The Closely Related Species Candida albicans and Candida dubliniensis Can Mate

Because Candida dubliniensis is closely related to Candida albicans,we tested whether it underwent white-opaque switching and matingand whether white-opaque switching depended on MTL homozygosityand mating depended on switching, as they do in C. albicans.We also tested whether C. dubliniensis could mate with C. albicans.Sequencing revealed that the MTL ${alpha}$ locus of C. dubliniensis washighly similar to that of C. albicans. Hybridization with theMTLa1, MTLa2, MTL ${alpha}$ 1, and MTL ${alpha}$ 2 open reading frames of C. albicansfurther revealed that, as in C. albicans, natural strains ofC. dubliniensis exist as a/ ${alpha}$ , a/a, and ${alpha}$ / ${alpha}$ , but the proportionof MTL homozygotes is 33%, 10 times the frequency of naturalC. albicans strains. C. dubliniensis underwent white-opaqueswitching, and, as in C. albicans, the switching was dependenton MTL homozygosis. C. dubliniensis a/a and ${alpha}$ / ${alpha}$ cells also mated,and, as in C. albicans, mating was dependent on a switch fromwhite to opaque. However, white-opaque switching occurred atunusually high frequencies, opaque cell growth was frequentlyaberrant, and white-opaque switching in many strains was camouflagedby an additional switching system. Mating of C. dubliniensiswas far less frequent in suspension cultures, due to the absenceof mating-dependent clumping. Mating did occur, however, athigher frequencies on agar or on the skin of newborn mice. Theincreases in MTL homozygosity, the increase in switching frequencies,the decrease in the quality of switching, and the decrease inmating efficiency all reflected a general deterioration in theregulation of developmental processes, very probably due tothe very high frequency of recombination and genomic reorganizationcharacteristic of C. dubliniensis. Finally, interspecies matingreadily occurred between opaque C. dubliniensis and C. albicansstrains of opposite mating type in suspension, on agar, andon mouse skin. Remarkably, the efficiency of interspecies matingwas higher than intraspecies C. dubliniensis mating, and interspecieskaryogamy occurred readily with apparently the same sequenceof nuclear migration, fusion, and division steps observed duringintraspecies C. albicans and C. dubliniensis mating and Saccharomycescerevisiae mating.

INTRODUCTION

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Introduction

Beginning in 1990, a number of studies of the genetic relatednessof Candida albicans isolates derived from human immunodeficiencyvirus (HIV)-positive individuals identified atypical strains(9, 29, 31, 33, 49). These strains were subsequently groupedinto the separate species C. dubliniensis in 1995 by Coleman,Sullivan, and coworkers (52). This new species shared a numberof phenotypic characteristics with C. albicans, including twothat in the past had been used to distinguish C. albicans fromother species, namely, chlamydospore and true hypha formation(52). Like C. albicans, C. dubliniensis was diploid, containeda homolog to the dispersed complex repeat sequence RPS, andunderwent the bud-hypha transition and colony-based 3153A-likephenotypic switching (10, 17, 19, 37, 50, 51). The basic phenotypiccharacteristics that distinguished C. dubliniensis from C. albicanswere different colony color on CHROM-agar, failure to fluoresceunder Wood's light on methyl blue-Sabouraud agar, chlamydosporeformation on Staib's agar, lack of expression of ß-glucosidase,and failure to grow at 42°C (1, 10, 37, 51, 52). More importantly,a number of genetic traits distinguished C. dubliniensis fromC. albicans, including diminished hybridization with the relatedDNA fingerprinting probes 27A (51, 52) and Ca3 (20, 31) anddifferences in restriction fragment length polymorphism patterns(52), randomly amplified polymorphic DNA patterns (52), multilocusenzyme electrophoresis patterns (9, 24, 33, 51), hybridizationwith microsatellite DNA sequences (49, 52), and ribosomal DNA(rDNA) sequences (52). It was subsequently demonstrated thatC. dubliniensis contained a dispersed species-specific midrepeatsequence, which represented a portion of the complex DNA fingerprintingprobe Cd25 (20). Furthermore, it was demonstrated that C. dubliniensiscontained on average twice as many full-length copies of thecommon repeat sequence RPS as did C. albicans and underwentRPS reorganization on average at twice the rate per genome asdid C. albicans (19). The presence of a potentially recombinogenicspecies-specific midrepeat sequence and twice as many recombinogenicRPS elements, both dispersed throughout the genome, explainedin part the high degree of karyotypic instability of C. dubliniensis(19). Together, these results suggested that C. albicans andC. dubliniensis had diverged into separate species but had doneso only recently in evolutionary time.

The unusual level of relatedness of these two species led usto test whether C. dubliniensis strains, like C. albicans strains,were normally MTL heterozygous and underwent MTL homozygosis,whether MTL zygosity regulated the white-opaque transition,whether mating was dependent on a switch from white to opaque,and whether skin facilitated mating. It also led us to testwhether C. albicans and C. dubliniensis could mate and undergokaryogamy.

MATERIALS AND METHODS

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Materials and Methods

Strain origin and maintenance. The collection of 82 C. dubliniensis strains used in this studywere DNA fingerprinted with the complex probe Cd25 (7, 11, 20).It included 61 from the group I clade and 21 from group II,a less closely related group of isolates than those of groupI (see Fig. 1). The 32 isolates analyzed for switching are listedin Table 1, with host disease state, geographical origin, andrelevant reference. The origin of C. albicans strains WO-1 ( ${alpha}$ / ${alpha}$ )and P37005 (a/a) and their mating characteristics have beenpreviously described (25, 26, 40). All strains were maintainedin 20% glycerol at –80°C. For experimental purposes,the cells were plated on agar containing Lee's amino acid-richmedium (23) supplemented with zinc and arginine (4). For differentiallystaining opaque phase sectors and colonies, 5 µg of phloxineB per ml was added to the agar (2).

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FIG. 1. The majority of a/a and ${alpha}$ / ${alpha}$ strains of C. dubliniensis are members of the group I clade. The dendrogram was generated from the similarity coefficients (S_AB) of DNA fingerprint patterns of 82 strains of C. dubliniensis, using the automated DENDRON program (43) and the unweighted pair group method (41). DNA fingerprinting was performed by Southern blot hybridization with the complex probe Cd25 (20). All 82 strains were analyzed for MTL zygosity by Southern blot hybridization (see Fig. 2) with the C. albicans MTLa1, MTL ${alpha}$ 1, and MTL ${alpha}$ 2 genes (15). Unshaded strains were a/ ${alpha}$ , while shaded strains were either a/a (a) or ${alpha}$ / ${alpha}$ ( ${alpha}$ ) as noted. While 43% of group I isolates are MTL homozygous, only 5% of group II isolates are MTL homozygous.

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TABLE 1. White-opaque switching and MTL zygosity of C. dubliniensis

Mating analyses. The method for testing mating in suspension cultures was similarto that of Lockhart et al. (26). In brief, 10⁷ opaque phasecells of two strains (a/a and ${alpha}$ / ${alpha}$ ) that had been grown to stationaryphase were mixed in 1 ml of fresh supplemented Lee's mediumin a 15-ml Falcon tube and rotated at 250 rpm in a water bathshaker at 25°C.

Isolation and sequence analysis of the MTL ${alpha}$ locus of C. dubliniensis. To obtain the full-length MTL ${alpha}$ locus and flanking sequences,5 x 10⁴ plaques of a C. dubliniensis genomic library of straind1558 (20) were screened with the 555-bp DNA fragment spanningthe MTL ${alpha}$ 2 open reading frame (ORF) of C. albicans (15). Six positiveclones were subjected to a secondary screen, and clones ${lambda}$ CDPL1.1and ${lambda}$ CDPL2.1 were chosen for further characterization. To generatethe first consensus sequence for locus walking, a 7.5-kb EcoRIfragment containing the MTL ${alpha}$ 2 regions was subcloned into pGEM-7Z(Promega Corp., Madison, Wis.). Using the initial consensusDNA sequence generated from the plasmid, the full-length MTL ${alpha}$ locus sequence was obtained by lambda clone walking with customprimers and an ABI sequencing apparatus (PE-ABI Inc., FosterCity, Calif.). Comparisons of protein and nucleotide sequenceswere performed using the multiple-alignment editor of ClustalW software (http://clustalw.genome.jp).

Analysis of mating-type zygosity. The mating type of each of the 82 C. dubliniensis strains wasanalyzed by Southern blot hybridization with the C. albicansMTLa1, MTL ${alpha}$ 1, and MTL ${alpha}$ 2 ORFs (13). DNA probes were generated byPCR. A subset of strains was also analyzed with the C. albicansMTLa2 ORF (53) and with C. dubliniensis MTL ${alpha}$ 1 and MTL ${alpha}$ 2 ORFs,generated by PCR. Southern analysis was performed as previouslydescribed with EcoRI-digested DNA (26).

Mating on skin. The methods for analyzing mating on skin were similar to thosepreviously described (21, 22). In brief, a cotton patch saturatedwith equal numbers of two test strains was taped to the skinon the back of a newborn White Swiss/Webster ND-4 mouse (Sprague-Dawley,Madison, Wis.). After 24 h, the animal was sacrificed, the patchwas removed, and the skin under the patch was excised and processedfor scanning electron microscopy.

Fluorescent staining of live cells. The methods for staining live cells were previously reportedin detail (26). In brief, cells from one strain were vitallystained with rhodamine-conjugated concanavalin A (ConA) andcells from the other strain were stained with fluorescein isothiocyanate(FITC)-conjugated ConA (Vector Laboratories, Burlingame, Calif.).After mating, cells were stained with calcofluor white M2R (MolecularProbes, Eugene, Oreg.). The cells were visualized with a Radiance2100 MP multiphoton laser-scanning confocal microscope (Bio-Rad,Hemel Hampstead, United Kingdom). For nuclear staining of fusants,cells stained with rhodamine- and FITC-conjugated ConA werefixed with 4% paraformaldehyde and stained with a solution of1 µg of Hoechst 33342 (Molecular Probes, Eugene, Oreg.)per ml.

Analysis of released chemoattractant. The methods for testing yeast cells for the release of chemoattractantfor polymorphonuclear leukocytes (PMNs) were presented in detailin previous publications (12, 13). In brief, cells were suspendedin buffer for 3 h and pelleted, and the supernatant (conditionedbuffer) was placed in one well of a quartz single-cell chemotaxischamber (38, 39) fashioned as described by Zigmond (54). Bufferalone was added to the other well. Human PMNs were placed overthe bridge between wells. After 5 min at 37°C, cell behaviorwas continuously videorecorded and analyzed with DIAS software(42, 47). Instantaneous velocity, chemotactic index, and percentpositive chemotaxis were computed as previously described (12,42).

RESULTS

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Results

Mating type. To assess the MTL zygosity (a/ ${alpha}$ , a/a, or ${alpha}$ / ${alpha}$ ) of natural C. dubliniensisstrains, Southern blots of EcoRI-digested DNA from natural isolateswere probed with the C. albicans MTLa1, MTL ${alpha}$ 1, and MTL ${alpha}$ 2 ORFs(15). Of the 82 tested isolates, 61 were members of the groupI clade and 21 were members of group II (Fig. 1) (7, 11, 20).EcoRI-digested DNA of all 82 isolates hybridized to either MTLa1alone (a/a), the two MTL ${alpha}$ probes alone ( ${alpha}$ / ${alpha}$ ), or all three probes(a/ ${alpha}$ ) (Fig. 2). EcoRI-digested DNAs from a subset of 10 a/a,10 ${alpha}$ / ${alpha}$ , and 10 a/ ${alpha}$ strains were subsequently probed with the recentlyidentified MTLa2 ORF (53). While all 20 a/a and a/ ${alpha}$ strains hybridizedto the probe, the 10 ${alpha}$ / ${alpha}$ strains did not, demonstrating that asin C. albicans (53), a1 and a2 are linked (data not shown).

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FIG. 2. Southern blot hybridization with the full-length C. albicans MTLa1 (a1), MTL ${alpha}$ 1, ( ${alpha}$ 1), and MTL ${alpha}$ 2 ( ${alpha}$ 2) genes demonstrated that all 82 tested C. dubliniensis strains were a/ ${alpha}$ , a/a, or ${alpha}$ / ${alpha}$ . Representative Southern blot hybridization patterns are presented for C. albicans to the left and C. dubliniensis to the right. Strains are identified at the top of the lanes.

Of the 82 natural C. dubliniensis strains tested, 55 (67%) wereMTL heterozygous (a/ ${alpha}$ ), 17 (21%) were MTLa homozygous (a/a),and 10 (12%) were MTL ${alpha}$ homozygous ( ${alpha}$ / ${alpha}$ ) (Fig. 1). The proportionof natural MTL-homozygous C. dubliniensis strains was therefore33%, which is approximately 10-fold higher than the proportionof natural C. albicans strains that are MTL homozygous (25).As is evident in the dendrogram in Fig. 1, all but one of theMTL-homozygous strains of the C. dubliniensis collection weremembers of the group I clade. While 43% of group I isolateswere MTL homozygous, only 5% of group II isolates were MTL homozygous.This difference was significant (P = 8 x 10^–4, Fisher'sexact test).

The hybridization results suggested a high level of identitybetween the MTLa and MTL ${alpha}$ genes of C. albicans and the C. dubliniensishomologs. To test this directly, the MTL ${alpha}$ locus was sequencedand compared to that of C. albicans strain SC5314 (a/ ${alpha}$ ) (15)(Fig. 3). The organization of the C. dubliniensis MTL ${alpha}$ locuswas identical to that of C. albicans (Fig. 3). The nucleotidesequences of the five genes in the locus were between 87.5 and90.0% identical to the C. albicans homologs (Fig. 3). The deducedamino acid sequences exhibited between 96.8 and 99.5% similarity(Fig. 3). The identity of the bands hybridizing to the C. albicansMTL ${alpha}$ 1 and MTL ${alpha}$ 2 probes was confirmed by using a select group of10 strains with PCR-generated C. dubliniensis MTL ${alpha}$ 1 and MTL ${alpha}$ 2probes (data not shown). Recently, data from the C. dubliniensisgenome sequencing project of the Pathogen Sequencing Unit atthe Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/Projects/C_dubliniensis/)revealed that the MTLa locus of C. dubliniensis also has highhomology and similar structure to the C. albicans MTLa locus.

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FIG. 3. Sequence analysis of the MTL ${alpha}$ locus of C. dubliniensis strain d1558 reveals the same organization of genes as in the MTL ${alpha}$ locus of C. albicans strain SC5314 (15) and high levels of gene homology. OBP ${alpha}$ , oxysterol-binding protein-like protein; PIK ${alpha}$ , phosphatidylinositol kinase; PAP ${alpha}$ , poly(A) polymerase; aa, amino acid; nt, nucleotide. Numbering of each locus begins with the first nucleotide of MTL ${alpha}$ 2. A 12,105-nucleotide sequence of C. albicans including the MTL ${alpha}$ locus plus flanking sequences was compared with the C. dubliniensis nucleotide sequence containing the MTL ${alpha}$ locus. The accession number for the C. dubliniensis MTL ${alpha}$ locus is AY622606.

White-opaque switching. To test whether natural strains of C. dubliniensis underwentthe white-opaque transition and whether it was regulated byMTL zygosity, as it is in C. albicans (25, 30), cells from 17a/a strains, 10 ${alpha}$ / ${alpha}$ strains, and 5 a/ ${alpha}$ strains were plated on phloxineB-containing agar. In C. albicans, phloxine B distinguishesopaque sectors and colonies by differentially staining themred, as demonstrated for C. albicans strain WO-1 in Fig. 4A(2). Over half of the natural C. dubliniensis a/a and ${alpha}$ / ${alpha}$ strainsproduced red sectors and/or red colonies that contained cellswith the signature opaque-cell phenotype, as described in thenext section (Table 1; Fig. 4B through F). However, two generalcharacteristics of white-opaque switching in C. dubliniensismade the process harder to study than in C. albicans. First,in contrast to switching in wild-type C. albicans (Fig. 4A),a second switching system appeared to be operational in at leasthalf of the strains, resulting in colony phenotypes that appearedto express the opaque phenotype as well as variant characteristicsof the second switching system (e.g., Fig. 4B and C). Second,high frequencies of reversibility from opaque to white werethe norm, such as at the edge of the opaque sectors of straind90006 in Fig. 4D and E. Among C. dubliniensis strains thatshowed a white-opaque phase transition, estimated frequenciesof the transition from the white to the opaque phase were similarto those of C. albicans (25). White-phase populations accumulatedopaque phase cells at frequencies of 10^–3 to 10^–4.In contrast, opaque-phase cell populations of most C. dubliniensisstrains accumulated white phase cells at a much higher frequency(>10^–1). We were able to identify only five strainswith opaque-phase cells stable enough (i.e., with low reversionrates to white) to perform many of the subsequent experimentsreported here. These strains, d90006 (a/a), d81217 (a/a), d88014(a/a), ANSA5 ( ${alpha}$ / ${alpha}$ ), and d126423 ( ${alpha}$ / ${alpha}$ ), exhibited switching frequenciesfrom opaque to white of approximately 10^–1, 10^–3,10^–3, 10^–3, and 10^–2, respectively. However,opaque-phase cells of strains d88014 and ANSA5 also switchedto additional phenotypes, presumably part of a second switchingsystem, at frequencies of ca. 10^–1 and 10^–2, respectively.

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FIG. 4. MTL-homozygous strains of C. dubliniensis undergo white-opaque switching at the colony level. (A) Switching in C. albicans strain WO-1 ( ${alpha}$ / ${alpha}$ ) for comparison. (B through F) Examples of switching are presented for individual a/a and ${alpha}$ / ${alpha}$ strains. In strains of d88014 (B) and ANSA5 (C), a second switching system ("smooth to rough edge" [RE]) is also active. Wh, white; Op, opaque.

Three of the five tested MTL-heterozygous (a/ ${alpha}$ ) strains (UP6a,UP16, and UP12) also formed red colonies and/or sectors on agarcontaining phloxine B (Table 1). For C. albicans, it was demonstratedthat a/ ${alpha}$ strains that underwent the white-opaque transition didso because they underwent high-frequency MTL homozygosis andthat it was in fact the MTL-homozygous offspring in every casethat were undergoing white-opaque switching (25). We thereforepicked white and opaque offspring of the three MTL-heterozygousstrains forming opaque sectors and tested them for MTL zygosityby Southern analysis. The offspring were demonstrated to berelated by fingerprinting with the C. dubliniensis species-specificprobe Cd25 (Fig. 5). For UP6a, the five selected clones thatwere white (W1 through W5) proved to be a/ ${alpha}$ while both clones(O1 and O2) that were opaque proved to be a/a (Fig. 5A). ForUP16, two of the four clones that were white (W1 and W4) provedto be a/ ${alpha}$ while the remaining two (W2 and W3) were a/a (Fig.5B). The three clones (O1 through O3) that were opaque werea/a (Fig. 5B). Finally, for UP12, the five clones that werewhite (W1 through W5) proved to be a/ ${alpha}$ while the two that formedrough-edged (RE) red colonies and sectors that contained bothlarge round (LR) cells and a minority of opaque cells provedto be a/a (Fig. 5C). These results are consistent with the conclusionthat C. dubliniensis cells undergoing white-opaque switchingare MTL homozygous, as with C. albicans (25, 30).

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FIG. 5. Apparent white-opaque switching in MTL-heterozygous strains is due to MTL homozygosis. White, opaque, and rough-edge (RE) colonies and sectors from three a/ ${alpha}$ strains that switched were analyzed for cellular phenotype (W, white; O, opaque; LR, large round) and MTL zygosity by Southern analysis with the C. albicans MTLa1 (a1) and MTL ${alpha}$ 2 ( ${alpha}$ 2) genes. Each clone was also DNA fingerprinted by Southern blot hybridization with the Cd25 probe (20) to demonstrate that all clones represented offspring of the same parent strain. Wh, white colony; Op, opaque colony; P, parent; W1, W2, etc., white progeny clones; O1, O2, etc., opaque progeny clones; RE1, RE2, rough- edge clones containing a large round variant cell type (LR) as well as opaque-phase cells. MTL Gen., MTL genotype; Col. Phen., colony phenotype; Cell. Phen., cellular phenotype.

Opaque-phase cell phenotype. In C. albicans, the surface of white cells is smooth and thecell shape round to slightly ellipsoidal while the surface ofopaque cells is pimpled and the shape is elongate (2, 40). Thesurface of MTL-homozygous C. dubliniensis cells in the whitephase was smooth, and the shape was round to ellipsoidal (Fig.6A), like white cells of C. albicans. The surface of the majorityof cells in the opaque phase was pimpled, and the shape waselongate (Figure 6B to D), like opaque cells of C. albicans(2). This opaque-phase signature phenotype was expressed byboth C. dubliniensis a/a and ${alpha}$ / ${alpha}$ cells. Opaque-phase cells ofC. dubliniensis sometimes grew into very large, elongate cellsthat tapered apically and were pimpled, suggesting that suchC. dubliniensis cells may have entered the apical mode of growthcharacteristic of hyphae while still expressing the opaque phenotype.This unusual phenotype has not been observed in C. albicans.In fact, it has been demonstrated that hyphae formed by C. albicansopaque cells are devoid of pimples (3). In addition, very large,round cells were sometimes found mixed with opaque cells inrough-edged red sectors or colonies (data not shown). Sincethese cells were usually found in sectors or colonies also exhibitingcharacteristics of a second switching system (e.g., Fig. 4C),we tentatively conclude that these aberrant cellular phenotypesmay have resulted from overlapping switching systems.

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FIG. 6. C. dubliniesis opaque-phase cells have pimples like C. albicans but can become abnormally large and long. (A) white cells; (B through D) opaque cells; (E and F) large, elongate opaque cells. Note the pimples on opaque cells. Bars, 2 µm.

C. dubliniensis opaque a/a and ${alpha}$ / ${alpha}$ cells can mate. To test whether C. dubliniensis a/a and ${alpha}$ / ${alpha}$ cells could mate andwhether mating depended on a switch, from white to opaque, mixturesof a/a and ${alpha}$ / ${alpha}$ cells were incubated in shaker cultures overnightand then examined for fusants by using differential interferencecontrast (DIC) optics. This method was used to obtain high frequenciesof fusants in mating mixtures of C. albicans (26). When whitea/a cells (strain d88014) and white ${alpha}$ / ${alpha}$ cells (strain ANSA5) wereincubated together, cells neither "shmooed" (i.e., extendedconjugation tubes constricted at the mother-tube junction) norfused (Fig. 7A and B). When opaque a/a cells (strain d88014)and opaque ${alpha}$ / ${alpha}$ cells (strain ANSA5) were incubated together, over80% formed shmoos after 4 h (Fig. 7C), and the shmoo extensionsgrew into conjugation tubes (Fig. 7D to F). Fusants formed after24 h (Fig. 7F to H) but were rare ( $~$ 0.1%). Similar results wereobtained with mixtures of opaque d81217 (a/a) and d126423 ( ${alpha}$ / ${alpha}$ )cells (data not shown).

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FIG. 7. Cell biology of C. dubliniensis mating. Equal numbers of opaque a/a and ${alpha}$ / ${alpha}$ cells were mixed and cultured in suspension for 5 h. (A and B) A cross between white a/a and ${alpha}$ / ${alpha}$ cells. (C) Shmooing in a cross between opaque a/a and ${alpha}$ / ${alpha}$ cells. (D and E) Conjugation tube formation in a cross between opaque a/a and ${alpha}$ / ${alpha}$ cells. (F) Example of a conjugation tube and fusant in a cross between opaque a/a and ${alpha}$ / ${alpha}$ cells after 7 h. (G and H) Examples of fusants in a cross between opaque a/a and ${alpha}$ / ${alpha}$ cells after 7 h. dc, daughter cell. Bar, 5 µm.

Therefore, although mixtures of C. dubliniensis opaque a/a and ${alpha}$ / ${alpha}$ cells in suspension cultures readily formed shmoos that grewinto long conjugation tubes, like C. albicans, the frequencyof fusion was less than 1/20 that of C. albicans (26). The reasonfor this appeared to be in the degree of cell clumping. Suspendedmixtures of C. albicans opaque a/a and ${alpha}$ / ${alpha}$ cells formed large,stable clumps that facilitated mating (26). However, C. dubliniensisopaque a/a and ${alpha}$ / ${alpha}$ cells did not clump. When samples were takenfrom suspended mating mixtures, the cells adhered loosely toone another, usually side by side as doublets, as in Fig. 7E.We therefore tested whether the frequency of fusion increasedin dense undisturbed cultures by simply incubating mixturesof a/a and ${alpha}$ / ${alpha}$ cells on nutrient agar. As expected, the frequencyincreased approximately fivefold over that in suspension cultures.It was still, however, far below that observed in comparablesuspension cultures of mating C. albicans cells (26).

To test unambiguously that fusions occurred only between C.dubliniensis a/a and ${alpha}$ / ${alpha}$ cells, a/a opaque cells were vitallystained green with FITC-conjugated ConA and ${alpha}$ / ${alpha}$ opaque cells werevitally stained red with rhodamine-conjugated ConA prior tomixing (26). If fusion occurred and if it was mating-type dependent,then 100% of fusants would include one green (a/a) and one red( ${alpha}$ / ${alpha}$ ) parent cell. The experiment was performed with both suspensionand standing dish cultures. All the fusants (20 fusants analyzed)included a green and a red parent cell (Fig. 8). None exhibiteda homogeneous color combination. These results demonstrate that,as in C. albicans (26), fusion occurs only between C. dubliniensisopaque a/a cells and opaque ${alpha}$ / ${alpha}$ cells.

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FIG. 8. Demonstration by laser-scanning confocal microscopy that only a/a and ${alpha}$ / ${alpha}$ cells fuse in mating cultures of C. dubliniensis. FITC-conjugated ConA (green)-stained a/a and rhodamine (Rho)-conjugated ConA (red)-stained ${alpha}$ / ${alpha}$ cells were allowed to mate in suspension cultures for 5 h. Crosses were between opaque d88014 a/a and ANSA5 ${alpha}$ / ${alpha}$ cells and between opaque d88014 a/a and d126423 ${alpha}$ / ${alpha}$ cells. (A and E) Fusant stained with calcofluor for cell wall to visualize the entire zygote. (B and F) Selective imaging of FITC-conjugated ConA. (C and G) Selective imaging of rhodamine-conjugated ConA. (D and H) Overlays of calcofluor, FITC-conjugated ConA, and rhodamine-conjugated ConA images. Bar, 5 µm.

Mating between species. Because of the high degree of genetic relatedness of C. albicansand C. dubliniensis, the possibility of interspecies matingwas entertained. We first tested whether C. dubliniensis opaquea/a cells responded to the C. albicans ${alpha}$ -pheromone peptide byshmooing and subsequent conjugation tube growth, like C. albicansa/a cells (6, 27, 32). When white a/a cells of C. dubliniensisstrains d81217 and d88014 were treated with 10^–5 M chemicallysynthesized C. albicans ${alpha}$ -pheromone (27), they did not shmoo(Figure 9A to C). However, when opaque a/a cells of the twostrains were treated with 10^–5 M C. albicans ${alpha}$ -pheromone,they formed unconstricted evaginations after 2 h in the processof shmooing (Fig. 9D to F). These evaginations elongated, formingconjugation tubes, which reverted apically to the budding-growthform after 5 h of incubation (Fig. 9G to I), as do the conjugationtubes of ${alpha}$ -pheromone-treated C. albicans a/a cells (27).

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FIG. 9. C. dubliniensis opaque a/a cells shmoo in response to C. albicans ${alpha}$ -pheromone (26). (A to C) White a/a cells do not shmoo after treatment with C. albicans ${alpha}$ -pheromone for 5 h. (D to F) Opaque a/a cells shmoo after treatment with C. albicans ${alpha}$ -pheromone for 2 h (arrows indicate unconstricted evaginations). (G to I) Opaque a/a conjugation tubes revert apically to the budding-growth form after a 5-h treatment (arrows indicate apical buds). Bar, 5 µm.

We then tested whether C. dubliniensis mated with C. albicansin a mating-type-dependent fashion. Mixtures of equal numbersof C. albicans opaque a/a cells (P37005) stained with FITC-conjugatedConA (green) and C. dubliniensis opaque ${alpha}$ / ${alpha}$ cells (d126423) stainedwith rhodamine-conjugated ConA (red) were incubated for 10 hin shaker cultures and then analyzed for fusants by laser-scanningconfocal microscopy. Fusants were found throughout the populationat frequencies approximately 10 times higher than in homospeciessuspension mixtures of C. dubliniensis a/a and ${alpha}$ / ${alpha}$ opaque cells.All the fusants (50 analyzed) exhibited the green and red colorcombination (Fig. 10), demonstrating that C. albicans and C.dubliniensis fused in a mating-type-dependent fashion. The higherfrequency of interspecies mating between C. albicans opaquea/a and C. dubliniensis opaque ${alpha}$ / ${alpha}$ cells in suspension cultureswas accompanied by clumping, as in mating mixtures of C. albicansa/a and ${alpha}$ / ${alpha}$ cells. Clumping and mating also occurred in mixturesof C. albicans opaque ${alpha}$ / ${alpha}$ and C. dubliniensis opaque a/a cells.

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FIG. 10. Demonstration of interspecies mating between opaque C. albicans a/a cells and opaque C. dubliniensis ${alpha}$ / ${alpha}$ cells. FITC-conjugated ConA (green)-stained C. albicans strain P37005 (a/a) and rhodamine (Rho)-conjugated ConA (red)-stained C. dubliniensis strain d126423 ( ${alpha}$ / ${alpha}$ ) cells were allowed to mate in suspension cultures for 5 h. (A and E) Fusants stained with calcofluor for cell wall visualization (B and F) Selective imaging of FITC-conjugated ConA. (C and G) Selective imaging of rhodamine-conjugated ConA. (D and H) Overlays of calcofluor, FITC-conjugated ConA, and rhodamine-conjugated ConA images. Bar, 5 µm.

Karyogamy in interspecies fusants. To visualize nuclei in fusants, cells from a 6-h culture containinga mixture of opaque C. albicans strain P57072 ( ${alpha}$ / ${alpha}$ ) cells stainedwith FITC-conjugated ConA and opaque C. dubliniensis straind90006 (a/a) cells stained with rhodamine-conjugated ConA werefixed and stained additionally with the nuclear dye Hoechst33342. Of the fusants that had not yet formed a daughter cell,the majority were in various stages of nuclear migration intothe conjugation tube (Fig. 11A). A minority contained two nucleiside by side in the tube (Fig. 11B). In the majority of fusantsthat had not yet formed a daughter bud, the nuclei were elongate.Thirty-nine fusants with daughter cells analyzed for the numberand spatial localization of nuclei exhibited the following fivemajor patterns of nuclear localization: (i) two nuclei sideby side in the bridge (8%) (Fig. 11C); (ii) one fused nucleusin the bridge (33%) (Fig. 11D); (iii) one nucleus in the bridgeand one in the daughter cell (21%) (Fig. 11E); (iv) two nucleiin the bridge and one in the daughter cell (8%) (Fig. 11F);and (v) two nuclei distributed in the bridge and mother cellsand two in the daughter cell (10%) (Fig. 11G). Other patternsrepresented 5% or less in the collection of fusants analyzedand therefore are not shown. From the major patterns, we havededuced a tentative dynamic sequence of nuclear migration andfusion, diagrammed at the bottom Fig. 11. A qualitative analysisof intraspecies C. dubliniensis fusants and C. albicans fusantsrevealed similar nuclear patterns (data not shown).

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FIG. 11. Nuclear migration, division, and localization during mating between opaque a/a C. albicans (P57072) and opaque ${alpha}$ / ${alpha}$ C. dubliniensis (d90006) cells. The former were vitally stained with FITC-conjugated ConA and the latter were stained with rhodamine- conjugated ConA prior to mixing. After 6 h, mating mixtures in suspension cultures were fixed and stained with the nuclear dye Hoechst 33342. Using a two-photon laser- scanning confocal microscope, FITC-conjugated ConA-stained C. albicans cells were visualized as green, rhodamine-conjugated ConA-stained C. dubliniensis cells were visualized as red, and nuclei were visualized as blue. For each fusant, three images are presented: the DIC image, the FITC, rhodamine, and Hoechst 33342 images overlaid, and the Hoechst 33342 image alone. Twenty fusants that had not formed daughter buds and thirty-nine that had formed daughter buds were scored for number and location of nuclei. The images shown in panels A through G represent the major patterns. Cells that were not part of the fusion are marked with an X in the DIC images and have been digitally removed from the fluorescent images. The sequence of stages was deduced from the major patterns, the quantitation of which is presented in the Results section. dc, daughter cell.

Mating on skin. Since newborn mouse skin has been demonstrated to facilitatemating between C. albicans opaque a/a cells and opaque ${alpha}$ / ${alpha}$ cells(22), we tested whether skin supported C. dubliniensis matingand then tested whether it supported interspecies mating. WhenC. dubliniensis opaque a/a cells (strain d88104) were incubatedalone for 24 h on skin, no shmoos, cells with conjugation tubes,or fusants were observed (Fig. 12A and B). However, when C.dubliniensis opaque a/a cells were incubated with opaque ${alpha}$ / ${alpha}$ cellson skin for 24 h in two different mating crosses (strains d88104[a/a] and d126423 [ ${alpha}$ / ${alpha}$ ] or strains d81217 [a/a] and ANSA5 [ ${alpha}$ / ${alpha}$ ]),cells with conjugation tubes and fusants were observed (Fig.12C and D). The frequency was 10 to 20 times higher than thatfor the same mixture in suspension cultures. When interspeciescrosses of C. dubliniensis strain d81217 a/a opaque cells andC. albicans strain WO-1 ${alpha}$ / ${alpha}$ opaque cells were incubated on skinfor 24 h, fusants were also observed at comparable frequencies(Fig. 12E to H). When C. dubliniensis strain d126423 ${alpha}$ / ${alpha}$ opaquecells were incubated with C. albicans strain L26 a/a opaquecells on skin for 24 h, fusants were also observed (data notshown).

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FIG. 12. Newborn mouse skin supports intraspecies C. dubliniensis mating and interspecies C. albicans and C. dubliniensis mating. (A and B) C. dubliniensis (C.d) a/a strain d88014 cells alone. (C and D) C. dubliniensis a/a strain d88014 and ${alpha}$ / ${alpha}$ strain d126423 cell mixture. (E to H) C. dubliniensis a/a strain d81217 and C. albicans ${alpha}$ / ${alpha}$ strain WO-1 cell mixture. The positions of junctions between mother and daughter cells are indicated in panels A and B by white arrowheads. Fusions are indicated in panels C through H by white arrowheads. Bars, 2 µm.

White-opaque switching in C. dubliniensis regulates the release of PMN chemoattractant. Recently, it was demonstrated by a single-cell chemotaxis assay(54) that white-opaque switching in C. albicans regulates therelease of a potent PMN chemoattractant (13). While white cellssecrete the attractant, opaque cells do not. It was suggestedthat the absence of attractant in opaque cultures may play arole in the overall mating strategy (13). We therefore testedwhether white-opaque switching in C. dubliniensis regulatedthe release of attractant by using a single-cell chemotaxisassay in which cells were placed on a gradient chamber bridgebordered by one trough containing buffer alone and one troughcontaining cell-conditioned buffer (12, 13). Gradients of low-molecular-weightmolecules form between 5 and 20 min of incubation (12, 13, 38,39). The "chemotactic index" (C.I.) was calculated as the distancemoved by a cell in the direction of the trough with conditionedbuffer divided by the total distance moved. A C.I. of +1.00represents persistent movement in a straight line toward thesource with conditioned buffer (positive chemotaxis), a C.I.close to 0.00 represents random movement (no chemotaxis), andC.I.s increasing from 0.00 to +1.00 reflect increasing efficienciesof positive chemotaxis. The parameter "percent positive chemotaxis"represents the proportion of cells exhibiting a positive chemotacticindex. A measure of 100% means that all test cells exhibitednet movement toward the source of chemoattractant. A measureof 50% positive chemotaxis reflects random movement (i.e., nochemotaxis). Conditioned buffer from white and opaque cellsof three different strains of C. dubliniensis (ANSA5, d81217,and d88014) was tested. In every case, conditioned medium fromwhite cells induced chemotaxis (average C.I. between +0.32 and+0.62; percent positive chemotaxis between 91 and 93%) (Table2). In contrast, conditioned medium from opaque cells did notinduce chemotaxis (average C.I. between –0.01 and +0.10;percent positive chemotaxis between 42 and 48%) (Table 2). Therefore,just as for C. albicans (13), switching in C. dubliniensis regulatesthe release of a PMN chemoattractant.

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TABLE 2. White, but not opaque, cells of C. dubliniensis release a PMN chemoattractant

DISCUSSION

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Discussion

The C. dubliniensis MTL locus and MTL zygosity. Using the C. albicans mating-type genes MTLa1, MTL ${alpha}$ 1, and MTL ${alpha}$ 2as probes (15), we have demonstrated by Southern analysis thata/ ${alpha}$ , a/a, and ${alpha}$ / ${alpha}$ strains of C. dubliniensis exist in nature, likeC. albicans (25). Conservation of the MTL locus was confirmedby comparing the sequence of the MTL ${alpha}$ locus of C. dubliniensiswith that of C. albicans (15). The arrangement of the five genesharbored by the MTL ${alpha}$ locus was the same as in C. albicans. Thenucleotide sequences and deduced amino acid sequences were highlysimilar. Recently, sequencing data provided by the WellcomeTrust Sanger Institute demonstrated that this was also the casefor MTLa and confirmed our results for MTL ${alpha}$ .

In the collection of 82 C. dubliniensis strains analyzed here,roughly one-third were MTL homozygous. This represents 10 timesthe frequency of MTL homozygosity found in natural C. albicansstrains (25), suggesting that MTL homozygosis occurs more frequentlyin C. dubliniensis. Recently, mitotic recombination has beendemonstrated to be one mechanism for spontaneous MTL homozygosisin natural strains of C. albicans (W. Wu, S. Lockhart, and D.R. Soll, unpublished data). The high frequency of MTL homozygotesin the C. dubliniensis collection is therefore consistent withprevious observations that C. dubliniensis undergoes genomicreorganization at high frequency, leading to karyotype instability(19). Presumably, the high rate of genomic reorganization results,at least in part, from the high rate of mitotic recombination,which in turn results from the twofold-larger number of recombinogenicRPS sequences (19) and the additional C. dubliniensis-specificrepeat sequence dispersed throughout the genome (20).

We also found twice as many a/a as ${alpha}$ / ${alpha}$ strains in the C. dubliniensiscollection, suggesting an adaptive hierarchy in nature of a/ ${alpha}$ > a/a > ${alpha}$ / ${alpha}$ . Finally, we found that MTL homozygotes werealmost exclusively members of the group I clade. Members ofthis clade have been shown by DNA fingerprinting to be geneticallymore closely related than members of the group II clade (20)and to be more frequently recovered from HIV-positive individualsthan group II strains (11). Clade-specific characteristics havebegun to emerge in C. albicans (46). Natural flucytosine resistancewas found to be restricted to one of the five clades of C. albicans(34). Flucytosine resistance represents the first identifiedclade-specific phenotypic characteristic of C. albicans (34,46). The high frequency of MTL homozygosis in group I representsa new clade-specific characteristic in C. dubliniensis.

White-opaque transition in C. dubliniensis. We have demonstrated for the first time here that MTL-homozygousstrains of C. dubliniensis undergo white-opaque switching, likeMTL-homozygous strains of C. albicans (25, 30). However, white-opaqueswitching in C. dubliniensis was less uniform and sometimesharder to assess than in C. albicans for two reasons. First,it appeared to occur sometimes concurrently with other high-frequency switching systems. These other systems affected bothcolony and cellular phenotype and in many cases appeared todistort or camouflage the opaque phenotype. The second reasonthat switching is less uniform in C. dubliniensis was the highfrequency of reversion from opaque to white, which was obviousat the edges of opaque sectors. In fact, we found that onlya minority of the MTL-homozygous C. dubliniensis strains thatundergo the white-opaque transition do so at frequencies lowenough to perform many of the experiments reported here.

The cellular phenotype of C. dubliniensis opaque cells was similarto that of C. albicans opaque cells, including unique opaque-specificpimples (2), selective adhesion to skin (21, 22), the absenceof secreted PMN chemoattractant (13), and mating competency(25, 30). However, opaque-phase cells of C. dubliniensis differedfrom those of C. albicans in their propensity to grow to enormoussizes by elongation while continuing to form pimples on theirsurface. The tapered, tubular shape of these cells suggestedthat they may have begun to grow in an apical mode, as do hyphae,even though they continued to form pimples in the wall. Theseaberrant opaque cell morphologies have not been reported forC. albicans, and hyphae formed by opaque cells are free of pimples(3).

Intraspecies and interspecies mating. We have presented evidence that C. dubliniensis mating followsthe same general rules as those for C. albicans mating (18,45, 48), namely, that mating-type zygosity regulates white-opaqueswitching, that cells must switch from the white to the opaquephase in order to mate, that fusion occurs only between oppositemating types, that ${alpha}$ -pheromone induces shmooing and conjugationtube formation in a/a cells, and that the cellular stages ofmating are similar to those in C. albicans. We have also demonstratedthat skin facilitates mating, as it does in C. albicans (22).However, we have also found that in suspension cultures, althoughthe majority of opaque a/a and ${alpha}$ / ${alpha}$ cells shmoo, fusion is infrequent,presumably because C. dubliniensis opaque a/a and ${alpha}$ / ${alpha}$ cells donot clump as do C. albicans opaque a/a and ${alpha}$ / ${alpha}$ cells in suspension.Clumping appears to facilitate mating by stabilizing cell-cellassociations (26). Low frequencies of fusion and the absenceof clumping were observed in crosses between all tested setsof strains. We therefore tested whether the absence of clumpingwas the reason for the low frequency of fusion by assessingfusion in undisturbed a/a and ${alpha}$ / ${alpha}$ mixes on agar. The frequencywas approximately fivefold higher than in suspension, supportingthis conclusion. Furthermore, skin facilitated higher frequenciesof fusion, again supporting the idea that the low frequenciesof fusion in suspension are due to the lack of clumping.

Finally, we have demonstrated that C. albicans and C. dubliniensiscan mate in suspension cultures. Surprisingly, the frequenciesof interspecies mating were higher than those of intraspeciesC. dubliniensis mating in suspension cultures, whether C. dubliniensiswas the a/a or the ${alpha}$ / ${alpha}$ partner. The higher efficiency of interspeciesmating correlated with increased clumping. The cellular characteristicsof interspecies mating in suspension or on skin were at leastsuperficially similar to those of intraspecies mating.

Karyogamy. We have found that in fusants that have not yet formed a daughtercell, the parent cell nuclei move into the bridge, ending upnext to each other at the site of daughter cell formation. Migratingnuclei were highly elongate, suggesting tension, presumablythe result of associated cytoskeleton involved in migration(8, 14, 35). The number of nuclei and their spatial localizationwere quantitatively analyzed in interspecies fusants and qualitativelyanalyzed in intraspecies C. dubliniensis and C. albicans fusants.From the proportions of cells exhibiting different patterns,we have deduced the tentative temporal and spatial program ofnuclear events accompanying interspecies mating. The nucleithat migrate into the bridge fuse and divide. One daughter nucleusthen enters the daughter cell, while the other remains in thebridge. The nucleus in the bridge divides, and then the nucleusin the daughter cell divides. This scheme is similar to thatdeduced from a qualitative analysis of intraspecies C. albicansand C. dubliniensis fusants (data not shown). The scheme isalso remarkably similar to that of Saccharomyces cerevisiae(36).

Because interspecies mating and karyogamy occur so readily invitro, it seems likely that the event may occur in nature, sincemixed mycotic infections readily occur (44). However, althoughthe two species can readily mate and undergo karyogamy, it seemsunlikely that recombination normally occurs in nature, giventhe divergence of the two species documented at the geneticlevel (10, 11, 19, 20, 49, 50, 51, 52). Experiments are nowin progress to demonstrate at the genetic level whether thefused cells of interspecies mating generate viable allotetraploidssimilar to the autotetraploids generated by C. albicans fusion(5, 16, 28) and whether these cells revert to diploids by chromosomeloss, as do C. albicans tetraploids generated by mating (5).

Conclusion. We have found that although C. dubliniensis possesses the samedevelopmental programs as C. albicans, these programs appearto be less uniform, more variable between strains, and lessefficient. It seems likely that this is a result of the highlevel of genetic instability characteristic of C. dubliniensis,due in part to increased frequencies of mitotic recombination(19). One may therefore consider C. dubliniensis to have lostthe balance between mitotic recombination and genomic stabilityexhibited by C. albicans. Although the higher levels of recombinationexhibited by C. dubliniensis could provide an advantage forrapid adaptation, it is obviously detrimental to the maintenanceof its developmental programs and MTL heterozygosity in nature.This may explain why C. albicans, not C. dubliniensis, remainsthe dominant Candida species in human commensalisms and disease.

We have also found that C. dubliniensis and C. albicans, whichcan cohabit in the same body niches in nature, readily matein vitro and on skin. The fact that these related species maintaingenetic integrity in the same geographical locales suggeststhat even if they readily mate, they may not undergo recombinationin nature. Alternatively, they may mate, undergo karyogamy,and recombine in nature, but the recombinants may be less competitivethan either parent. These alternatives are now being tested.

ACKNOWLEDGMENTS

We thank Randy Nessler of the Central Electron Microscopy ResearchFacility at Iowa for technical assistance. Images were obtainedin the W. M. Keck Dynamic Image Analysis Facility at Iowa.

This research was supported by NIH grants AI2392 and DE14219.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Sciences, 302 BBE, The University of Iowa, Iowa City, IA 52242. Phone: (319) 335-1117. Fax: (319) 335-2772. E-mail: david-soll{at}uiowa.edu.

REFERENCES

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