In vivo and in vitro anaerobic mating in Candida albicans

Abstract

Candida albicans cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at 37 degrees C, block production of farnesol, and permit in vitro mating at 37 degrees C at efficiencies of up to 84%. Aerobically, farnesol prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized gastrointestinal (GI) tract, could serve as niches for C. albicans mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells, confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear, and prototrophic, and they contained the heterologous hisG marker from one of the parental strains.

FIG. 1.

Evans blue staining of aerobically grown opaque MTLa/a and MTLα/α C. albicans. MTLα/α (strain 3740) (A and B), MTLa/a (strain 3745) (C and D), and WO-1 opaque cells were grown separately at 25°C in SD medium supplemented with the necessary amino acids, either with 40 μM farnesol (B and D) or without farnesol (A and C). Cells were assessed for viability after 4 h with 1 mg/ml Evans blue. The cells were analyzed by fluorescence microscopy; cells that stain are dead (12). Both phase-contrast (left panels) and fluorescence (right panels) microscopy pictures are shown. Bar, 10 μm. (E) Graph with the percentage of cell viability for opaque and white cells after treatment with 40 μM farnesol. Data presented are averages for two independent experiments. Similar values were obtained when the cells were treated with 50 μg/ml propidium iodide.

FIG. 2.

In vitro anaerobic mating in C. albicans. MTLa/a (strain 3745) cells were stained with rhodamine-ConA, and MTLα/α (strain 3740) cells were stained with FITC-ConA by the method of Lockhart et al. (16). The stained cells were mixed, incubated in anaerobic medium (6) for 72 h at 25°C, and analyzed by confocal microscopy. The left panel is the fluorescent image, whereas the right panel merges the phase-contrast and fluorescent images to visualize the unstained conjugation tube. Excitation and emission wavelengths were 543 and 560 nm for rhodamine-ConA and 488 and 505 to 525 nm for FITC-ConA. Bar, 2 μm.

FIG. 3.

(A) Growth curves for anaerobically grown opaque MTLa/a cells (strain 3745) and opaque MTLα/α cells (strain 3740) at 25°C and 37°C. Opaque MTLa/a and MTLα/α cells were inoculated separately in 10 ml of anaerobic GPP medium (6) supplemented with the auxotrophic requirements and incubated for 1 week at either 25°C or 37°C. The OD₆₀₀ was monitored every 24 h. (B) Growth curves for aerobically grown opaque MTLa/a and MTLα/α cells at 25°C. Opaque cells were inoculated separately in 10 ml of aerobic GPP medium supplemented with the required amino acids. The OD₆₀₀ was monitored every hour.

FIG. 4.

RT-PCR analysis of white phase- and opaque phase-specific gene expression for strain 3740 (MTLα/α) (A) and strain 3745 (MTLa/a) (B). White-phase cells (W) were grown aerobically at 37°C in GPP medium for 24 h, and opaque cells (O) were grown anaerobically at 37°C for 7 days. Total RNA was prepared from harvested cultures, and mRNA expression of a white phase-specific gene (WH11), an opaque phase-specific gene (OP4), and a control gene, β-tubulin (TUB), was performed using RT-PCR analysis as described in Materials and Methods. The negative control was no cDNA (−).

FIG. 5.

PCR detection of hisG in the mating progenies. PCR was done on 10 randomly selected prototrophic colonies recovered from the large intestine. The cells were restreaked onto YPD plates and grown for 2 days at 25°C whereupon PCR was done on individual colonies. The hisG gene from S. enterica serovar Typhimurium was recovered in all 10 prototrophs tested (the first five [X1 to X5] are shown). P1, 3740 parent; P2, 3745 parent; X, progeny. For a control, the tubulin (TUB) gene was used. M, molecular size markers.

FIG. 6.

FACS analysis of the DNA content of mating progenies and parental strains. Progeny cells derived from the in vivo mating experiment are mononuclear and have twice the DNA of control diploid cells. The control tetraploid (DDCA45) was made by aerobic mating of 3740 × 3745 (Table 1). Bar, 10 μm.