Cryptococcus neoformans mates on pigeon guano: implications for the realized ecological niche and globalization

Abstract

The ecological niche that a species can occupy is determined by its resource requirements and the physical conditions necessary for survival. The niche to which an organism is most highly adapted is the realized niche, whereas the complete range of habitats that an organism can occupy represents the fundamental niche. The growth and development of Cryptococcus neoformans and Cryptococcus gattii on pigeon guano were examined to determine whether these two species occupy the same or different ecological niches. C. neoformans is a cosmopolitan pathogenic yeast that infects predominantly immunocompromised individuals, exists in two varieties (grubii [serotype A] and neoformans [serotype D]), and is commonly isolated from pigeon guano worldwide. By contrast, C. gattii often infects immunocompetent individuals and is associated with geographically restricted environments, most notably, eucalyptus trees. Pigeon guano supported the growth of both species, and a brown pigment related to melanin, a key virulence factor, was produced. C. neoformans exhibited prolific mating on pigeon guano, whereas C. gattii did not. The observations that C. neoformans completes the life cycle on pigeon guano but that C. gattii does not indicates that pigeon guano could represent the realized ecological niche for C. neoformans. Because C. gattii grows on pigeon guano but cannot sexually reproduce, pigeon guano represents a fundamental but not a realized niche for C. gattii. Based on these studies, we hypothesize that an ancestral Cryptococcus strain gained the ability to sexually reproduce in pigeon guano and then swept the globe.

FIG. 1.

Growth and pigmentation of Cryptococcus species on medium containing pigeon guano. C. neoformans var. grubii, C. neoformans var. neoformans, and C. gattii strains were grown overnight at 30°C in YPD medium, washed with PBS, and 10-fold serially diluted (10³ to 10⁶ dilutions). (A) Two microliters of each diluted cell suspension was spotted directly onto YNB or pigeon guano medium containing 25%, 12%, or 2.5% pigeon guano and incubated at 25°C for 7 days. To examine pigmentation, a sterilized 14-kDa-cutoff dialysis membrane was placed on the medium surface and then 0.5 μl of each diluted cell suspension was spotted onto the membrane. After 7 days of incubation at 25°C, membranes were removed from the medium and placed on moist filter paper to examine colony pigmentation. (B) To quantify growth, the 10³ dilutions of JEC21α (neoformans), KN99α (grubii), and B4546α (gattii) grown on membranes were removed after 7 days and placed in PBS, and CFU were counted by serial dilution on YPD. Numbers of CFU resulting from growth on the various media are expressed as percentages of the number for the strain grown on YNB.

FIG. 2.

Laccase mutant strains retain partial pigmentation on medium containing pigeon guano but not on medium containing l-DOPA. C. neoformans var. grubii H99 (wild type [WT]), lac1 and lac2 single mutants, a lac1 lac2 double mutant, and a Candida albicans strain were washed with PBS, and 2 μl of each cell suspension was spotted directly onto minimal medium (medium lacking l-DOPA [−l-DOPA]), Niger seed medium, medium containing l-DOPA to induce melanin production, or onto medium containing 25%, 12%, or 2.5% pigeon guano (PG). To examine the pigmentation of the strains grown on the pigeon guano medium, a sterilized 14-kDa-cutoff dialysis membrane was placed on the medium surface and then 0.5 μl of each diluted cell suspension was spotted onto the membrane. After 7 days of incubation at 25°C, membranes were removed from the medium, placed on moist filter paper, and photographed.

FIG. 3.

Cryptococcus neoformans var. grubii and var. neoformans mate robustly on pigeon guano. Opposite-mating-type strains of C. neoformans var. grubii and var. neoformans were washed with PBS, and equal volumes of each mating type were combined. The mixture was placed as a 10-μl drop onto V8 medium (pH 5 for var. grubii and pH 7 for var. neoformans) or medium containing 25%, 12%, or 2.5% pigeon guano (PG). Plates were incubated in the dark at 25°C for 7 days. (A) Filamentation (×20) and sporulation (×400) of var. neoformans and var. grubii strains on media containing 25% and 12% pigeon guano. (B) Comparison of C. neoformans var. neoformans mating colonies on V8 (pH 7) medium or pigeon guano medium containing increasing levels of pigeon guano.

FIG. 4.

Cryptococcus gattii mating is inhibited on pigeon guano medium (PG). C. gattii strains of opposite mating types were washed with PBS, and equal volumes of each mating type were combined. The mixture was placed as a 10-μl drop onto MS medium or medium containing 25%, 12%, or 2.5% pigeon guano. Plates were incubated in the dark at 25°C for 3 days and then photographed at a ×20 magnification. The top panel shows a random patch at the edge of the mating colony on MS medium. The lower three panels show patches of the mating colonies where filamentation was observed on media containing 2.5%, 12%, and 25% pigeon guano, respectively.

FIG. 5.

Regulation of C. neoformans and C. gattii mating on V8 medium compared to that on medium containing pigeon guano (PG). (A) Schematic diagram of Cryptococcus mating. (B) Cells (10⁸) of opposite mating types, genetically marked with a NAT or NEO resistance marker, were mixed, and 5-μl spots were placed onto V8 medium (pH 5 for C. neoformans var. grubii and pH 7 for C. neoformans var. neoformans and C. gattii) or medium containing 25%, 12%, or 2.5% pigeon guano. After 24 h of incubation, the resulting cells were screened for fusion products containing both markers. The numbers of fusion events on the various media are expressed as percentages of the number of fusion events occurring on V8 medium for each mating pair. (C) Six diploid strains for each variety/species were washed with PBS, inoculated onto V8 medium (pH 5 for C. neoformans var. grubii and pH 7 for C. neoformans var. neoformans and C. gattii) or pigeon guano medium contain differing levels of pigeon guano, and incubated in the dark for 7 days. Filament length is based on the average distance from the edge of the colony to the outer edge of filamentation and is expressed as a percentage of the average filament length on V8 medium.