FIG. 4.

Sexual development requires nitrogen, salts, and metals. Crosses between wild-type strains were carried out with agar medium containing the full complement of defined components for DV8 medium (DV8), no defined components (core), or defined components tested individually (nitrogen, salts, or metals). The panels show the periphery of a cross under ×100 magnification.

FIG. 5.

Copper induces sexual development in DV8 medium. Crosses between wild-type strains were carried out with agar medium containing the full complement of defined components for DV8 medium, DV8 medium without any metals, or DV8 medium containing only a single metal (iron, zinc, or copper). The panels show the periphery of a cross under ×200 magnification.

FIG. 6.

Copper enhances mate recognition, cell fusion, and filamentation and upregulates pheromone gene expression. (A) Left panels show wild-type a cells (left) and wild-type α cells (right) in confrontation assays in the presence and absence of copper. Magnification, ×200. The middle panels show colonies on petri plates resulting from cell fusion in the presence and absence of copper. The right panels show the peripheries of crosses between wild-type cells in the presence and absence of copper. Magnification, ×200. (B) Northern blot on RNA from strains under a variety of conditions after 24 h of incubation: Lane 1, wild-type cross on DV8 medium without metals; lane 2, wild-type cross on DV8 with 100 μM copper; lane 3, haploid α on DV8 medium; lane 4, haploid α on DV8 with 100 μM copper; lane 5, haploid a on DV8 medium; lane 6, haploid a on DV8 medium with 100 μM copper. The probes are listed to the right of each panel.