1 Department of Microbiology, Mail Code 7758, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA
2 Laboratorio di Micologia Medica, Instituto di Igiene e Medicina Preventiva, Universita degli Studi di Milano, IRCCS Ospedale Maggiore, Milano, Italy
Correspondence
B. L. Wickes
wickes{at}uthscsa.edu
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ABSTRACT |
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The GenBank accession number for the MFa1A pheromone sequence reported in this paper is AY129299.
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INTRODUCTION |
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The life-cycle of C. neoformans has been well defined. Mating occurs when MATa and MAT cells physically contact one another and fuse to form a diploid, which then differentiates into a dikaryotic hypha with fused clamp connections. Basidia eventually form at the tips of these hyphae and provide the site where meiosis and sporulation occur. Haploid spores of each mating type form in four random chains on the basidial surface by successive mitotic divisions. Once released from the spore chains, individual basidiospores eventually germinate to form haploid budding yeast cells, completing the life-cycle (Kwon-Chung, 1975
, 1976
).
After the life-cycle was described, it was used to help classify C. neoformans into two varieties, which were further subdivided into serotypes (Kwon-Chung et al., 1982a). C. neoformans can be separated into four serotypes (A, B, C and D), with a fifth serotype (AD) occasionally observed. These serotypes have been grouped into two varieties, neoformans (A, D) and gattii (B, C) (Evans, 1949
, 1950
; Evans & Kessel, 1951
; Vogel, 1966
), although a new variety designated C. neoformans var. grubii has recently been proposed for serotype A isolates (Franzot et al., 1999
). The four serotypes are not equally distributed throughout the world. The gattii variety is found in tropical and subtropical areas while the neoformans variety is found globally (Kwon-Chung, 1987
). Serotype A is the most frequent serotype recovered from clinical isolates (Kwon-Chung & Bennett, 1992
). This frequency is reduced, however, in tropical and subtropical areas where the gattii variety is found, as well as in parts of Europe where serotype D is frequently found (Bennett et al., 1977
; Viviani et al., 2000
). In spite of some of these geographical variations, serotype A isolates cause the overwhelming majority of cryptococcal infections throughout the world (Kwon-Chung & Bennett, 1992
).
Similar to the bias in serotype prevalence, a number of studies have also documented a bias in mating-type representation in clinical specimens. These studies have found that MAT isolates comprise more than 95 % of all clinical isolates (Bennett et al., 1977
; Hironaga et al., 1983
; Jong et al., 1982
; Madrenys et al., 1993
; Yan et al., 2002
). Although this bias suggests that the MAT
mating type is more virulent than the MATa mating type, this assumption is complicated. On the one hand, Kwon-Chung et al. (1992)
showed that MAT
cells are more virulent than MATa cells by using congenic strains, which only differ at the mating-type locus, in a mouse survival model. On the other hand, the MAT
bias in clinical isolates also extends into environmental samples (Kwon-Chung & Bennett, 1978
), which allows for the possibility that exposure frequency may play a role in the clinical bias. Therefore, although most evidence points to MAT
cells being innately more virulent than MATa cells, additional studies need to be performed to confirm this.
Presently, most genetic studies of C. neoformans are conducted in serotype D strains because of the existence of both MATa and MAT strains as well as a congenic pair of strains, which serves as a standard laboratory pair. The inability to identify a serotype A MATa strain suggested that these strains were either extinct or existed in an ecological niche that remained to be found. In 2000, Lengeler reported the identification of a serotype A MATa clinical isolate from a Tanzanian AIDS patient (Lengeler et al., 2000
). This report provided the first evidence that a serotype A MATa locus existed; however, the strain was sterile. Another study designed to establish the origin of serotype AD strains, which are diploid or aneuploid hybrids derived from a fusion of serotype A and D parents, showed that several of these unique strains harboured a serotype A MATa locus (Lengeler et al., 2001
). The high frequency of the serotype A MATa mating-type locus in diploid strains suggested that these strains do, in fact, exist in nature and can be readily recovered (Cogliati et al., 2001
). It is, therefore, curious as to why haploid serotype A MATa isolates still remain elusive.
Here we report the detailed characterization of a fertile serotype A MATa C. neoformans isolate. This isolate, designated IUM 96-2828, was initially recovered as an environmental isolate from Italy (Viviani et al., 2001). We show in this study that IUM 96-2828 mates in the laboratory at high frequency and produces recombinant progeny, which demonstrates that it is MATa in mating type. Serotypic analysis by agglutination and phylogenetic analysis of genes not linked to mating type revealed that the strain is most closely related to serotype A strains, confirming that it is a true serotype A isolate. Analysis of a variety of phenotypic traits revealed that this strain had no unusual characteristics with regard to phenotype, as all were wild-type. Taken together, these results demonstrate that IUM 96-2828 is a true, fertile serotype A MATa isolate.
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METHODS |
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Serotyping.
Serotyping was performed using the Crypto Check slide agglutination kit (Iatron Labs) according to the manufacturer's instructions.
TA cloning, sequencing and oligonucleotide synthesis.
PCR products were TA cloned into pCR2.1 (Invitrogen) according to the manufacturer's instructions. Templates were prepared for sequencing using the Wizard miniprep kit (Promega) and sequenced at the Advanced Nucleic Acids Core Facility at the University of Texas Health Science Center at San Antonio. Data were analysed and aligned using MACVECTOR and ASSEMBLYLIGN (Oxford Molecular Group). Oligonucleotides were obtained from the Advanced Nucleic Acids Core Facility at the University of Texas Health Science Center at San Antonio.
PCR.
All reactions were performed in a GeneMate thermocycler (Techne) using a 50 µl reaction volume with 2·5 U Taq DNA polymerase (Invitrogen BRL) and 2550 ng DNA as template. Primers are listed in Table 2. The serotype D MATa pheromone (MFa1) was amplified as an
800 bp fragment from JEC-20 template DNA using primers All.MFa.5' and All.MFa.3'. Amplification conditions were 94 °C for 2 min, an annealing temperature of 62 °C for 30 s and an extension step of 72 °C for 60 s, which was then repeated 29 times except that the initial 94 °C denaturation step was reduced to 30 s. The final cycle included a 3 min extension step at 72 °C. The serotype A MATa pheromone (MFa1A) was amplified as an
800 bp fragment from IUM 96-2828 DNA using the same primers and conditions as for the serotype D MFa1 gene, and then TA cloned and sequenced. The serotype A MAT
pheromone gene coding sequence was amplified from H99 using primers H99.MF.F-2 and H99.MF.R. Amplification conditions were 94 °C for 2 min, an annealing temperature of 56 °C for 15 s and an extension step of 72 °C for 30 s, which was then repeated 29 times except that the initial 94 °C denaturation step was reduced to 15 s. The final extension was extended for an additional 3 min at 72 °C. The
100 bp H99 A MF
1 fragment was then TA cloned and sequenced.
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Accession number.
The MFa1A pheromone sequence has been deposited in GenBank under accession number AY129299.
Electrophoretic karyotyping.
Seed cultures of 50 ml YPD were inoculated with approximately 1x106 cells and grown at 30 °C for 16 h with shaking at 300 r.p.m. before 100 ml pre-warmed YPD broth was added. Shaking and incubation continued for 46 h, and then cells were harvested. Plugs were prepared as described previously (Wickes et al., 1994), except that sphaeroplasting took place in solution prior to agarose addition using 950 mg lysing enzymes from Trichoderma harzianum (Sigma) per 1 ml buffer. Samples were run in a 0·7 % PFGE-grade agarose (Bio-Rad) gel with 0·5xTBE running buffer using the following protocol: 110 V, switch times of 50130 s for 23 h followed by 170400 s for 47 h, at a gel temperature of 10 °C. Southern blots were prepared as follows. After staining in ethidium bromide for 30 min, the gel was nicked in a Stratalinker (Stratagene) set at 60 mJ. Following a 15 min wash in 0·4 M NaOH/1·5 M NaCl, a standard capillary transfer was set up using a Hybond-N+ nylon membrane. Transfers continued until 2 l buffer had passed through the gel, or approximately 48 h. The membranes were neutralized after transfer by washing in 0·5 M Tris/HCl (pH 7) for 5 min and then rinsed briefly in 2xSSC. Hybridizations of karyotype blots followed Hybond-N+ membrane instructions, except that Rapid-Hyb buffer (Amersham Pharmacia) was used.
Phylogeny study.
Several genes were chosen for phylogenetic analysis based on the availability of sequences from all four serotypes for comparison. Accession numbers are listed in Table 3. C. neoformans CAP10, CNLAC1 and URA5 genes were amplified from IUM 96-2828 using primers to conserved regions identified from alignments of sequences from the other serotypes (Table 2
). A 480 bp CAP10 fragment was amplified with primers CAP10.F and CAP10.R using an initial step of 94 °C for 2 min, 63 °C for 12 s, 72 °C for 30 s, followed by 29 additional cycles where the 94 °C cycle was reduced to 12 s and a final extension of 1 min at 72 °C. A 650 bp CNLAC1 fragment was amplified with primers LAC.F and LAC.R using an initial step of 94 °C for 2 min, 50 °C for 30 s, 72 °C for 1 min 30 s, followed by 29 additional cycles where the 94 °C step was reduced to 30 s and then a final extension of 3 min at 72 °C. A 780 bp URA5 fragment was amplified using primers URA5.F and URA5.R with an initial step of 94 °C for 2 min, 58 °C for 20 s, 72 °C for 45 s, followed by 29 additional cycles where the 94 °C step was reduced to 20 s and a final extension of 2 min at 72 °C. All PCR products were TA cloned and then sequenced.
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Virulence studies.
Virulence assays utilized 10 female BALB/c mice per isolate, which were injected with either H99 or IUM 96-2828 via the lateral tail vein. Inocula were prepared as previously described (Kwon-Chung et al., 1992) and then used to inject each mouse with 0·2 ml of a suspension containing 5x106 organisms ml-1. Mice were observed daily and scored for mortality. Data were analysed by KaplanMeier survival estimates using JMP software (SAS Institute). Egg yolk agar was used for testing phospholipase activity (Price et al., 1982
; Vidotto et al., 1998
). Strains were inoculated onto egg yolk agar as a suspension (1x108 cells ml-1) after growth on YPD for 24 h. Cultures were observed for the formation of a precipitate around the colony for up to 7 days incubation at 30 °C. Melanin production was tested on asparagine agar (Kwon-Chung et al., 1982b
; Rhodes et al., 1982
). A 24 h seed culture was grown on YPD at 30 °C. A suspension of approximately 1x108 cells ml-1 was prepared and 510 µl of this were dropped onto the agar. Positive reactions consisted of a dark-brown colony colour within 2448 h incubation at 30 °C. Capsule production was assayed as described using a modified LIM (Wills et al., 2001
). Plates were inoculated from a 24 h YPD culture and grown at 25 °C for 57 days. Cells were then mounted with India ink to visualize the capsule microscopically.
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RESULTS |
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Progeny of H99xIUM 96-2828 are recombinant
PFGE was used to compare the karyotypes of IUM 96-2828, H99, one F1 MATa and one F1 MAT progeny (Fig. 4
). The karyotypes of these four strains were highly conserved and were also conserved when compared to karyotype patterns previously reported for serotype A strains (Wickes et al., 1994
). The chromosomes in these karyotypes ranged from 770 kb to 3·8 Mb in size, which agrees with data previously reported for serotype A karyotypes (Wickes et al., 1994
). Both F1 progeny displayed non-parental karyotypes with at least one band being non-parental. The karyotype of the F1 MATa strain contained one band that was derived from the H99 (MAT
) parent, while the karyotype of the F1 MAT
strain contained one band derived from the IUM 96-2828 (MATa) parent, but lacked another band present in the H99 karyotype. The F1 progeny were themselves fertile when backcrossed to either compatible parent. In Southern blots of whole karyotypes, IUM 96-2828 and the F1 MATa strain from the cross between IUM 96-2828 and H99 only hybridized to the MFa1A probe from IUM 96-2828 (Fig. 4
). Conversely, H99 and the F1 MAT
strain from the cross between IUM 96-2828 and H99 only hybridized to the MF
1A probe from H99. The probes hybridized to the band corresponding to the MAT chromosome (Clarke et al., 2001
), which is approximately 1·8 Mb in size. The size and location of the MAT chromosome in the karyotypes of these strains is conserved in both serotypes.
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DISCUSSION |
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The first confirmed serotype A MATa isolate was identified from a Tanzanian clinical isolate. Although the strain was shown to be virulent in mice, it did not produce mating pheromone, lacked one or more components of the signal transduction pathway required for mating and consequently, was sterile (Lengeler et al., 2000). IUM 96-2828, the second putative serotype A MATa strain to be identified, was initially recovered during a study of European isolates and identified by PCR (Viviani et al., 2001
). The results of the present study provide strong evidence that IUM 96-2828 is a true serotype A MATa isolate, which is fertile. The purity of the progeny from the cross between IUM 96-2828 and H99 with regard to parental serotype suggested that there is no evidence of serotype D antigenicity in the IUM 96-2828 genomic background. Importantly, the clustering of IUM 96-2828 with other serotype A strains in the phylogenetic analysis of genes not linked to mating type also argues against any serotype D ancestry in this strain, especially since this analysis is molecular-based and independent of the agglutination assay. Therefore, by multiple techniques IUM 96-2828 always behaved unambiguously as a serotype A strain in our hands.
We also used multiple approaches to confirm that IUM 96-2828 was MATa in mating type. Southern blots of both mating types of all four serotypes revealed that the IUM 96-2828 MFa1A pheromone probe hybridized to genomic DNA from IUM 96-2828 and JEC-20, but did not hybridize to DNA from representative strains of the gattii variety (serotypes B and C). This pattern matches the hybridization pattern of the serotype D MATa pheromone (McClelland et al., 2002). In contrast, the MF
1A pheromone gene from H99 hybridized to all four serotypes. This hybridization pattern matched the serotype D MAT
pattern (McClelland et al., 2002
). Xu et al. (2000)
suggested that C. neoformans var. neoformans diverged from C. neoformans var. gattii approximately 37 million years ago and then separated into serotypes A and D approximately 18·5 million years ago. Although little is known about the evolution of mating types in C. neoformans, the MATa pheromones may have evolved after the divergence into the two varieties, and almost certainly after the MAT
pheromone. The hybridization pattern of MFa1A and the sequence conservation when compared to the serotype D MATa pheromone would be consistent with this hypothesis. Based on the sequence of the IUM 96-2828 pheromone, this strain would be predicted to mate as a MATa strain. In a standard mating reaction, IUM 96-2828 mated with H99 and produced abundant basidiospores. It also mated with a serotype D MAT
strain (JEC-21) but did not mate with a serotype D MATa strain (JEC-20). In a standard mating reaction with strains in which one or both backgrounds are unknown, it is possible to produce basidiospores that are not meiotic products of the two input parents by two ways. The first way is that the spores can be products of monokaryotic fruiting and therefore, are non-recombinant because they arise from a single strain in the absence of mating (Wickes et al., 1996
). The second way is that the spores can be produced from a cross to a diploid or aneuploid self-fertile strain, which contains both a MATa and MAT
mating-type locus. In this case, although the spores could technically be recombinant, they would not be the product of a mating with the intended parent. These possibilities were excluded by the 1 : 1 mating-type segregation pattern of the progeny from the cross to H99, as well as the hybridization results with the pheromone probes and finally, the STE20 PCRs. The karyotype results in which PFGE was used to compare karyotypes from IUM 96-2828, H99 and the F1 progeny were also consistent with IUM 96-2828 being a fertile serotype A MATa. Both F1 progeny displayed non-parental karyotypes, providing evidence that the progeny were meiotic products. These results demonstrate the utility of IUM 96-2828 as a compatible tester strain to H99 and confirm that IUM 96-2828 is MATa in mating type.
Since IUM 96-2828 is serotype A and MATa, and has been shown to mate efficiently in a standard mating reaction, we have begun the construction of congenic strains in both the IUM 96-2828 and H99 backgrounds. Both strains mate efficiently and we are currently at generation F8. During mating analyses, we have observed that matings between IUM 96-2828 and H99 do not produce the intense hyphal fringe observed in crosses between the serotype D congenic strains JEC-20 and JEC-21 (see Fig. 1). However, the number of basidia produced in IUM 96-2828xH99 crosses is much higher and the spore chains produced appear to be substantially longer that those formed during JEC-20xJEC-21 crosses. Although mating efficiency can vary greatly depending on strain, our results suggest that IUM 96-2828 and H99 will be useful for most genetic analyses. To enhance their use for basic genetic analyses, we have also begun to insert various genetic markers into the strains and have succeeded in recovering an ura5 auxotroph, which can be complemented by transformation with the native gene. In the serotype D congenic pair, mating is markedly enhanced when two auxotrophs are crossed. Therefore, having marked strains will provide genetic flexibility for the study of serotype A strains and will also provide the appropriate hosts for transformation.
In addition to the utility of a congenic pair for future genetic studies, the two congenic pairs will provide an opportunity to test the role of mating type in the virulence of serotype A strains. In 1992, Kwon-Chung reported an association between mating type and virulence during a study of the serotype D congenic strains JEC-20 and JEC-21 (Kwon-Chung et al., 1992). The study concluded that the MAT
strain was significantly more virulent than the MATa strain. We were interested in determining the virulence of IUM 96-2828 in a mouse model in order to have background data for a future congenic virulence study. Our results demonstrated that IUM 96-2828 was virulent; however, it was significantly less virulent than H99. Interestingly, the Tanzanian isolate reported by Lengeler et al. (2000)
was also significantly less virulent than H99. A number of factors are required for virulence in C. neoformans, which include, but are not restricted to, the production of capsule, melanin and phospholipase and growth at 37 °C (Buchanan & Murphy, 1998
; Hamilton & Goodley, 1996
; Kozel, 1995
). IUM 96-2828 showed no difference from H99 in any of these phenotypic traits. Therefore, an explanation for the reduced virulence of IUM 96-2828 does not include a mutation in one of the above virulence factors. While it is tempting to conclude that reduced virulence is linked to the MATa mating-type locus of IUM 96-2828, in reality there could be many other reasons independent of mating type that explain the virulence difference. We identified slight differences in carbon assimilation and isozyme patterns; however, since C. neoformans strains are variable in these phenotypic traits, it is unclear what effect, if any, these differences would have on virulence. To address the association of mating type with virulence in serotype A strains, we plan to repeat the serotype D congenic strain study with the serotype A strains. The congenic strain strategy circumvents the problem of assembling a large, diverse collection of MATa and MAT
isolates because it allows the testing of each mating type in a normalized genetic background. By preparing two pairs of strains, one set in each parental background, the role of mating type in virulence can be more rigorously tested. Since H99 is a clinical isolate and IUM 96-2828 is an environmental isolate, if the MAT
strain in both backgrounds is significantly more virulent, this outcome in combination with the previous serotype D congenic virulence study should provide conclusive evidence for the role of mating type in the virulence of C. neoformans var. neoformans.
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ACKNOWLEDGEMENTS |
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Received 31 July 2002;
revised 20 September 2002;
accepted 24 September 2002.