Environmental induction of white-opaque switching in Candida albicans - PubMed (original) (raw)

Environmental induction of white-opaque switching in Candida albicans

Bernardo Ramírez-Zavala et al. PLoS Pathog. 2008.

Abstract

Candida albicans strains that are homozygous at the mating type locus (MTLa or MTLalpha) can spontaneously switch at a low frequency from the normal yeast cell morphology (white) to an elongated cell type (opaque), which is the mating-competent form of the fungus. The ability to switch reversibly between these two cell types also contributes to the pathogenicity of C. albicans, as white and opaque cells are differently adapted to specific host niches. We found that in strain WO-1, a strain in which genomic alterations have occurred, but not in other tested strains, switching from the white to the opaque phase can also be induced by environmental conditions. Transient incubation of white cells under anaerobic conditions programmed the cells to switch en masse to the opaque phase. The anaerobic induction of white-opaque switching was controlled by the transcription factor CZF1, which in heterozygous MTLa/alpha cells regulates filamentous growth under embedded, hypoxic conditions. Intriguingly, passage of white cells of strain WO-1 through the mouse intestine, a host niche in which the cells are likely to be exposed to anaerobic conditions, resulted in a strongly increased frequency of switching to the opaque phase. These results demonstrate that white-opaque switching is not only a spontaneous process but, in combination with genomic alterations, can also be induced by environmental signals, suggesting that switching and mating of C. albicans may occur with high efficiency in appropriate niches within its human host.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Anaerobic conditions induce white-opaque switching in C. albicans strain WO-1.

(A) White-phase cells were spread on Lee's agar plates with phloxine B, incubated for 2 days at 25°C under anaerobic conditions, and then grown for 7 days under aerobic conditions (left) or grown for 7 days under aerobic conditions without a previous anaerobic incubation (right). (B) Microscopic appearance of white-phase cells that had been incubated for 2 days at 25°C on Lee's agar plates under anaerobic (left) or aerobic conditions (right). Cells in both panels are shown at the same magnification. (C) Expression of the opaque-phase-specific OP4 and SAP1 genes and the white-phase-specific WH11 gene after anaerobic induction of white-opaque switching. Derivatives of strain WO-1 expressing the GFP reporter gene under control of the OP4, SAP1, or WH11 promoter were incubated under anaerobic (left) or aerobic (right panels) conditions as described in (A). Cells from the resulting opaque colonies after the anaerobic incubation and from white colonies on the control plates were grown to log phase in liquid Lee's medium at 25°C and observed by microscopy. Shown are corresponding differential interference contrast (top panels) and epifluorescence micrographs (bottom panels) of the cells. (D) Percentage of white (white bars), opaque (black bars), and mixed white/opaque colonies (grey bars) after incubation of white-phase cells on Lee's agar plates for 12 h, 24 h, and 48 h under anaerobic conditions at 25°C (left) or 37°C (right). (E) Percentage of white, opaque, and mixed white/opaque colonies after incubation of white-phase cells for 48 h in liquid Lee's medium containing the indicated amounts of lovastatin or ketoconazole and subsequent growth of the cells on Lee's agar plates. (F) Percentage of white, opaque, and mixed white/opaque colonies after incubation of white-phase cells on agar plates with or without 5 µg ml−1 ketoconazole for 24 h or 48 h at 25°C under anaerobic conditions. Control plates were incubated under aerobic conditions.

Figure 2

Figure 2. The transcription factor Czf1p is required for anaerobically induced white-opaque switching.

White-phase cells of the wild-type strain WO-1 (CZF1/CZF1) and two independently constructed heterozygous (CZF1/_czf1_Δ) and homozygous (_czf1_Δ/_czf1_Δ) czf1 mutants and complemented strains (_czf1_Δ/_czf1_Δ+CZF1) were incubated for 2 days under anaerobic conditions at 25°C on Lee's agar plates and subsequently grown for one week under aerobic conditions to determine the percentage of opaque (black bars) and mixed white/opaque colonies (grey bars).

Figure 3

Figure 3. Tetracycline-induced CZF1 expression results in WOR1 induction.

Strains expressing CZF1 (Ptet-CZF1) or GFP (control) from the Tet-inducible promoter were grown for 18 h at 30°C in liquid Lee's medium with (+) or without (−) 50 µg ml−1 doxycycline (Dox). RNA was isolated from the cultures and WOR1 transcript levels were quantified by real-time RT-PCR and normalized to ACT1 transcript levels. Two independently constructed strains (A and B) were used in each case.

Figure 4

Figure 4. WOR1 copy number and inducibility of white-opaque switching in different stocks of strain WO-1 and mutant derivatives.

(A) Southern hybridization of _Eco_RI/_Xho_I-digested genomic DNA of various strains with a probe from the WOR1 upstream region. Lanes 1–4 show the hybridization pattern of the wild-type strain WO-1 (WOR1/WOR1/WOR1) and mutants in which one (WOR1/WOR1/_wor1_Δ), two (WOR1/_wor1_Δ/_wor1_Δ), or all three WOR1 alleles (_wor1_Δ/_wor1_Δ/_wor1_Δ) were deleted. Lanes 5–8 show the hybridization pattern of mutants of additional WO-1 stocks in which one WOR1 allele was replaced by the SAT1 flipper cassette. The identity of the hybridizing DNA fragments is indicated. The size of the fragment representing the _wor1_Δ allele remains the same after excision of the SAT1 flipper cassette due to an introduced _Xho_I site. (B) White-phase cells of the wild-type strain WO-1 and two independently constructed series of mutants lacking one, two, or all three WOR1 alleles were incubated for two days under anaerobic conditions at 25°C on Lee's agar plates and subsequently grown for one week under aerobic conditions to determine the percentage of opaque (black bars) and mixed white/opaque colonies (grey bars). Stars (*) indicate that no opaque colonies were observed in these strains. (C) White-phase cells of different WO-1 stocks and mutant derivatives were incubated for two days under anaerobic conditions at 37°C on Lee's agar plates and the percentage of opaque and mixed white/opaque colonies was determined as described in (B). Results are from two experiments performed in parallel for each strain. WT: wild-type parent, M1: mutant in which one WOR1 allele was deleted.

Figure 5

Figure 5. White-opaque switching is induced in the mammalian gastrointestinal tract.

(A) Mice were inoculated intragastrically with 106 white-phase cells of strain WO-1. Cells from the inoculum and cells recovered from the feces of the animals after 24 h (experiment 1 with mouse 1 and mouse 2) or 20 h (experiment 2 with mice 1 to 4) were grown on Lee's agar plates to determine the percentage of opaque (black bars) and mixed white/opaque colonies (grey bars). The stars indicate that mice 1 and 2 were infected a second time. In both experiments, no opaque colonies were present on the plates containing cells from the inoculum (243 and 194 white colonies). (B) Five antibiotic treated mice were inoculated intragastrically with 5×107 white-phase cells of strain WO-1. Cells were recovered from the feces of the animals at one, two, or three days post infection (p.i.). The cells were grown on Lee's agar plates to determine the percentage of opaque (black bars) and mixed white/opaque colonies (grey bars). No opaque colonies were present on plates containing cells from the inoculum (436 white and 2 mixed white/opaque colonies).

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