Impact of Mating Type, Serotype, and Ploidy on the Virulence of Cryptococcus neoformans

Abstract

Hybridization with polyploidization is a significant biological force driving evolution. The effect of combining two distinct genomes in one organism on the virulence potential of pathogenic fungi is not clear. Cryptococcus neoformans, the most common cause of fungal infection of the central nervous system, has a bipolar mating system with a and α mating types and occurs as A (haploid), D (haploid), and AD hybrid (mostly diploid) serotypes. Diploid AD hybrids are derived either from a-α mating or from unisexual mating between haploid cells. The precise contributions of increased ploidy, the effect of hybridization between serotypes A and D, and the combination of mating types to the virulence potential of AD hybrids have remained elusive. By using in vitro and in vivo characterization of laboratory-constructed isogenic diploids and AD hybrids with all possible mating type combinations in defined genetic backgrounds, we found that higher ploidy has a minor negative effect on virulence in a murine inhalation model of cryptococcosis. The presence of both mating types a and α in AD hybrids did not affect the virulence potential, irrespective of the serotype origin. Interestingly, AD hybrids with only one mating type behaved differently, with the virulence of αADα strains similar to that of other hybrids, while aADa hybrids displayed significantly lower virulence due to negative epistatic interactions between the Aa and Da alleles of the mating type locus. This study provides insights into the impact of ploidy, mating type, and serotype on virulence and the impact of hybridization on the fitness and virulence of a eukaryotic microbial pathogen.

Intra- and interspecies hybridization is a driving force in evolution that promotes emergence of new species more fit for novel or changing environments (18, 64). The evolutionary advantage of hybridization with increased ploidy is evident in the plant kingdom, where a significant proportion of plant species are polyploid and originated via hybridization of different species (19, 67, 76). In contrast, little is known about the impact of hybridization with increased ploidy on the virulence of pathogenic fungi.

Cryptococcus neoformans is a ubiquitous human fungal pathogen that causes the most common fungal infection of the central nervous system, predominantly in immunocompromised patients (9). Although cases of cryptococcosis in AIDS patients have decreased in developed countries due to the introduction of highly active antiretroviral therapy in the mid-1990s, this disease remains responsible for up to 30% of the attributable mortality in AIDS patients in regions where highly active antiretroviral therapy is not readily available, such as Southeast Asia and Africa (7, 9, 12, 24, 25, 27, 28, 37).

C. neoformans is classified into three serotypes based on capsular agglutination reactions: serotype A (C. neoformans var. grubii), serotype D (C. neoformans var. neoformans), and AD hybrids (6). In Europe, ∼5 to 30% of the infections are infections with AD hybrids (5, 8, 15, 20, 49, 69), and this is likely an underestimate due to the failure to identify all AD hybrids with capsular antigen-based serotyping techniques (4, 8, 53, 72). The fact that AD hybrids are common in both clinical and environmental samples may reflect an impact of hybridization on C. neoformans fitness and virulence (49, 59, 72). A recent report hypothesized that hybrid fitness may have contributed to the worldwide distribution of AD hybrids (50).

C. neoformans has a bipolar mating system, and traditional mating involves cells of opposite mating types, mating types a and α. A filamentous dikaryon is produced after cell-cell fusion, and nuclear fusion occurs later in the fruiting body basidium, resulting in a transient a/α diploid state that immediately undergoes meiosis and sporulation, producing a and α haploid progeny (38, 39). Although an opposite mating partner is preferred, C. neoformans can undergo unisexual mating, especially between α cells, to produce stable α/α diploids and also α haploid progeny (46-48, 77). When serotype A and D strains mate, cell-cell fusion proceeds normally, but meiosis is impaired due to genetic differences between the two divergent serotypes (∼10 to 15% nucleotide polymorphism at the whole-genome level [35]), resulting in few viable haploid progeny (34, 42). Despite impaired meiosis, the AD hybrid cells resulting from these intravarietal matings can propagate mitotically as uninucleate diploid yeast cells after nuclear fusion events. Thus, many natural AD hybrids remain in the diploid (or aneuploid) state (42, 68).

Both serotypes and mating types have been implicated as virulence factors in C. neoformans. Serotype A is associated with a high level of pathogenicity, and it causes the majority of infections worldwide (95%) (9). In addition to epidemiological evidence, serotype A isolates are usually associated with better growth in vitro and higher virulence potential in animal models than serotype D isolates. For example, two widely used laboratory reference strains, Aα strain H99 and Dα strain JEC21, exhibit considerable differences in stress responses, and H99 is dramatically more virulent than JEC21 (3, 14, 48, 70; this study).

In contrast to the contributions of serotype, the contributions of mating type to virulence potential are more elusive. The predominance of the α mating type in clinical isolates (>98 to 99.9%) (9, 40) may simply reflect the overwhelming presence of this mating type in the environment. In addition, congenic α and a cells (genetically identical except at the mating type locus) of both serotype A and serotype D strains grow equally well under various laboratory conditions. Congenic α and a strains in the serotype A H99 background display the same pathogenicity level in various invertebrate and mammalian models (56, 57), and the same is true for congenic α and a strains in the serotype D B3501 background (58). However, in some backgrounds or under certain conditions, the α mating type locus may enhance virulence. For example, nonisogenic α strains in general were found to be more virulent than a strains (3, 36, 44). α strains in the serotype D strain JEC21 and NIH433 backgrounds are more virulent than congenic a strains in a murine model of cryptococcosis (41, 58), and α cells in the serotype A strain H99 background preferentially disseminate to the central nervous system during coinfection with congenic a cells (56).

AD hybrid isolates have been used to address the relationship between serotype, mating type, and virulence in several previous studies (3, 14, 42, 70). The abundance of AD hybrids in both clinical and environmental samples and their unique evolutionary position as intervarietal hybrids underscore the importance of studies on their virulence potential (20, 49, 59). The fact that all possible combinations of mating types can be isolated in AD hybrids provides a unique opportunity to study interactions between serotype and mating type during infection. Previous studies on the virulence of C. neoformans AD hybrids reported variable virulence potential depending on the isolate and route of infection (3, 14, 42, 70). For example, markedly reduced virulence of natural AD hybrids was observed compared to the Aα H99 reference strain in a murine inhalation model of cryptococcosis (42). The virulence of most natural αADa hybrids (7/8) was slightly less than but similar to that of the Aα H99 reference strain in a murine intravenous model (14). In an intracisternal rabbit model of cryptococcal meningitis, αADa and aADα hybrids were similar to haploid Aα or Aa cells in terms of virulence (70). Recently, the pathogenicities of 15 C. neoformans strains, including isolates with the Dα, Da, Aα, Aa, αADa, and aADα genotypes, were characterized using a murine cryptococcosis intravenous model and also an inhalation model for a subset of isolates. Wide variation in the virulence of these strains was observed (3). Aα (one natural strain plus the H99 reference strain) and αADa (three natural isolates) strains were highly pathogenic, leading the authors to conclude that virulence is associated with the Aα mating type (3).

Thus, the virulence potential of AD hybrids is unclear, and several confounding factors may contribute. First, most AD hybrids are diploid, and the effect of ploidy per se on C. neoformans virulence is unknown. Effects of ploidy on virulence could be species specific based on studies of other pathogenic fungi. For example, diploid Aspergillus nidulans strains are more virulent than the prototrophic haploid parent in mice (63), and some diploid hybrids between Beauveria bassiana and Beauveria sulfurescens are hypervirulent in insects (71). In contrast, tetraploid Candida albicans strains are similarly or less virulent than diploid strains (29, 33, 65). Second, different animal models and routes of infection were used in previous studies, and these different methods could affect strains differently. Third, most of the AD hybrids used are natural isolates that are genetically diverse and exhibit phenotypic variation, likely including virulence.

To elucidate the contributions of mating type, serotype, and ploidy to the virulence of C. neoformans, diploid serotype A strains (αAAα) and AD hybrids with all possible mating type combinations in defined genetic backgrounds were generated and characterized. These diploid strains and reference haploid serotype A or D strains were analyzed for defined C. neoformans virulence factors in vitro. All hybrid strains behaved similarly in vitro regardless of the mating type combination and route of derivation. AD hybrid strains were more thermotolerant and more resistant to UV irradiation than either serotype A or D parental strains. Increased ploidy was associated with larger cell size and exerted modest adverse effects on melanization, tolerance to high temperature, and virulence. In a murine inhalation model of cryptococcosis, all strains were much more virulent than the parental serotype D strains. Most hybrids, including αADa, aADα, and αADα hybrids, were similar to diploid αAAα strains in terms of virulence. In contrast, the aADa hybrids were less virulent than all other AD hybrids. The nearly isogenic aAAa diploid isolate was highly virulent, providing evidence that the presence of two copies of the a mating type locus alleles does not have a detrimental effect on pathogenicity, and the reduced virulence potential of aADa hybrids is likely due to negative epistatic interactions between the Aa and Da mating type alleles with each other and/or other genetic loci in the AD hybrid background. Our observations demonstrate (i) the benefits of hybridization in C. neoformans, (ii) that there is little or no impact on the virulence of AD hybrids due to the presence of both a and α mating types, (iii) that the serotype origin of the mating type has no effect on AD hybrid virulence, and (iv) that the virulence of AD hybrids is reduced when both the Aa and Da mating types are present due to negative epistasis.

MATERIALS AND METHODS

Strains and growth conditions.

Strains used in this study and their sources are listed in Table 1. Cells were maintained on YPD medium (1% yeast extract, 2% Bacto Peptone, and 2% dextrose) or YNB medium (yeast nitrogen base medium; Difco, Detroit, MI). For marker screening, YPD medium containing nourseothricin (NAT) (YPD+NAT medium) and YPD medium containing neomycin (NEO) (YPD+NEO medium) were used for strains with dominant drug resistance markers, while synthetic complete media without adenine (SC-ade), uracil (SC-ura), or lysine (SC-lys) were used for strains with auxotrophic markers. Mating or cell fusion was conducted on V8 medium (pH 7.0 or 5.0) with or without 50 μM CuSO₄ in the dark at 22°C.

TABLE 1.

Strains used in this study

Strain	Genotype and/or phenotype	Reference(s) or source
JEC21	Dα	26, 41
JEC20	Da	26, 41
B3502	Da	26, 41
JEC30a	Dalys1	54
JEC156a	Daade2 ura5	54
JEC157a	Dα ade2 lys1 ura5	54
XL342a	Dα ade2	This study
XL465a	Dagsy1::NATr	45
XL467a	Dα gsy1::NATr	45
XL141a	Dα	47
XL143a	αDDα	47
XL370b	Da	47
XL374b	aDDa	47
H99	Aα	60
KN99α	Aα	57
KN99a	Aa	57
F99	Aα ura5	73
JF99	Aaura5	55
YSB119	Aα aca1::NATrura5 ACA1-URA5	2
YSB121	Aaaca1::NEOrura5 ACA1-URA5	2
KN99α NAT13	Aα NATr marker inserted between BSP1 and RPL39 in MAT	56
KN99α NEO1	Aα NEOr marker inserted between BSP1 and RPL39 in MAT and ectopic integration	This study
KN99a NEO1	Unstable NEOr marker	This study
XL1462	αADα ade2/ADE2 ura5/URA5	48
XL1495	aADaura5/URA5 lys1/LYS1	Fusion product of JF99 and JEC30
XL1500	αAAα NATr NEOr	Fusion product of KN99α NAT13 and KN99α NEO1
XL1501	αAAα NATr NEOr	Fusion product of YSB119 and KN99α NEO1
XL1511	aADa NATr NEOr	Fusion product of KN99aNEO1 and XL465
XL1514	aADa NATr NEOr	Fusion product of YSB121 and XL465
XL1548	aADα NATr NEOr	Fusion product of YSB121 and XL467
XL1552	αADa NATr NEOr	Fusion product of KN99αNEO1 and XL465
KN4B7#16	aAAa	Eighth-backcross progeny of H99c

^aStrain isogenic with JEC21 and JEC20.

^bStrain isogenic with B3502.

^cSee reference 57.

Determination of mating type.

To determine the mating type, isolates were grown on YPD medium for 1 day at 30°C and separately cocultured with the reference tester strains, JEC20 (a) and JEC21 (α), on V8 medium in the dark at 22°C (41). An isolate and tester strains were cultured alone on the same plate as controls. The mating reaction plates were examined after 1 week for the formation of mating hyphae, which signaled the initiation of sexual reproduction. Mating types were also determined by PCR with SXI1α, SXI2a, and STE20α/a gene primers that yield mating type- and serotype-specific amplicons (42, 48).

Determination of ploidy by fluorescence flow cytometry.

Cells were processed for flow cytometry as described previously (47, 66). Briefly, cells were harvested from YPD medium, washed once in phosphate-buffered saline (PBS), and fixed in 1 ml of 70% ethanol overnight at 4°C. Fixed cells were washed once with 1 ml of NS buffer (10 mM Tris-HCl [pH 7.6], 250 mM sucrose, 1 mM EDTA [pH 8.0], 1 mM MgCl₂, 0.1 mM CaCl₂, 0.1 mM ZnCl₂) and then stained with propidium iodide (10 mg/ml) in 0.2 ml of NS buffer containing RNase A (1 mg/ml) at 4°C for 4 to 16 h. Then 0.05 ml of stained cells was diluted into 2 ml of 50 mM Tris-HCl (pH 8.0) and sonicated for 1 min. Flow cytometry was performed with 10,000 cells, and the results were analyzed on the FL1 channel with a Becton-Dickinson FACScan.

Determination of cell size.

Cells were grown overnight in YNB medium at 37°C with shaking. An aliquot of the cells was analyzed by forward scatter flow cytometry, and ∼10,000 cells were employed. Another aliquot of the cells was fixed in 3.7% formaldehyde in PBS and photographed by using microscopy. The diameters of mother yeast cells (43 to 50 cells per genotype) were measured using Photoshop measurement tools.

Preparation of genomic DNA.

Strains were grown in 50 ml YPD medium at 30°C overnight with shaking. The cells were washed three times with distilled water and harvested by centrifugation at 4,000 × g for 8 min. Each cell pellet was frozen immediately at −80°C, lyophilized overnight, and stored at −20°C until genomic DNA was prepared using the cetyltrimethylammonium bromide protocol as described previously (61).

Construction of αAAα and AD hybrid diploid strains.

To construct the αAAα diploid strains, two independent fusion procedures were performed. First, strains KN99αNAT13 (Aα NAT^r) and KN99αNEO1 (Aα NEO^r) marked with dominant drug resistance markers were cocultured together with JEC156 (Da ade2 ura5) as a pheromone donor on V8 medium (pH 5.0) containing 50 μM CuSO₄ in the dark at 22°C. Addition of copper to the medium enhances mating or fruiting of C. neoformans (46). After 24 h of coculture on V8 medium, cells were collected and spread on YPD medium containing NAT and NEO (YPD+NAT+NEO medium) at 37°C to select for fusion products. Two types of fusion products were obtained: the desired diploid αAAα diploid strains and triploid Da/Aα/Aα strains, which were distinguished by mating behavior and analysis of ploidy by fluorescence-activated cell sorting (FACS). The chosen diploid αAAα hybrid strains were further confirmed by performing mating type- and serotype-specific PCR analyses. Independently, YSB119 (Aα NAT^r) and KN99αNEO1 (Aα NEO^r) were also used to isolate αAAα diploids as described above.

To construct αADa strains, KN99αNEO1 (Aα NEO^r) and XL465 (Da NAT^r) were cocultured on V8 agar (pH 5.0) in the dark at 22°C. After 20 h of coculture, cells were collected and spread on YPD+NAT+NEO medium at 37°C to select for fusion products. αADa strains were confirmed to be diploid by FACS analysis. The genotype was confirmed by performing mating type- and serotype-specific PCR analyses.

To construct aADα strains, strains YSB121 (Aa NEO^r) and XL467 (Dα NAT^r) were cocultured on V8 agar (pH 5.0) in the dark at 22°C. aADα diploid strains were validated as described above.

To construct aADa strains, three independent fusion procedures were performed. First, the auxotrophic strains JF99 (Aa ura5) and JEC30 (Da lys1) were cocultured together with JEC157 (Dα ade2 lys1 ura5) as the pheromone donor on V8 medium (pH 5.0) containing 50 μM CuSO₄ in the dark at 22°C. These three strains are unable to grow on minimal media without addition of uracil, lysine, and uracil plus adenine plus lysine, respectively. After 24 h of coculture, cells were collected and spread on YNB minimal medium at 37°C to select for prototrophic fusion products. Two types of fusion products were obtained: the desired diploid aADa hybrid strains and triploid Aa/Da/Dα strains, which were distinguished by mating behavior and analysis of ploidy by FACS. The chosen diploid aADa hybrid strains were further confirmed by performing mating type- and serotype-specific PCR analyses. Independently, strains KN99aNEO1 (Aa NEO^r) and XL465 (Da NAT^r) were cocultured together with strain JEC157 (Dα ade2 lys1 ura5) as the pheromone donor on V8 medium (pH 5.0) containing 50 μM CuSO₄ in the dark at 22°C. After 24 h of coculture, cells were collected and spread on YPD+NAT+NEO medium at 37°C to select for fusion products. The genotypes and ploidy of the desired aADa hybrid strains were confirmed as described above. Finally, strains YSB121 (Aa NEO^r) and XL465 (Da NAT^r) were cocultured together with strain JEC170 (Dα ade2 lys2) as the pheromone donor on V8 medium (pH 5.0) containing 50 μM CuSO₄ in the dark at 22°C. After 24 h of coculture, cells were collected and spread on YPD+NAT+NEO medium at 37°C to select for fusion products. The genotypes and ploidy of the desired aADa hybrid strains were confirmed as described above.

All fusion products were selected at 37°C, the temperature at which even dikaryotic hyphae undergo nuclear fusion and transform morphologically to uninucleate yeast cells (66). Thus, all the selected cells were uninucleate yeasts. This was confirmed by examination with fluorescence microscopy prior to analysis of ploidy by FACS.

In vitro assay of virulence factors.

Virulence factors were assayed as previously described (46, 48). Briefly, yeast cells were grown on YPD medium overnight and washed three times with water. Cell density was determined by determining the optical density at 600 nm, and cells were serially diluted. To examine melanin production, cells were spotted on melanin-inducing media containing l-dihydroxyphenylalanine (l-DOPA) (100 mg/liter) (13) and incubated at 22 and 37°C in the dark for 2 to 6 days. Melanization was observed as the colonies developed a brown color. To analyze growth at different temperatures, cells were spotted on YPD medium and incubated at the indicated temperatures. Cell growth was assessed from day 2 to day 4. To determine sensitivity to UV irradiation, cells were spotted on YPD medium and exposed to UV irradiation in a Stratalinker (Stratagene, La Jolla, CA) for 0, 6, and 12 s and then incubated at 22°C. Cell growth was monitored daily from day 2 to day 4. To characterize capsule production, equal numbers of C. neoformans cells were transferred to Dulbecco's modified Eagle's medium (Invitrogen, California) and grown for 3 days at 37°C. Cells were then suspended in India ink, and the capsule was visualized as a white halo surrounding the yeast cell due to exclusion of ink particles.

Growth competition assay.

Strains were grown in YPD liquid medium at 30°C overnight and then washed three times with sterile water. Strains having the same cell density based on hemocytometer counting were paired (XL1501 and KN99αNEO1, XL1501 and YSB119, XL1511 and KN99aNEO1, XL1511 and YSB121, XL1552 and KN99αNEO1), and each mixture was inoculated into YPD liquid medium (final density, 8 × 10⁴ cells/ml) and cultured at both 39°C and room temperature. An aliquot of each culture was removed at 0, 24, and 48 h, plated on YPD medium plates, and incubated for 2 days before it was replicated onto YPD, YPD+NAT, and YPD+NEO medium plates. The colonies growing on each plate were counted. The following formulas were used to calculate haploid/diploid ratios: KN99αNEO1/XL1501 ratio = (NEO^r − NAT^r NEO^r)/NAT^r NEO^r; YSB119/XL1501 ratio = (NAT^r − NAT^r NEO^r)/NAT^r NEO^r; KN99aNEO1/XL1511 ratio = (YPD − NAT^r)/NAT^r; YSB121/XL1514 ratio = (NEO^r − NAT^r NEO^r)/NAT^r NEO^r; and KN99αNEO1/XL1552 ratio = (NEO^r − NAT^r NEO^r)/NAT^r NEO^r.

Murine inhalation model of cryptococcosis and recovery of fungal cells.

Animals were infected essentially as previously described (16). Groups of 6- to 8-week-old female A/J mice were anesthetized by intraperitoneal injection of phenobarbital (∼0.035 mg/g). Animals were infected intranasally with 5 × 10⁴ fungal cells in 50 μl PBS. The yeast cell inocula were confirmed by determining the number of CFU after serial dilution. To verify the identity of a strain in an inoculum, 100 to 300 colonies for diploid and hybrid strains and 50 to 200 colonies for the controls were tested for markers (auxotrophic or dominant). Three or four colonies of each diploid or hybrid strain were randomly chosen, and ploidy was checked by fluorescent flow cytometry. After inoculation of fungal cells, animals were monitored twice daily, and the animals showing signs of severe morbidity (weight loss, extension of the cerebral portion of the cranium, abnormal gait, paralysis, seizures, convulsions, or coma) were sacrificed by CO₂ inhalation. The rates of survival of animals were plotted against time, and P values were calculated with the Mann-Whitney U test (see Table 4). To examine the reduction in ploidy in vivo, the lungs and brains from two sacrificed animals infected with the hybrids or the diploids were removed, weighed, and homogenized in 2 ml of sterile PBS. Serial dilutions of the organ samples were plated on YPD agar plates containing 100 μg/ml chloramphenicol and incubated at 37°C overnight. Randomly picked colonies (50 colonies for each organ per mice) were tested for markers (auxotrophic or dominant). Two to four colonies from each organ were randomly chosen, and ploidy was analyzed by fluorescent flow cytometry. To examine increases in ploidy in vivo, mice were infected with KN99α as described above. At 21 days postinfection the animals were sacrificed, and 96 colonies from the lungs and brain were analyzed by FACS as described above. The animal use in this research was in compliance with all relevant federal guidelines and institutional policies.

TABLE 4.

P values for the virulence studies

Strain	Parent 1		Parent 2		Ploidy control
	Strain	P value	Strain	P value	Strain	P value
XL1552 (αADa)	KN99αNEO1 (Aα)	0.0010	XL465 (Da)	0.0001	XL1501 (αAAα)	0.7394
XL1548 (aADα)	YSB121 (Aa)	0.0340	XL467 (Dα)	0.0001	XL1501 (αAAα)	0.5128
XL1462 (αADα)	F99 (Aα)	0.0115a	XL342 (Dα)	0.0001a	XL1501 (αAAα)	0.4813
XL1495 (aADa)	JF99 (Aa)	0.0001a	JEC30 (Da)	1.000a	KN4B7#16 (aAAa)	0.0002
XL1511 (aADa)	KN99aNEO (Aa)	0.0004	XL465 (Da)	0.0001	KN4B7#16 (aAAa)	0.0002
XL1514 (aADa)	YSB121 (Aa)	0.0010	XL465 (Da)	0.0001	KN4B7#16 (aAAa)	0.0002
XL1500 (αAAα)	KN99αNAT13 (Aα)	0.0001	KN99αNEO1 (Aα)	0.2480
XL1501 (αAAα)	YSB119 (Aα)	0.0021	KN99αNEO1 (Aα)	0.0355
XL1501 (αAAα)	KN99α (Aα)	0.0013b	KN99a (Aa)	0.0013b
KN4B7#16 (aAAa)	KN99a (Aa)	0.7128
XL143 (αDDα)	XL141 (Dα)	0.4206
XL374 (aDDa)	XL370 (Da)	0.0952

^aBecause parental strains are auxotrophic, the virulence of strains was compared to the virulence of isogenic prototrophic strains.

^bStrain XL1501 was compared to the unmarked isogenic haploid strain KN99α and the unmarked congenic strain KN99a.

Murine tail vein injection model of cryptococcosis.

Animals were infected essentially as previously described (58). Six- to 8-week-old female DBA mice were inoculated with 1 × 10⁶ fungal cells in 50 μl of PBS in the lateral tail vein. The sizes of the yeast cell inocula were confirmed by determining the numbers of CFU after serial dilution. After inoculation of fungal cells, animals were monitored twice daily, and the animals showing signs of severe morbidity (weight loss, extension of the cerebral portion of the cranium, abnormal gait, paralysis, seizures, convulsions, or coma) were sacrificed by CO₂ inhalation. The survival rates of the animals were plotted against time, and P values were calculated with the Mann-Whitney U test (see Table 4). The experiment was terminated at day 178. The statistical results did not change if it was assumed that the animals surviving at the termination of the experiment survived for 1,000 days.

Comparative genome hybridization and data analysis.

An experiment was performed as previously described (1). Basically, genomic DNA was sonicated to generate ∼500-bp fragments and purified with a DNA Clean and Concentrator kit (Zymo Research, California). Five micrograms of DNA was used for Cy-3 dUTP or Cy-5 dUTP labeling reactions using the random primer/reaction buffer mixture (Invitrogen BioPrime array comparative genome hybridization [CGH] genomic labeling system). Labeled DNA from the sample and the control was competitively hybridized to microarray slides containing 70-mer oligonucleotides for the C. neoformans JEC21 whole genome and serotype- and mating type-specific genes in the MAT locus (46). After hybridization, the arrays were scanned with a GenePix 4000B scanner (Axon Instruments, Foster City, CA) and analyzed using GenePix Pro v 4.0 and BRB array tools (developed by Richard Simon and Amy Peng Lam at the National Cancer Institute; http://linus.nci.nih.gov/BRB-ArrayTools.html).

RESULTS

Generation of isogenic αAAα and AD hybrids with all possible mating type combinations.

To study the effects of ploidy, isogenic haploid and diploid strains were isolated and compared. Although isogenic α/α and a/a diploid serotype D strains in the JEC21 and B3502 background were generated in previous studies (30, 32, 47), this background is considerably attenuated in virulence and not ideal for pathogenicity studies. Because serotype A is highly virulent and the most prevalent serotype in clinical isolates, it is important to study ploidy effects on virulence in this genetic background. To generate αAAα diploid strains in the H99 background, two Aα strains harboring either the NAT or NEO dominant drug resistance marker were cocultured together with a counterselectable a pheromone donor on mating medium. The desired αAAα diploid fusion products were isolated and validated by FACS analysis and PCR amplification of serotype- and mating type-specific genes (Fig. 1A) (see Materials and Methods).

FIG. 1.

Scheme for isolation of isogenic diploid and AD hybrid strains. (A) Two Aα strains marked with the NAT and NEO dominant drug resistance markers were cocultured with an auxotrophic Da pheromone donor strain. Doubly drug-resistant strains (diploid αAAα and triploid Da/Aα/Aα) were selected on YPD medium containing NAT and NEO. (B) Two strains (Aa and Dα or Aα and Da) marked with NAT and NEO resistance markers were cocultured on mating medium. Doubly drug-resistant strains (hybrid aADα or αADa) were selected on YPD+NAT+NEO medium. (C) Auxotrophic Aa and Da strains requiring different nutritional supplements were cocultured with an auxotrophic pheromone donor Dα strain. Prototrophic strains (diploid aADa hybrid and triploid Aa/Da/Dα) were selected on YNB minimal medium. Alternatively, dominantly marked Aa and Da strains were cultured together with a pheromone donor Dα strain. Doubly resistant strains (diploid aADa hybrid and triploid Aa/Da/Dα) were selected on YPD+NAT+NEO medium. The outer ellipses represent cells, and the inner ellipses represent nuclei. a and α indicate the mating type. Different shades of nuclei indicate different serotypes.

To study the contribution of interactions between mating type and serotype to the virulence of AD hybrids, AD hybrids were constructed based on the H99 (haploid Aα) and JEC21 (haploid Dα) backgrounds (see Materials and Methods). H99 and JEC21 are well characterized, with completed genome sequences (http://cneo.genetics.duke.edu/; http://www.broad.mit.edu/annotation/genome/cryptococcus_neoformans/Home.html) and readily available congenic strain pairs, and both of these strains are widely used for genetic and pathogenesis studies (26, 41, 52, 57). Here, AD hybrids with all possible mating type combinations, including the common genotypes aADα and αADa, and hybrids with only one mating type, αADα (48) or aADa, were generated (Fig. 1B and 1C). All of the desired products were genotyped using mating type- and serotype-specific PCR, and ploidy was assessed by FACS analysis (data not shown). To ensure that the phenotypes of the resulting hybrid strains were not affected by differences in the markers of the parental strains, AD hybrids of some genotypes were obtained by independent fusion procedures (Fig. 1 and Table 1) (see Materials and Methods). Strains generated by different fusion procedures behaved similarly in vitro (Table 1) (X. Lin and J. Heitman, unpublished observations).

Higher ploidy is associated with increased cell size in C. neoformans.

An association between higher ploidy and larger cell size has been observed in other fungal species (23, 29, 62). To establish whether this also occurs in C. neoformans, laboratory-constructed diploid strains were observed microscopically. Diploid cells (either diploid αAAα or diploid AD hybrids) appeared to be larger than haploid cells of Aα H99 or Dα JEC21 strains (Fig. 2A and C). Because cell size is heterogeneous in a population, scattered forward flow cytometry was also performed on 10,000 cells to determine the cell size of these populations. The scattered forward FACS profiles of haploids and diploids also indicated that the cells of diploid strains were larger than the cells of haploid controls (Fig. 2B), consistent with microscopic observations. To further confirm this association and to avoid variations caused by differences in developmental stages, cell size was directly determined by measuring the diameters of mother yeast cells from photographs (Fig. 2D). Again, the results showed that diploid cells were larger than haploid cells. Increased cell size was also observed for other diploid AD hybrids (data not shown). These findings are in accord with a previous study reporting cell size (diameter) variation among haploid Aα H99 and Da NIH430 and eight natural αADa strains (14, 66).

FIG. 2.

Higher ploidy is associated with larger cell size in C. neoformans. Haploid JEC21 (Dα) and H99 (Aα) and diploid XL1500 (αAAα) and XL1462 (αADα) cells were grown overnight in YNB medium. Cells were observed by light microscopy (A), and the diameters of mother yeast cells (43 to 50 cells per genotype) were measured with Photoshop using its measurement tool (D). Cells were also analyzed by flow cytometry using the forward scatter channel (B). For ploidy analysis, cells were processed with propidium iodide staining to examine the cellular DNA content by FACS (C). For the forward scatter flow cytometry profile, the x axis indicates the forward scatter area, which is positively correlated with cell size, and the y axis indicates the cell count. For the fluorescence flow cytometry profile, the x axis indicates the fluorescence intensity, which is positively correlated with the DNA content, and the y axis indicates the cell count. Scale bar, 10 μm.

Laboratory-constructed AD hybrids exhibit hybrid vigor in vitro.

Laboratory-generated hybrids and diploids were tested in vitro for several well-established C. neoformans virulence factors. Here, we tested three classic C. neoformans virulence factors (melanization, capsule production, and the ability to grow at high temperature) and also resistance to UV irradiation.

In vitro virulence attributes of the laboratory-constructed diploid and hybrid strains were compared to attributes of the haploid control strains belonging to both serotype A and serotype D. All isolates, including haploid, diploid, and AD hybrid isolates, produced capsule based on microscopic observations (data not shown). The serotype A haploid strains (Aα and Aa) were more resistant to UV irradiation than the serotype D haploid strains (Dα and Da) (Fig. 3). Diploid isolates (αAAα and αDDα) exhibited slightly increased UV resistance compared to the haploid strains (Aα and Dα). AD hybrid strains were modestly more resistant to UV irradiation, which was indicative of hybrid vigor. This finding is consistent with a previous observation of hybrid vigor for UV resistance of laboratory-generated αADα hybrids (48).

FIG. 3.

In vitro assay of survival and virulence traits. Yeast cells of C. neoformans strains (haploid Aα, Aa, Dα, and Da strains and diploid αAAα, αDDα, αADα, αADa, aADα, and aADa hybrids) were grown in liquid YPD medium overnight and washed three times with distilled water. Cell concentration was determined by determining the optical density at 600 nm. Three-microliter serial dilutions (10-fold) of cells were spotted onto media for phenotypic characterization. Cells were grown on YPD medium at 22°C for 3 days as a control for growth (first column from the left); cells on YPD medium were subjected to UV irradiation for 12 s (∼48 mJ/cm²) and then incubated at 22°C for 3 days (second column); cells were grown on YPD medium at 39°C for 3 days (third column); cells were grown on medium containing l-DOPA at 22°C for 6 days (fourth column); and cells were grown on medium containing l-DOPA at 37°C for 6 days (fifth column).

The serotype A haploid strains also grew better at 39°C than the serotype D haploid strains (Fig. 3). Again, all AD hybrid strains grew significantly better at 39°C than either serotype A or D haploid control strains. The enhanced growth of AD hybrid cells at 39°C was due to hybrid fitness instead of higher ploidy as isogenic diploid strains (αAAα or αDDα) showed modestly poorer growth at 39°C than their parental haploid strains (Aα or Dα) (Fig. 3).

The observation that higher ploidy exerted a negative effect on growth at high temperature while hybridization between serotype A and D strains conferred hybrid vigor was further established by performing growth competition assays at 39°C. Diploid αAAα strain XL1501, aADa hybrid strains XL1511 and XL1514, and αADa hybrid strain XL1552 were coinoculated with their respective serotype A parental strains in liquid YPD medium and incubated at 39°C with shaking. Because serotype D strains grow poorly at this higher temperature, they were not included in the competition assay. Aliquots of each culture were taken at the indicated times and plated on different media to calculate the ratios of haploid cells to diploid cells. As shown in Fig. 4A, the diploid αAAα strain XL1501 was outcompeted by both of its haploid parental Aα strains (KN99αNEO and YSB119), which was indicative of a negative effect of higher ploidy on growth at this temperature. In contrast, the AD diploid hybrids tested outcompeted their haploid serotype A parental strains (Aα and Aa) when they were cultured together at 39°C, which was indicative of hybrid vigor at this higher temperature (Fig. 4B). However, at a lower temperature, 22°C, diploid AD hybrids were less competitive than their haploid serotype A parental strains in coculture (see Fig. S1 in the supplemental material). These observations indicate the specificity of hybrid vigor at a higher temperature but a negative effect at a lower temperature. Therefore, higher ploidy per se can negatively affect growth, while hybridization offers growth advantages to diploid AD hybrid isolates at a higher temperature but is a disadvantage at a lower temperature.

FIG. 4.

Hybrids exhibit hybrid vigor, while diploidy per se has a negative effect on growth at 39°C. Paired strains were cocultured in YPD liquid medium at 39°C. The ratios of haploid serotype A parental strains to the diploid αAAα strain or AD hybrid strains at 0, 24, and 48 h are shown for one representative experiment. The experiment was repeated with similar results (not shown). (A) Isogenic αAAα diploid strain XL1501 competing with the haploid parental Aα strain KN99αNEO or YSB119. (B) AD diploid hybrid strain XL1511 (aADa), XL1514 (aADa), or XL1552 (αADa) competing with the haploid Aα or Aa parent strain.

C. neoformans produces melanin by oxidizing diphenolic substrates, including the neurotransmitter l-DOPA (10), resulting in the formation of darkly pigmented yeast colonies. At 22°C, the haploid Aα and Aa strains, the diploid αAAα strain, and all of the AD hybrids produced significant amounts of melanin compared to serotype D haploid and diploid strains (Dα, Da, and αDDα) (Fig. 3). At 37°C, however, the melanization of the AD hybrids was drastically reduced and was more comparable to that of the less melanized serotype D strains (Fig. 3). Although higher ploidy appears to have a slightly negative effect on melanization (the production of melanin by the diploid αAAα strain was modestly reduced compared to the production of melanin by the haploid Aα and Aa strains), the reduction in melanization was much more dramatic in all of the AD hybrids, indicating that the reduced melanization of AD hybrids at a higher temperature was largely caused by hybridization rather than by increased ploidy (Fig. 3). This observation indicates that there is a complicated interaction of different virulence attributes (temperature and melanization) in the hybrids.

In conclusion, serotype A strains were more fit than serotype D strains in these in vitro assays, and mating type α or a did not appear to affect their phenotypes. Higher ploidy alone has modest negative effects on melanization and growth. AD hybrid strains displayed hybrid vigor for some virulence traits, such as resistance to UV irradiation and tolerance to high temperature, whereas the effect of hybridization on melanization was temperature dependent. Differences in the mating type combinations in these AD hybrids did not affect their in vitro phenotypes.

Ploidy of C. neoformans is stable during infection.

In the obligate commensal and opportunistic pathogenic fungus C. albicans, ploidy alteration can occur during infection, with tetraploids reduced to diploid/aneuploid, the normal ploidy of C. albicans (33). Whether the ploidy of C. neoformans is stable during cryptococcal infection was not known.

To determine if any ploidy increase occurs during cryptococcal infection, we examined the ploidy of fungal cells recovered from the lungs (the initial site of infection) and brains (lethal infection) of mice that were inoculated intranasally with the haploid Aα strain H99. Ninety-six colonies were randomly chosen from fungal cells recovered from lungs and brains (a total of 192 colonies) and analyzed by fluorescence flow cytometry to determine ploidy. All 192 colonies tested remained haploid regardless of the recovery organ (data not shown), indicating that increases in ploidy either do not occur in vivo or are below the limit of detection (<0.5%). These observations indicate that host conditions do not stimulate efficient diploidization of C. neoformans.

Conversely, to determine if ploidy reduction occurs during cryptococcal infection, animals were infected with diploid αAAα or AD hybrid strains, and the ploidy of recovered fungal isolates following infection was examined (see Materials and Methods). As shown in Tables 2 and 3, the ploidy of fungal cells recovered from infected lungs and brains was the same as the original ploidy of the strain used for inoculation. Thus, diploid isolates are also stable under host conditions. It is interesting to note, however, that dominant markers in two of the diploid strains (XL1500 and XL1511) could be lost in vivo. A similar loss of the dominant marker was also observed in one haploid parental strain when cultured in vitro without selective pressure (such as in YPD medium), indicating that marker loss was not due to positive selection in vivo and did not affect ploidy (Table 2).

TABLE 2.

Ploidy is stable during cryptococcal infection: strain characteristics at inoculation

Strain	Genotype and/or phenotype	Marker test						Ploidy
		% of colonies					n
		Medium with NAT	Medium with NEO	SC-ade	SC-ura	SC-lys		% of diploid colonies	% of haploid colonies	na
XL1511	aADa NEOr NATr	100	80				200	100b		8
XL1514	aADa NEOr NATr	100	100				150	100		3
XL1501	αAAα NEOr NATr	100	100				200	100		3
XL1500	αAAα NEOr NATr	100	100				100	100		3
XL1548	aADα NEOr NATr	100	100				300	100		4
XL1552	αADa NEOr NATr	100	100				300	100		4
YSB121	Aa NEOr	0	100				150		100	2
KN99αNEO1	Aα NEOr	0	100				200		100	2
KN99αNAT13	Aα NATr	100	0				50		100	2
YSB119	Aα NATr	100	0				150		100	2
XL1462	αADα ade2/ADE2 ura5/URA5			100	100		300	100		4
XL1495	aADaura5/URA5 lys1/LYS1				100	100	300	100		4

^aNumber of colonies that were randomly chosen for FACS analyses.

^bFour colonies with the wild-type phenotype (NEO^r NAT^r) tested were all diploid, and four colonies that had lost the NEO^r marker tested were also all diploid.

TABLE 3.

Characterization of fungal cells recovered after infection

Strain	Genotype and/or phenotype	Organ (mouse)	Marker test			Ploidy
			% of colonies		n
			Medium with NAT	Medium with NEO		% of diploid colonies	na
XL1511	aADa NEOr	Brain (m1)	100	0	50	100	2
	NATr	Brain (m2)	100	0	50	100	2
		Lung (m1)	100	2	50	100	3
		Lung (m2)	100	4	50	100	4
XL1514	aADa, NEOr	Brain (m1)	100	100	50	100	2
	NATr	Brain (m2)	100	100	50	100	2
		Lung (m1)	100	100	50	100	2
		Lung (m2)	100	100	50	100	2
XL1501	αAAα NEOr	Brain (m1)	100	100	48	100	2
	NATr	Brain (m2)	100	100	50	100	2
		Lung (m1)	100	100	50	100	2
		Lung (m2)	100	100	50	100	2
XL1500	αAAα NEOr	Brain (m1)	98	100	50	100	3
	NATr	Brain (m2)	100	100	50	100	2
		Lung (m1)	100	100	50	100	2
		Lung (m2)	98	100	50	100	3
XL1548	aADα NEOr	Brain (m1)	100	100	50	100	2
	NATr	Brain (m2)	100	100	50	100	2
		Lung (m1)	100	100	50	100	2
		Lung (m2)	100	100	50	100	2
XL1552	αAda NEOr	Brain (m1)	100	100	50	100	2
	NATr	Brain (m2)	100	100	50	100	2
		Lung (m1)	100	100	50	100	2
		Lung (m2)	100	100	50	100	2

^aTwo colonies of fungal cells recovered from each organ were randomly chosen for FACS analysis. When there were colonies that underwent marker loss, two colonies with marker loss together with two randomly chosen colonies that retained all of the markers were tested by FACS. When there was only one colony tested that underwent marker loss, three fungal colonies were tested for ploidy. A large fraction of the cells recovered from mice inoculated with strain XL1511 lost the dominant NEO^r marker, which did not appear to affect ploidy as hybrid cells that lost this marker were still diploid (see Table 2). It was noted that the marker in the parental haploid strain KN99aNEO1 is also unstable, which may explain the instability of this marker in the hybrid strain derived from it (unpublished data). Some of the AD hybrids were generated through fusion of two auxotrophic haploid strains (XL1462 and XL1495), and so the maintenance of diploidy could potentially be caused by positive selection as haploids may become auxotrophic and thus less competitive in nutrient-limiting conditions, such as the host environment. Thus, strains derived from auxotrophic parental strains were excluded, and only strains derived from parental strains with dominant markers were included.

Higher ploidy exerts a minor negative effect on virulence in a murine inhalation model of cryptococcosis.

To assay the impact of ploidy on virulence, animals were intranasally infected with haploid parental Aα strains and the constructed diploid αAAα strains. Animal survival was monitored and plotted against time. The diploid αAAα strain XL1500 was modestly less virulent than its parental strain KN99αNAT13, but the decreased virulence compared to the other parental strain, KN99αNEO1, was not statistically significant (Fig. 5A and Table 4). The other diploid αAAα isolate, XL1501, was also modestly less virulent than its parental strains, and the differences were statistically significant (Fig. 5B and Table 4). Because the marked haploid parental strains displayed a modest difference in virulence (KN99αNAT and YSB119 were more virulent than KN99αNEO1 [Fig. 5A and B]), this could have resulted from the insertion of dominant markers. Because the diploid strains derived from these haploids retain a wild-type copy of the insertion site in the genome, any effect of the insertion of the dominant markers on the virulence of diploids is likely to be minimal. Therefore, comparing the diploids to the unmarked isogenic parental haploid strains provides a more robust comparison for ascertaining the possible effects of ploidy on virulence. Thus, the virulence of diploid strain XL1501 was compared with the virulence of the unmarked congenic haploid strains KN99α and KN99a. As shown in Fig. 5C, the diploid αAAα strain XL1501 was modestly but significantly less virulent than the unmarked haploid strains, indicating that higher ploidy exerts a minor negative effect on virulence in the murine inhalation model of cryptococcosis.

FIG. 5.

Higher ploidy exerts a negative effect on virulence in the murine inhalation model. Cells were grown in liquid YPD medium overnight at 30°C and washed three times with PBS. (A to C) Animals were infected intranasally with 5 × 10⁴ fungal cells and monitored for 40 days. (D) Animals were infected intravenously with 1 × 10⁶ fungal cells and monitored for 178 days. Animal survival was plotted against time after inoculation. P values are shown in Table 4.

Because serotype D strains in the JEC21 background (Dα and Da strains) were avirulent in this model under the same conditions (48; data not shown), the effect of ploidy on virulence in serotype D strains was tested using the intravenous murine cryptococcosis model, where fungal cells were injected directly into the lateral tail vein. Although the onset of illness was later in animals infected with the diploid αDDα strain, the virulence of the αDDα strain XL143 was similar to the virulence of the haploid Dα strain (Fig. 5D). This could have been due to the difference in the routes of infection or to differences between the two divergent serotypes (see Discussion).

All AD hybrids are highly virulent, with the exception of aADa hybrid isolates.

To assay the virulence potential of AD hybrids in the murine inhalation model, animals were intranasally infected with haploid parental serotype A strains and the constructed AD hybrid strains. Dα and Da strains examined under the same conditions were found to be avirulent in this model (48; data not shown) and were not included. Animal survival was monitored and plotted against time. The AD hybrid strains with both the a and α mating types present (αADa and aADα) were highly virulent. Both αADa and aADα hybrids were slightly less virulent than the parental haploid Aα or Aa strains (Fig. 6A and B and Table 4). The slightly lower virulence of these AD hybrids than of the haploid parental strains was most likely due to higher ploidy and not to hybridization. Because their virulence is comparable to that of diploid αAAα strains (Table 4), the presence of opposite mating types, a and α, does not affect the virulence potential of the AD hybrid strains, and hybridization between highly virulent serotype A and avirulent serotype D strains results in an AD hybrid strain with virulence potential close to that of the haploid serotype A parental strains.

FIG. 6.

aADα and αADa hybrids are highly virulent, whereas aADa hybrids are less virulent. Cells were grown in liquid YPD medium overnight at 30°C and washed three times with PBS. Animals were infected intranasally with 5 × 10⁴ fungal cells and monitored for 40 days. Survival was plotted against time after inoculation. P values are shown in Table 4.

To examine the virulence potential of AD hybrid strains in which only a single mating type is present, animals were also infected with αADα and aADa strains. As previously reported, the αADα strain was highly virulent (48) and exhibited a level of pathogenicity similar to that of αAAα diploid and other AD hybrid strains (Fig. 6C). Surprisingly, this was not the case for the aADa strains. Instead, all three aADa strains behaved differently in vivo, and the virulence of all these strains was considerably and statistically significantly lower than the virulence of other AD hybrids and the diploid αAAα strains (Fig. 6C). One aADa strain (XL1495) was derived from auxotrophic parental strains (the diploid hybrid is prototrophic), whereas two other aADa strains (XL1511 and XL1514) were generated from two separate fusion events using parental strains with different dominant markers. As each aADa strain was derived from an independent fusion event and the in vitro phenotypes of the strains are similar to the phenotypes of other AD hybrids, the reduced virulence in vivo of aADa strains was not likely caused by the differences in the markers of the parental strains. The aADa strains grew well at high temperature in vitro compared to Aa parental strains and other AD hybrids (Fig. 3 and 4), suggesting that the lower virulence of these strains is not due to impaired growth.

Although their virulence varied, the fact that all three aADa strains exhibited reduced virulence suggests that the lower virulence potential was likely due to the presence of the Aa and Da mating types. There have been no previous studies of aADa hybrids because no natural or laboratory-constructed aADa hybrids have been reported thus far. Our observations indicate that the presence of either the Aa or Da mating type alone in AD hybrids does not impair pathogenicity because aADα and αADa hybrids were as virulent as the diploid αAAα strain. Two hypotheses may explain the lower virulence potential of aADa strains: either the presence of two copies of the a mating type (regardless of the serotype origin) reduces the full virulence potential of the diploid strain (copy number model), or specific interactions between the Aa and Da mating type alleles interfere with the full expression of virulence during animal infection (e.g., there are antagonistic or negative genetic interactions between the two loci) (negative epistasis model).

The diploid aAAa isolate is highly virulent.

To distinguish between the two models for the lower virulence potential of aADa hybrids (copy number model and negative epistasis model), it would be ideal to compare the virulence of aAAa diploid strains to that of aADa hybrid strains. If the reduced virulence of aADa hybrid strains is caused by the presence of two copies of the a mating type (copy number model), then a similar reduction in virulence for aAAa diploid strains would be predicted. If negative epistasis exists between Aa and Da alleles, then aAAa diploid strains would exhibit a level of pathogenicity similar to that of other diploids and hybrids.

Although attempts to construct an aAAa diploid strain by fusion of two marked Aa strains in the H99 background have been unsuccessful thus far, serotype A MATa strain KN4B7#16 (based on serotype- and mating type-specific PCR of SXI1/2 and STE20 genes in the MAT locus) was found to be a diploid by FACS analysis (X. Lin, S. Patel, A. Litvintseva, A. Floyd, R. Hicks, T. G. Mitchell, and J. Heitman, unpublished data). This strain is estimated to be ∼99.61% genetically identical to the KN99a reference strain based on the fact that it was isolated as one of the eighth-backcross progeny generated during construction of a congenic pair (KN99α and KN99a) in the H99 background (57). This strain displays typical isogenic diploid phenotypes in vitro similar to those of αAAα strains (Lin et al., unpublished data).

To establish that KN4B7#16 is indeed an aAAa diploid strain and not an unrecognized heterozygous αAAa strain or an abnormal AAa strain with only one mating type locus allele, the gene content of the mating type locus of this strain was analyzed by CGH. Genomic DNA of strain KN4B7#16 and a control (either KN99a [Aa] or H99 [Aα]) were labeled with fluorescent dyes and competitively hybridized to a genomic microarray slide that contained mating type- and serotype-specific 70-mers for the MAT locus and 70-mers for all open reading frames in the serotype D JEC21 genome. To confirm that KN4B7#16 is a MATa strain, the intensity of hybridization to MATα and MATa genes was analyzed. Hybridization was strong for all MATa genes and weak for the vast majority of MATα genes (Fig. 7A). Cross-hybridization between a and α alleles for highly conserved genes, such as RPO41, BSP2, and ETF1, was also observed, and cross-hybridization between a and α alleles of these genes was also documented in another study (Lin et al., unpublished data). The log₂ ratio of fluorescence intensity between KN4B7#16 and control strain KN99a (Aa) for all serotype A MATa genes and MATα genes was close to 0 (the average log₂ ratios of fluorescence intensity ranged from −0.533 to ∼0.330, meaning that the differences between the sample and control ranged from 0.7-fold to ∼1.26-fold [Fig. 7B]), indicating that the genetic contents of the control and sample are similar at the mating type locus. Similarly, only minor variations between the sample and the Aa control in non-MAT genomic regions were observed (data not shown). As a diploid, KN4B7#16 has twice the genomic DNA content per cell as the haploid strain KN99a, and the CGH analysis also indicated that KN4B7#16 has twice the genetic content at the mating type locus. These observations indicate that KN4B7#16 contains two copies of MATa alleles. Because there were only a genes and no α genes in the control, this result also indicates that KN4B7#16 contains only a genes, consistent with the PCR analysis.

FIG. 7.

Comparative genome hybridization of the mating type locus of the diploid aAAa strain KN4B7#16. Fragmented genomic DNA from the sample isolate KN4B7#16 and genomic DNA from control strains H99 (Aα) and KN99a (Aa) were labeled with fluorescent dyes and competitively hybridized to a 70-mer array. (A) Average fluorescence intensity for the KN4B7#16 sample for the serotype A MAT locus a and α alleles. (B) Average log₂ ratio of fluorescence intensity for the sample and the control for the serotype A a and α MAT locus alleles. The fluorescence signal level was normalized across the genome, and hybridization was repeated three times for KN4B7#16/KN99a and twice for KN4B7#16/H99. (C) Schematic representation of the serotype A MATa locus (22). Blue indicates intergenic regions, and yellow indicates highly conserved genes.

To ensure that the lack of hybridization to Aα probes was not due to a failure of the Aα 70-mers on the microarray slides, hybridization of KN4B7#16 with H99 (Aα) was also performed. As expected, the levels of hybridization of fluorescence-labeled KN4B7#16 genomic DNA to all Aa alleles were higher (most log₂ ratios of fluorescence intensity were >2, meaning that the KN4B7#16/Aα ratio was >4), and the levels of hybridization to all Aα alleles were lower (most log₂ ratios of fluorescence intensity were less than −2, meaning that the KN4B7#16/Aα ratio was <0.25) (Fig. 7B), indicating that strain KN4B7#16 contains opposite mating type genes compared to the control Aα strain H99. This result supports the conclusion that strain KN4B7#16 is a diploid aAAa isolate containing two copies of the Aa allele and no Aα allele.

To compare the virulence potential of aAAa and aADa hybrids in the murine inhalation model, animals were intranasally infected with the aAAa strain KN4B7#16 and the aADa hybrid XL1511. The Aa (KN99a), Aα (KN99α), and diploid αAAα (XL1501) strains were also included for comparison. Animal survival was monitored and plotted against time. The congenic haploid Aα and Aa reference strains were similarly virulent, consistent with previous studies (57), and they were also the most virulent strains studied (Fig. 8A). The aADa hybrid (XL1511) was the least virulent strain, as observed earlier in this study. The diploid αAAα strain (XL1501) was slightly less virulent than either haploid strain, consistent with earlier observations in this study. The diploid aAAa strain KN4B7#16 was almost as virulent as the haploid controls and was much more virulent than the aADa hybrid (Fig. 8A).

FIG. 8.

The presence of two MATa alleles does not reduce the virulence potential. Cells were grown in liquid YPD medium overnight at 30°C and washed three times with PBS. (A) Animals were infected intranasally with 5 × 10⁴ fungal cells of serotype A strains (KN4B7#16 [aAAa], KN99a [Aa], XL1501 [αAAα], KN99α [Aα], XL1511 [aADa]) and monitored for 40 days. (B) Animals were infected intravenously with 1 × 10⁶ fungal cells of serotype D strains (XL370 [Da] and XL374 [aDDa]) and monitored for 178 days. Survival was plotted against time after inoculation. P values are shown in Table 4.

The virulence of the diploid aDDa strain XL374 was also tested and compared to that of the isogenic haploid Da strain XL370 in an intravenous murine cryptococcosis model. Similar to what was observed for serotype A strains, the diploid aDDa strain displayed the same pathogenicity level as the haploid Da strain (Fig. 8B). Together, these observations indicate that the presence of two copies of the MATa allele does not reduce the virulence potential of diploid strains. Thus, specific interactions between the Aa and Da alleles are hypothesized to be responsible for the reduced virulence potential of aADa hybrids, supporting the negative epistasis model.

These in vivo assays indicate that the haploid serotype A strains are the most virulent strains regardless of their mating type. Diploid strains, including isogenic αAAα and aAAa strains and most AD hybrids (aADα, αADa, and αADα), are slightly less virulent but still highly virulent. The aADa strains are significantly less virulent than other AD hybrids, indicating that a detrimental effect is associated with the presence of both the Aa and Da mating type alleles in the same strain. All strains tested are much more virulent than serotype D strains, indicating that the serotype D strains benefit from hybridization with a serotype A partner.

DISCUSSION

To study the effect of ploidy on virulence, laboratory-constructed αAAα diploids and AD hybrids with all possible combinations of mating types in defined serotype A and D backgrounds were generated. In vitro assays indicated that all of these diploid AD hybrids exhibit similar phenotypes regardless of the mating type combination. Hybrids exhibit hybrid vigor and are more resistant to UV and high temperature. However, they are less melanized at high temperature than an isogenic diploid strain or the serotype A parental strain. Higher ploidy per se, however, appears to have a minor negative effect on the fitness of strains as diploid cells in general melanize less well and are modestly less tolerant at a higher temperature (39°C). Increased ploidy does confer modest resistance to UV irradiation.

The in vivo assay of virulence showed that the diploid serotype A strain and AD hybrids are slightly less virulent than haploid serotype A strains, suggesting that higher ploidy may modestly reduce virulence in the host. Toffaletti et al. found that AD hybrids are at least as virulent as, or even more virulent than, haploid serotype A parental strains in the rabbit model of cryptococcal meningitis (70), whereas other workers have found that AD hybrids are attenuated to different extents (3, 14, 42). Two factors might explain the difference between these two observations. First, the body temperature of rabbits is higher than that of mice (39°C instead of 37°C), and hybrid vigor was observed at 39°C, which may explain the competitiveness (better growth) of hybrids over the haploid serotype A strain in the rabbit central nervous system model. Second, fungal cells were inoculated intranasally or intravenously in murine models of cryptococcosis, while fungal cells were inoculated intracisternally in the rabbit model of cryptococcal meningitis (70). The different routes of infection could contribute to the differences in the observed virulence of the AD hybrids. Hybridization between serotype D and A strains results in laboratory AD hybrid strain virulence potential that is close to that of the haploid serotype A parental strains. This observation, however, is different from some previous observations, where the virulence of natural aADα strains was found to be much lower (3, 42). The difference is most likely due to the different genetic backgrounds of natural isolates that originated independently and in which there was considerable mitotic expansion in nature (79).

Our study and a previous study (14) showed that diploid cells are larger than haploid cells. It is conceivable that the larger cell size could impede penetration and infection of the lungs by diploid fungal cells, where the fungus establishes an initial infection. This could result in a smaller effective inoculum for diploid cells and eventually lead to the slightly lower virulence observed for isogenic diploid αAAα cells and AD hybrids in the inhalation model of cryptococcosis. While cell size may be an important factor in the inhalation model of host infection, it may be less of a factor in the intracisternal or tail vein model. The observation that the virulence of serotype D diploids was similar to the virulence of serotype D haploids in a tail vein model supports this hypothesis.

The role of mating type in the virulence of pathogenic fungi is being actively investigated. Recent studies with the diploid fungus C. albicans indicate that homozygosis of the MTL locus in natural diploid a/α strains, which produces diploid a/a or α/α strains, can result in reduced virulence and competitiveness in the host (51, 78). Specific mating type locus gene deletions (a1, α2, or the entire MATa or MATα locus) result in modest reductions in virulence compared to the parental a/α strains (78). The a1-α2 heterodimer that controls cell identity is proposed to confer competitiveness to the a/α heterozygous diploid strains over a/a or α/α homozygous strains (51, 78).

The mating type locus of C. neoformans is far more complex than that of C. albicans and contains more than 20 genes, including genes encoding not only homeodomain sexual development regulators but also pheromones, pheromone receptors, and pheromone response pathway elements (22, 43). Although the sexual regulator Sxi1α does not contribute to the virulence of C. neoformans (30, 31), previous studies have suggested that pheromone production and sensing may occur during infection (17, 21, 56, 74). It is possible that when both mating types are present in a single AD hybrid strain, some portion of the cells could behave as a cells, while other cells behave as α cells, mimicking coinfection with α and a cells. This could occur in a population with the same genotype through differential transcriptional regulation of cell identity genes that could potentially influence cellular behavior. Thus, an AD hybrid bearing both the α and a mating types could contain some cells expressing α genes while other cells express a genes stochastically. The level of pathogenicity of αADa and aADα hybrids bearing both mating types that is similar to the level of pathogenicity of an αADα hybrid harboring only the α mating type suggests that a coinfectionlike situation may not occur with AD hybrids or that, if it does occur, it does not affect the survival of the animals. The reduced pathogenicity of aADa hybrids compared to other AD hybrids does suggest that mating type has a role in virulence. Either the presence of the α mating type confers an advantage in virulence, or the a mating type plays a negative role. Given that aADα and αADa hybrid strains that contain one copy of the a mating type are as virulent as αAAα diploid and αADα hybrid strains, the presence of an a mating type allele does not appear to have a negative impact. This hypothesis is supported by the observation that the virulence of the aAAa and aDDa strains is similar to that of their haploid counterparts.

The serotype origin of mating types (for example, α is from serotype A in the αADa hybrid, whereas α is from serotype D in the aADα hybrid) has been suggested in previous studies to contribute to the difference in virulence between natural αADa and aADα hybrids (3, 14), with the α allele from serotype A associated with higher virulence potential in AD hybrids (3). If the serotype origin of a specific mating type indeed affects virulence significantly, this should be manifested as a difference in virulence between αADa and aADα strains. However, as we showed in this study and also demonstrated in a previous study using AD hybrids in the H99 and JEC21 backgrounds (70), αADa and aADα hybrids in congenic genetic backgrounds are similarly virulent, indicating that the serotype origin of the α mating type does not affect virulence significantly.

Genetic background can determine whether a difference in virulence between congenic a and α cells is observed (41, 56-58), and thus interactions between mating type and other genomic regions affect virulence in a serotype- or genotype-specific manner. Functional studies of homologues of mating type genes in the serotype A H99 and D JEC21 backgrounds also suggest that serotype- or genotype-specific interactions of the mating type locus with other genomic regions impact virulence (11, 80). For example, Ste20α, the PAK kinase in the mating response pathway, is essential for full virulence of serotype A strains but not of serotype D strains (75). This effect is likely attributable to the fact that ste20 mutants are temperature sensitive, but only in the serotype A background (55, 75). Interestingly, the STE20α gene from a serotype D strain complements a serotype A ste20α mutant not only for the mating defect but also for the temperature-sensitive growth defect, and the wild-type serotype A STE20α allele restores mating in serotype D ste20α mutants (55). These findings suggest that homologues encoded by the mating type locus may have similar functions even though they have different serotype origins, but physiological actions (phenotypes) can differ significantly depending on the genotype of other genomic regions. In this study, the AD hybrids had genetic backgrounds that were identical except for the mating type locus since congenic strains were used to make the AD hybrid strains.

The observation that the pathogenicity levels of αADa, aADα, and αADα hybrids were similar while aADa hybrids displayed a lower pathogenicity level in the inhalation model of cryptococcosis supports models in which negative epistatic interactions resulting from the presence of both the Aa and Da mating types are responsible for the reduction in virulence. Future investigations will be directed toward elucidating the genetic interactions of different mating type alleles, either with each other or with other genomic regions, in order to obtain further insight into the role of mating type in virulence.