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. 2012 Jun;11(6):773–782. doi: 10.1128/EC.00047-12

N-Acetylglucosamine Induces White-to-Opaque Switching and Mating in Candida tropicalis, Providing New Insights into Adaptation and Fungal Sexual Evolution

Jing Xie a,b, Han Du a, Guobo Guan a, Yaojun Tong a,b, Themistoklis K Kourkoumpetis c, Lixin Zhang d, Feng-yan Bai a,✉, Guanghua Huang a,✉
PMCID: PMC3370467  PMID: 22544905

Abstract

Pathogenic fungi are capable of switching between different phenotypes, each of which has a different biological advantage. In the most prevalent human fungal pathogen, Candida albicans, phenotypic transitions not only improve its adaptation to a continuously changing host microenvironment but also regulate sexual mating. In this report, we show that Candida tropicalis, another important human opportunistic pathogen, undergoes reversible and heritable phenotypic switching, referred to as the “white-opaque” transition. Here we show that N-acetylglucosamine (GlcNAc), an inducer of white-to-opaque switching in C. albicans, promotes opaque-cell formation and mating and also inhibits filamentation in a number of natural C. tropicalis strains. Our results suggest that host chemical signals may facilitate this phenotypic switching and mating of C. tropicalis, which had been previously thought to reproduce asexually. Overexpression of the C. tropicalis WOR1 gene in C. albicans induces opaque-cell formation. Additionally, an intermediate phase between white and opaque was observed in C. tropicalis, indicating that the switching could be tristable.

INTRODUCTION

Fungal infections caused by Candida species have increased dramatically over the past decades. Although Candida albicans remains the single most important causative agent, non-albicans species of Candida are increasingly isolated in nosocomial samples (25, 31). Candida tropicalis, which is frequently associated with nosocomial candidemias, appears to be more prominent among patients with hematologic malignancies (31).

Phenotypic transitions and sexual mating play a critical role in the adaptation of fungi to host environments and promote the evolution of fungal pathogenic traits (16, 39). The ability to switch between different morphologies is believed to be associated with virulence (43). Sexual reproduction is pervasive in eukaryotes and has many advantages over asexual reproduction (9). First, it produces recombinant types that can be more environmentally adaptable. Second, it provides an efficient way to eliminate harmful mutations. Of note, white-opaque switching and sexual mating are two tightly linked biological processes in C. albicans, C. dubliniensis, and C. tropicalis (26, 33, 34).

The white-opaque transition in C. albicans was first identified in the clinically isolated strain WO-1 in 1987 (38). The switching between white and opaque cells is heritable and reversible (3). White and opaque cells have different cellular morphologies and form two distinct colony appearances on solid media. Microscopically, white cells are small and round to ovoid and bud in a manner similar to that seen with Saccharomyces cerevisiae cells, while opaque cells are twice as large, elongated, and bean shaped. Macroscopically, white cells form white, shiny, and dome-shaped colonies, while opaque cells form relatively darker and flatter colonies on agar (3, 40). On special phloxine B-containing media, opaque cells are selectively stained red (3).

Opaque cells also differ from white cells with respect to their vulnerability to immune cells, virulence, and mating competence (39). For example, opaque cells are more virulent in cutaneous infections whereas white cells are better at tissue colonization, as shown in a murine systemic virulence model (18). Interestingly, only opaque cells are mating competent. Specifically, opaque cells mate ∼106 times more frequently than white cells (26). In order to mate, C. albicans white cells first have to undergo homozygosis at the mating type locus (MTL) and then switch to the opaque phenotype. Of note is that the MTL locus controls mating as well as white-to-opaque switching in C. albicans (26). The MTLa/α complex inhibits the expression of the master regulator gene WOR1 (white-opaque regulator 1), which activates white-to-opaque switching via a positive-feedback loop (14, 41, 44). Wor1 is a conserved transcription factor in fungi, with two DNA-binding domains at its N-terminal end (24).

A variety of host microenvironmental cues, including N-acetylglucosamine (GlcNAc) and CO2, induce white-to-opaque switching in C. albicans (1, 13, 15, 23, 28, 39). GlcNAc functions through the Ras-cyclic AMP/PKA (Ras-cAMP/PKA) pathway and finally activates Wor1 to promote switching (15). Inactivation of RAS1 or CDC35, encoding a small GTPase or adenylyl cyclase, respectively, remarkably reduces GlcNAc-induced switching (15).

Several Candida species, including C. albicans and C. tropicalis, can also grow in filamentous forms in response to environmental changes (29, 43). The underlying mechanism that regulates filamentous growth in C. albicans has been intensively investigated. For example, a plethora of environmental factors, including serum, high temperature (37°C), GlcNAc, and CO2, induce filamentation (4, 8). GlcNAc also promotes filamentous growth via the Ras-cAMP/PKA pathway (4, 6). The relationship between white-opaque switching and yeast-filamentous growth transition is not clear, although a variety of environmental inducers and genes have been found to regulate both phenotypic transition systems (39). Of note is that we have recently proposed that white-opaque switching is a newly evolved developmental process (15).

In this study, we investigated whether other Candida species could also undergo similar white-opaque switching to uncover the evolutionary trajectory of the opaque phenotype and the relationship between switching and mating. Here, we demonstrate the white-to-opaque switching and sexual mating of C. tropicalis. GlcNAc induces opaque-cell formation and thus promotes mating in C. tropicalis. By using a similar assay, we tested for the presence of any potential white-to-opaque switching ability of other species of the CTG clade, including Candida parapsilosis, Lodderomyces elongisporus, Candida guilliermondii, Candida lusitaniae, and Debaryomyces hansenii; however, we did not observe any opaque- or sectored-colony formations. Bennett and colleagues have recently reported a similar discovery of white-opaque switching and mating in C. tropicalis (33). They found that two MTLa/a and α/α strains engineered from a natural MTLa/α strain can switch and mate. Our study not only validates their discoveries but also provides some new findings, as described in the following sections.

MATERIALS AND METHODS

Strains and growth conditions.

The strains used in this study are listed in Table S1 in the supplemental material. YPD (20 g/liter glucose, 20 g/liter peptone, 10 g/liter yeast extract) was used for routine growth. Lee's plus glucose and Lee's plus GlcNAc media were used for mating and white-opaque switching assays (15, 20).

Construction of plasmids and C. tropicalis ura3/ura3 mutant.

The primers for construction of pNIM-ctWOR1 (TETp-ctWOR1) were ctWOR1F (5-AATCTTGTCGACATGTCGGCGTCTAGATTATCA-3) and ctWOR1R (5-AATCTTAGATCTTCAATTTGCAGTGGTGTAATATGG-3). The PCR product of C. tropicalis WOR1 (ctWor1) was digested with BglII and SalI and subcloned into the BglII-SalI site of pNIM1 (30), generating pNIM-ctWOR1. To construct the plasmid pSFS2A-ctURA3 for URA3 gene disruption in C. tropicalis, two PCR fragments of the 5′ and 3′ ends of ctURA3 genes were subcloned into pSFS2A. The primers used for PCR were primer pair ctURA3apaF (5-AATACAGGGCCCAGATGAAGAGGTTACAAGTTTTG-3) and ctURA3xhoR (5-AATACACTCGAGTGATACCTTTGGGTCTTCCTC-3) and primer pair ctURA3sacIIF (5-AATACACCGcGGCACCAATAATGCAATAGAAGTAG-3) and ctURA3sacIR (5-AATACAGAGCTCATGGGATGATGATCAAGTTGATG-3).

The first copy of the URA3 gene was deleted by using the plasmid pSFS2A-ctURA3. Then, the URA3/ura3::SAT1 heterozygous transformants were streaked onto 5-fluoroorotic acid (5-FOA)-containing plates to screen auxotrophic isolates for uridine.

White-opaque switching and mating assays.

White-opaque switching was analyzed as described previously (15). Cells were patched or plated onto Lee's plus glucose and Lee's plus GlcNAc agar and incubated at 25 or 37°C for 4 to 7 days as indicated in the main text. Mating experiments were performed according to previous reports with slight modification (14, 26). Briefly, a mixture containing 106 cells of each of the two opposite MTL strains (one with white cells and the other with opaque cells) were dropped onto Lee's plus glucose and Lee's plus GlcNAc agar and incubated for 4 days. The resulting mating mixture was then used for further analysis.

The primers used for C. tropicalis MTLa1, MTLα2, and MDR1 PCR were as follows: MTLa1F (5-GCTCAAAAGGAAGAGGAGGAA-3), MTLa1R (5-TCAATTCTCTTTCCCGTCTGTT-3), MTLal2F (5-CCGGTTTTCGACTCAAAGAG-3), MTLal2R (5-AGCAAGTTCCGGAGACACCT-3), MDR1F (5-TGTTGGCATTCACCCTTCCT-3), and MDR1R (5-TGGAGCACCAAACAATGGGA-3).

SEM assay.

The scanning electron microscopy (SEM) assay was performed according to our previous publication (11). Briefly, the samples were gently washed with 1× PBS and fixed with 2.5% glutaraldehyde. Then, the samples were washed with 0.1 M Na3PO4 buffer (pH 7.2), dehydrated with increasing concentrations of ethanol, and coated with gold. The images were obtained with a scanning electron microscopy (FEI Quanta 200).

Invasive and filamentous growth assays.

Lee's plus glucose and Lee's plus GlcNAc medium plates were used for invasive and filamentous growth. A 3-μl volume of liquid medium containing ∼2 × 104 cells was dropped onto the agar for 4 days (at 37°C) or 7 days (at 25°C) of incubation. Images of plates were taken before and after washing with double-distilled water (ddH2O).

RESULTS

Comparative analysis of MTL locus genes.

Our analysis of its genomic sequence indicated that C. tropicalis contains a conserved MTL locus similar to those of C. albicans and C. dubliniensis (5, 34). The MTLa locus of C. tropicalis contains the MTLa1, a2, OBPa, PIKa, and PAPa genes, while the MTLα locus contains the MTLα1, α2, OBPα, PIKα, and PAPα genes. All four MAT genes (a1, a2, α1, and α2) remain intact in the C. tropicalis genome, although the degrees of identity and similarity of the protein sequences of C. tropicalis and C. albicans (identity, 46% to 82%; similarity, 69% to 92%) are lower than those of the protein sequences of C. dubliniensis and C. albicans (identity, 83% to 95%; similarity, 91% to 98%) (see Fig. S1 in the supplemental material). We also searched and analyzed the sequences of a number of C. tropicalis genes whose homologues are involved in white-opaque switching or mating in C. albicans. These genes are conserved in C. albicans, C. dubliniensis, and C. tropicalis, indicating that C. tropicalis can possibly undergo phenotypic switching and mating similar to that of C. albicans and C. dubliniensis.

Overexpression of the C. tropicalis WOR1 (ctWor1) gene in C. albicans induces opaque-cell formation.

We found the homologue of the C. albicans WOR1 gene, a master regulator of white-opaque switching, in C. tropicalis by a BLAST search in the Candida genome database (Broad Institute). Sequence analysis demonstrated that the two DNA binding domains of Wor1 are highly conserved between C. albicans Wor1 and ctWor1 (24). To overexpress ctWOR1, we replaced the native ctWOR1 promoter with a ctACT1 promoter in a C. tropicalis MTLa/a strain. However, no obvious effects on the induction of opaque-cell formation were observed. To test whether ctWor1 plays a similar role in white-opaque switching, we then turned to examination of the inducing effect of opaque-cell formation in C. albicans by constructing an overexpression strain containing a plasmid with an inducible TETp promoter-controlled ctWOR1 gene (30). Overexpression of ctWOR1 in a C. albicans MTLa/a strain obviously induced opaque-sector formation (switching frequency = 100%) in the presence of doxycycline at 50 μg/ml (inducing condition), while the switching frequency remained very low (<1%) in the absence of doxycycline (noninducing condition). The switching frequencies of the WT positive (WT+) vector control were also very low (<1%) under both inducing and noninducing conditions (Fig. 1). The findings described above, coupled with the observation that the deletion of ctWOR1 locks C. tropicalis cells in the white phase (33), indicate that ctWOR1 could play a similar role in promoting white-to-opaque switching.

Fig 1.

Fig 1

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Overexpression of C. tropicalis WOR1 in a C. albicans MTLa/a strain promotes opaque-cell formation. WT, GH1012. White cells of the strains WT+ vector and WT+TETp-ctWOR1 were plated onto Lee's plus glucose plates and cultured at 25°C for 5 days under noninducing (0 μg/ml doxycycline) or inducing (50 μg/ml doxycycline) conditions.

White-opaque switching in C. tropicalis.

Since only MTL homozygous strains undergo white-to-opaque switching in C. albicans,we hypothesized that this could be the same case in C. tropicalis. Therefore, we first assessed the MTL zygosity (a/a, α/α, or a/α) of 150 natural C. tropicalis strains. As revealed by testing these strains with PCR, 2 were MTLa homozygous (a/a), 3 were MTLα (α/α), and 145 were MTL heterozygous (a/α).

To test whether the natural C. tropicalis strains underwent white-to-opaque switching, we patched and plated 2 a/a and 2 α/α strains onto Lee's plus glucose plates and cultured them for 7 days at 25°C. C. albicans WO-1 (α/α) and 2 C. tropicalis a/α strains served as controls. We observed that C. tropicalis a/a and α/α strains indeed underwent white-to-opaque switching and formed sectored colonies, although the switching frequency was extremely low (∼0.1%). On the other hand, we did not observe opaque-sector formation in the tested C. tropicalis a/α strains on Lee's plus glucose plates. As expected, the reference strain C. albicans WO-1 exhibited a higher white-to-opaque switching frequency (∼5.0%).

Since we previously showed that GlcNAc is a potent inducer of white-to-opaque-cell formation in C. albicans (15), we tested whether GlcNAc can also regulate white-to-opaque switching in C. tropicalis. More specifically, we patched or plated several natural C. tropicalis strains onto Lee's plus glucose as well as Lee's plus GlcNAc plates and cultured them both at 25°C. The observed switching frequency of the GlcNAc-containing plates was much higher than that of the glucose-containing ones (Fig. 2). Also, the switching frequency of the MTLa/a strain (JX1374) was over 50.0% when cultured on GlcNAc medium. For a more detailed review of the images of switching in natural strains, please refer to the Fig. 3A legend. Surprisingly, we found that the MTL heterozygous strain (JX1016) underwent white-to-opaque switching in the GlcNAc medium whereas white-to-opaque switching on glucose medium was absent. Of note, C. albicans opaque cells undergo mass conversion to white at 37°C (the host temperature) (3). Here we showed that C. tropicalis can undergo the white-to-opaque transition at 37°C on GlcNAc medium as well as on glucose medium (Fig. 3B; see also Fig. S2 in the supplemental material). Similar results of switching at 37°C have been reported by the Bennett group (33). Notably, on phloxine B-containing GlcNAc medium (5 μg/ml), aged opaque sectors were stained red (Fig. 2A and B and Fig. 3), while they could hardly be stained on glucose medium (see Fig. S2 in the supplemental material). As we expected, the C. albicans reference strain WO-1 showed a high switching frequency on GlcNAc medium as well. These data suggest that GlcNAc can induce opaque-cell formation not only in C. albicans but also in C. tropicalis.

Fig 2.

Fig 2

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A comparison of white-to-opaque switching in C. albicans and C. tropicalis. (A) Morphology of white (wh)-opaque (op) switching in C. albicans. White cells of C. albicans were patched or plated onto Lee's plus glucose or GlcNAc plates and cultured at 25°C for 7 days. (B) Morphology of white-opaque switching in C. tropicalis. White cells of C. tropicalis were patched or plated onto Lee's plus glucose or GlcNAc plates and cultured at 25°C for 7 days. Scale bar, 5 μm for panels A and B. (C) Examples of SEM images of C. albicans white and opaque cells. (D) Examples of SEM images of C. tropicalis white and opaque cells. Scale bar, 4 μm for panels C and D.

Fig 3.

Fig 3

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GlcNAc induces opaque-cell formation and inhibits filamentous growth of natural C. tropicalis strains. (A) Experiments at 25°C. White cells of 5 natural strains were patched or plated onto Lee's plus glucose or Lee's plus GlcNAc plates and cultured for 7 days. Opaque (op) sectors are indicated. Strains used in the top two panels: top left, JX1002; bottom left, JX1004; top right, JX1001; middle right, JX1003; bottom right, JX1005. (B) Experiments at 37°C. White cells of 3 natural strains were patched plated onto Lee's plus glucose or Lee's plus GlcNAc plates and cultured for 4 days. Opaque (op) sectors were indicated. Strains used in the top two panels: top left, JX1009; top right, JX1008; bottom right, JX1010. Scale bar, 5 μm.

White- and opaque-cell features of C. tropicalis.

To confirm that the sectors formed by C. tropicalis contained true opaque cells, we used microscopy and scanning electron microscopy (SEM) assays. Our findings revealed that the cellular shape of C. tropicalis was similar to that of C. albicans. For example, the white cells of C. tropicalis were round and small and showed a strong tendency to undergo filamentous growth. On the other hand, the opaque cells of C. tropicalis were large and elongated and contained one or more vacuoles (Fig. 2B). Interestingly, the cell wall surface of a few opaque C. tropicalis cells was pimpled, while it was smooth in others. Of note is that the smooth surface is a prominent feature of white cells. Consistent with one previous report (3), the surface of most C. albicans opaque cells was pimpled, whereas white cells were smooth (Fig. 2C and D). The observation of a cellular form that shares both white and opaque C. tropicalis cell features probably suggests the existence of a transitional cellular state between the two forms. We tried to verify this hypothesis by culturing C. tropicalis cells for 4 instead of 7 days. In this experiment, some colonies contained a majority of cells that shared some characteristics with opaque cells (large and elongated but with small or no obvious vacuoles) and could not be stained red on phloxine B-containing plates. Whether these cells represented the intermediate state remains to be further investigated.

Mating in C. tropicalis.

It is well known that the white-to-opaque transition serves as a mating prerequisite in C. albicans (26, 39). Thus, our next step was to investigate whether C. tropicalis cells had to switch from white to opaque in order to mate similarly to C. albicans cells. To achieve that, we used natural C. tropicalis strains sensitive to nourseothricin (ClonNAT). The strategy of quantitative mating is shown in more detail in Fig. 4A. In brief, we first deleted a copy of the URA3 gene in a C. tropicalis MTLa/a strain by using a plasmid containing a dominant nourseothricin resistance marker, SAT1 (35). The URA3/ura3::SAT1 heterozygous transformants were then streaked onto 5-fluoroorotic acid (5-FOA)-containing plates in order to screen auxotrophic isolates for uridine. The resulting isolates (JX1374u) could not grow in uridine-depleted media and remained nourseothricin resistant because of the introduction of a SAT1 gene. The natural MTLα/α strain (JX1003) was prototrophic for uridine and sensitive to nourseothricin. Therefore, quantitative mating of the a/a and α/α strains could be monitored by selection for Uri-positive (Uri+) and nourseothricin resistance. A mixture containing 106 cells of the a/a strain (JX1374u) and 106 cells of the α/α strain (JX1003) was dropped onto Lee's plus glucose or GlcNAc plates and cultured at 25°C for 4 days. A total of 107 cells of the mating mixture were plated onto the selectable uridine-depleted plates (containing nourseothricin at 150 μg/ml). After 2 days of incubation at 37°C, the colonies formed by the conjugants arose from the selectable plates. To confirm that the resulting colonies were the actual mating products, colonies were restreaked onto new selectable plates. The cells were then used for analysis in a fluorescence-activated cell sorter (FACS). As shown in Fig. 4B, the DNA content of the mating product was about twice as much as that of the diploid mating partners (JX1374u and JX1003), indicating that the mating product was tetraploid. To confirm that the mating products contained DNA from both the JX1374u and JX1003 strains, we tested for the existence of the MTLa1 and α2 genes by PCR (Fig. 4C). We also found a single nucleotide polymorphism (SNP; indicated in bold) at the MDR1 gene locus of the parental strains JX1374u (5′-CATCATTTCAG-3′) and JX1003 (5′-CATCATTCCAG-3′). This sequence of the multidrug resistance protein MDR1 gene in the mating products was 5′-CATCATTYCAG-3′ (Y = T or C), suggesting that the genome contained the MDR1 gene sequences of both parents. Mating zygotes formed by fusion were observed in the mating mixture on both glucose- and GlcNAc-containing media by light microscopy (Fig. 4D). SEM analysis of fusion cells showed the formation of conjugation tubes between opaque a/a and α/α cells (Fig. 5). These results indicate that the mating process of C. tropicalis was highly similar to that of C. albicans (21). Of note, the white-opaque transition-regulated sexual mating has also been observed by Porman et al. (33).

Fig 4.

Fig 4

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Mating in C. tropicalis. (A) Strategy for quantitative mating in C. tropicalis. JX1374u, MTLa/a ura3-, nourseothricin resistant (Clonr); JX1003, WT, MTLα/α URA3/URA3, nourseothricin sensitive (Clons). Total volumes of 106 a/a and α/α cells were mixed and dropped onto Lee's plus glucose or Lee's plus GlcNAc plates and cultured for 4 days at 25°C. Then, 107 of the mating mixture cells were plated onto selectable plates containing nourseothricin (150 μg/ml) and depleted of uridine. The colonies growing on selectable plates were used for further experiments. (B) FACS analysis indicated the mating fusants from selectable plates were tetraploid. (C) PCR of MTLa and MTLα genes. Parental strains (a/a, α/α) and two random colonies of the mating fusant (Mat1, Mat2) were tested. (D) Cell microscopy of mating. S, shmoos; C, conjugation tubes. Scale bar, 5 μm.

Fig 5.

Fig 5

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Examples of SEM images of mating in C. tropicalis. Strains used for mating: JX1374u (MTLa/a) and JX1003 (MTLα/α).

GlcNAc increases C. tropicalis mating efficiency.

In C. albicans, efficient mating requires the conversion of white cells to opaque (26). Interestingly, we found that this is also the case in C. tropicalis. More specifically, we observed that only opaque cells of C. tropicalis formed shmoos and mating conjugations in a mixture of MTL white and opaque cells (Fig. 4D and Fig. 5). On the other hand, white cells retained their cellular shape without any change.

Since GlcNAc could induce white-to-opaque cell switching in C. albicans as well as in C. tropicalis, we then tested whether GlcNAc promoted mating in C. tropicalis. Our quantitative mating assay revealed that the mating efficiency on GlcNAc medium was over 100 times higher than that on glucose medium both at 25°C and at 37°C (Fig. 6). Surprisingly, on the medium that contained the same carbon source, the mating efficiencies at 25°C and 37°C were similar. These results were consistent with our previous observation of white-to-opaque switching at 37°C, suggesting that the opaque cells of C. tropicalis were not sensitive to high temperatures. However, in C. albicans, an increase of the temperature to 37°C remarkably reduced mating efficiency. A likely explanation is that at host temperatures (37°C), opaque cells are unstable and undergo mass conversion to the white state, which is largely incompetent with respect to mating (3, 26).

Fig 6.

Fig 6

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GlcNAc increases C. tropicalis mating efficiency. White cells of MTLa/a and α/α were first grown on glucose or GlcNAc medium at 25°C for 5 days and then mixed and cultured on corresponding plates for 4 days at 25 or 37°C. A total of ∼107 of the mating mixture cells were plated onto selectable plates. The images represent three independent experiments.

GlcNAc inhibits filamentous and invasive growth in C. tropicalis.

Interestingly, GlcNAc has been previously found to be a potent inducer of filamentous development in C. albicans (37). Other studies have shown that the conserved Ras-cAMP/PKA pathway likely mediates GlcNAc-induced filamentous growth as well as white-to-opaque transition in C. albicans (6, 15). Although GlcNAc played a role in the induction of opaque-cell formation in C. tropicalis similar to that seen with C. albicans, we found that GlcNAc had an opposite effect on the filamentous growth of C. tropicalis. On Lee's plus glucose medium, some C. tropicalis strains developed robust filamentous colonies even at 25°C (Fig. 2B and 3A), a temperature unfavorable for filamentous growth in C. albicans. However, when we replaced glucose with GlcNAc we observed inhibition of filamentous growth in a number of natural C. tropicalis strains, including all the three mating types (a/a, α/α, and a/α) at both 25°C and 37°C (Fig. 2B and 3). By examining all 6 natural strains (2 a/a, 2 α/α, and 2 a/α), we found that GlcNAc robustly reduced their invasive growth ability at 25°C as well as 37°C (Fig. 7). These results suggest that GlcNAc plays a negative role in the filamentous growth of C. tropicalis.

Fig 7.

Fig 7

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GlcNAc inhibits invasive growth in C. tropicalis. A total of 2,000 cells of 6 clinically independent C. tropicalis strains were dropped onto Lee's plus glucose or Lee's pGlcNAc plates and cultured for 7 (at 25°C) or 4 days (37°C). Images of plates were taken before and after washing with ddH2O. Strains used: JX1004 (a/a) (top row, left) and JX1374 (a/a) (bottom row, left); JX1369 (α/α) (top row, middle) and JX1003 (α/α) (bottom row, middle); JX1016 (a/α) (top row, right) and JX1009(a/α) (bottom row, right).

Analysis of white-to-opaque switching of other Candida species in the CTG clade.

It has been previously shown that the MTL locus controls white-opaque switching and mating in C. albicans (26). The organizations of MTL loci in the CTG clade (the species in this clade translate CUG codons as serine instead of leucine [17]) are very similar and conserved among Candida species (5). Therefore, we examined whether other species in the Candida clade could also undergo white-opaque switching. Three to 5 natural strains of each species were examined on Lee's plus glucose medium and Lee's plus GlcNAc medium at 25°C and 37°C. Notably, we observed white-to-opaque transition in C. albicans, C. dubliniensis, and C. tropicalis in both media but not in Candida parapsilosis, Lodderomyces elongisporus, Candida guilliermondii, Candida lusitaniae, or Debaryomyces hansenii under any conditions tested (Table 1).

Table 1.

White-to-opaque switching in the CTG clade speciesa

Species name White-to-opaque switching
Glucose
 GlcNAc
25°C 37°C 25°C 37°C
C. albicans Y N Y Y
C. dubliniensis Y N Y Y
C. tropicalis Y Y Y Y
C. parapsilosis N N N N
L. elongisporus N N N N
C. guilliermondii N N N N
C. lusitaniae N N N N
D. hansenii N N N N
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a

Lee's glucose and Lee's GlcNAc media were used for this experiment. For the species that grew very slowly on Lee's glucose and Lee's GlcNAc media, SD-glucose and SD-GlcNAc were used to verify their phenotypes. Y, yes (switching observed); N, no (switching not observed).

DISCUSSION

In the present report, we show that C. tropicalis has the ability to undergo white-opaque switching and mating. More importantly, we have found that GlcNAc induces white-opaque switching as well as mating in C. tropicalis. Although these two biological processes are highly similar in C. tropicalis and C. albicans, C. tropicalis exhibits some unique features. For instance, we show that C. tropicalis undergoes white-to-opaque switching and mating at both 37°C and 25°C, while C. albicans opaque cells become extremely unstable and mate poorly at 37°C.

A comparative analysis of C. tropicalis and C. albicans indicates that genes involved in mating and white-to-opaque switching are generally conserved. Notably, C. tropicalis ctWor1 is homologous and highly similar to the master regulator Wor1 of C. albicans. For example, the overexpression of Wor1 in a wild-type (WT) C. albicans strain promotes opaque-cell formation, a finding that pinpoints Wor1 as a key regulator of white-to-opaque switching in C. albicans through a positive-feedback loop (14, 41, 44). In this report, we demonstrate that the overexpression of ctWOR1 in C. albicans induces white-to-opaque switching (Fig. 1), suggesting that Wor1 may have similar functions in both species. We also tried to overexpress ctWOR1 in a C. tropicalis MTLa/a strain by replacing the native ctWOR1 promoter with a ctACT1 promoter. We did not observe any obvious effects on the induction of opaque-cell formation in the overexpressing C. tropicalis strain. A possible reason could be that ctWor1 does not activate its own expression in C. tropicalis whereas Wor1 does so via a self-feedback loop in C. albicans. This might explain why the white-to-opaque switching frequency in C. tropicalis is relatively low. Porman et al. have reported that deletion of ctWOR1 locked C. tropicalis cells in the white phase (33). These findings suggest that ctWor1 is indeed involved in the regulation of white-opaque switching in C. tropicalis, whereas the underlying regulatory mechanisms could be different.

We subsequently showed that a number of natural C. tropicalis strains, including all the three mating types (a/α, a/a, and α/α), have the ability to undergo white-to-opaque switching and mating at 25°C and 37°C. The MTLa1 and α2 genes were not lost in the opaque cells of a/α strains with PCR confirmation. Neither MTLa/a nor α/α opaque cells could mate with the opaque cells formed by MTLa/α strains (data not shown). These results exclude the possibility that the MTLa/α strains underwent a spontaneous conversion to an MTL homozygous state before switching to opaque. We also showed that the phenotypes of C. tropicalis white and opaque cells are similar to those of C. albicans. For instance, most C. tropicalis opaque cells are elongated and have a big vacuole just as in C. albicans cells. Also, the cell wall surface of white cells in both species is smooth. A small percentage of C. tropicalis opaque cells exhibit pimpled cell wall surfaces, whereas most C. albicans opaque cells are pimpled (21). Although aged C. tropicalis opaque sectors could be stained red by phloxine B, the newly formed opaque colonies remained white or slightly pink. These findings suggest that an intermediate form between the white and opaque phases might exist. Whether the elongated cells with smooth cell wall surfaces and the nonstained cells are in the intermediate state remains to be investigated. As with C. albicans, only opaque C. tropicalis cells can mate efficiently. Since the mating efficiency of C. tropicalis opaque cells remained robust at low (25°C) and high (37°C) temperatures, we can assume that the opaque cells of C. tropicalis can remain stable at both temperatures (Fig. 6).

GlcNAc is a well-known component of the bacterial cell wall and is also found in the mucus of the human gastrointestinal (GI) tract, the ultimate human reservoir for Candida infections (12, 29). Notably, Candida species are abundant in the GI tract, where they can be found in more than 21% of humans (7, 42). In one previous study, we reported that GlcNAc induced white-to-opaque transition in C. albicans (15). In the present study, we found that GlcNAc can also induce opaque-cell formation and promote mating in C. tropicalis. Our sequence analysis demonstrated that the C. tropicalis genome contains most genes required for GlcNAc sensing and metabolism, including the Ras-cAMP/PKA pathway, the transporter NGT1, and glucosamine-6-phosphate deaminase NAG1 genes. These results indicate that the two Candida species, as successful commensals and pathogens in the human body, have evolved to respond to the signals produced by the gut microbiota and by the GI epithelial cells.

In C. albicans, white cells can differentiate into filamentous or opaque cells in response to different stimuli. A variety of environmental cues, including GlcNAc and CO2, not only can induce opaque-cell formation but also promote filamentous development in C. albicans (8, 13, 15). Filamentous cells are genetically white and express a set of white-specific genes. Once the white cells switch to opaque, they lose the ability to form filaments under regular conditions (2). To undergo filamentous growth, opaque cells have to switch back to white. Interestingly, compared to its effect on C. albicans, GlcNAc plays a contrasting role in the filamentation of C. tropicalis. More specifically, we found that it inhibited the filamentation ability of all tested natural C. tropicalis strains. We propose that the opaque cellular state and filamentation represent two directions of white cell differentiation. To differentiate into the opaque phase, the white cells have to first block the expression of the regulatory machinery of initiating filamentation and then increase the expression of opaque-promoting genes such as the master regulator WOR1. This hypothesis has been confirmed in C. albicans (data not shown). Induction and maintenance of the opaque phase needs a Wor1-involved positive-feedback loop. The C. albicans wor1/wor1 mutant carrying a copy of an ACT1 promoter that controls the WOR1 gene cannot switch to the opaque phase and, surprisingly, also loses the ability to grow into the filamentous form even under inducing conditions (data not shown). These results suggest that moderate expression of WOR1 under the control of ACT1 promoter is not sufficient to induce the opaque phenotype but is sufficient to inhibit filamentous growth.

Given that UDP-GlcNAc is a constituent of the GI tract as well as the blood (32, 36), inhibition of C. tropicalis filamentation by GlcNAc may also have a potential clinical significance. Compared to C. albicans, natural C. tropicalis shows a stronger tendency to undergo filamentous growth, especially at the host temperature (37°C). Although filamentous cells are better at the initial phase of tissue penetration, yeast cells can be more easily disseminated through the bloodstream. Therefore, the inhibiting effect of GlcNAc on filamentous growth in C. tropicalis may facilitate dissemination in the host and play an important role in infections.

Since GlcNAc facilitates the phenotypic switching of C. albicans and C. tropicalis, we aimed to figure out whether this was possible with other Candida species in the CTG clade. Although we have not observed white-to-opaque switching in the natural strains tested, these species may undergo other forms of switching. For example, C. lusitaniae switches spontaneously among three colored phenotypes on CuSO4-containing medium: white, light brown, and dark brown (27).

In our opinion, our discovery of white-to-opaque switching regulating mating in C. tropicalis has revived an important question: why do white C. tropicalis and C. albicans cells have to switch to opaque to achieve mating (39)? Based on the observation of pheromone-induced white cell response in C. albicans, Daniels et al. have proposed that pheromone stimulation may lead to a chain event where a minority of opaque cells drive white cells to form a biofilm in which opaque cells are able to mate (10). The underlying mechanism of white-opaque switching and mating regulation could be much more complex than the hypothesis proposed by Daniels et al. (10). It is possible that the unique biological features in C. tropicalis and C. albicans not only provide mechanistic plasticity and diversity of sexual reproduction but might also improve the fitness and adaptation in their natural environments. For example, white cells can better survive in systemic infections and are more resistant to antifungals than opaque cells (39). Under natural conditions, sexual reproduction may not be necessary for Candida species, since the costs of sexual reproduction are considerable. Maintaining a white state could be an efficient way to shut down all the mating-related gene machinery. A similar strategy for sexual mating adopted by the yeast Clavispora opuntiae is to enter stationary phase to become maximally mating competent (19).

The present report not only validates the recent report published by Porman et al. (33) but also provides new insights into the evolution of white-opaque switching and mating in pathogenic Candida species. Both studies have found that C. tropicalis can undergo white-opaque switching and sexual mating. Only opaque cells of C. tropicalis mate efficiently, and the switching and mating are not sensitive to high temperatures. The switching frequency of white to opaque in this organism is extremely low in glucose-containing medium. However, several findings make our project of special novelty. First, natural MTL homozygous strains were used in our study, while MTLa/a and α/α strains engineered from an MTLa/α strain were used in the Porman et al. report (33). Second, we identified a possible intermediate phase between white and opaque. Third, we have shown that GlcNAc induces white-to-opaque switching, thus facilitating mating. Aged opaque colonies or sectors could be stained red on phloxine B-containing media. Fourthly, we have found that not only MTL homozygous but also MTL heterozygous strains undergo switching in the presence of GlcNAc. Therefore, it is very possible that most natural strains have the ability to undergo switching in ecological niches where inducing factors such as GlcNAc are present (e.g., physiological conditions). However, this was not the case with C. albicans; only a minority (8%) of natural C. albicans strains, which are homozygous at the MTL locus, switched from white to opaque (22). This finding indicates that white-opaque switching very possibly evolved before the association with mating in Candida species. Finally, inhibition of filamentous growth by GlcNAc in C. tropicalis provides new insights into the evolution of the opaque phenotype. Taken together, these two studies on the topic of white-opaque switching and mating in C. tropicalis are significant and provide new implications to aid in understanding the evolutionary trajectory of sexual reproduction in pathogenic Candida species.

Supplementary Material

Supplemental material
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ACKNOWLEDGMENTS

The authors are indebted to David Soll, Joachim Morschhauser, and Joseph Heitman for generous gifts of plasmids and strains.

This project is supported by a “100 Talent Program” grant from the Chinese Academy of Sciences and Chinese National Natural Science Foundation grant 31170086 to G.H.

Footnotes

Published ahead of print 27 April 2012

Supplemental material for this article may be found at http://ec.asm.org/.

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