Many globally isolated AD hybrid strains of Cryptococcus neoformans originated in Africa

Abstract

Interspecific and intervarietal hybridization may contribute to the biological diversity of fungal populations. Cryptococcus neoformans is a pathogenic yeast and the most common fungal cause of meningitis in patients with AIDS. Most patients are infected with either of the two varieties of C. neoformans, designated as serotype A (C. neoformans var. grubii) or serotype D (C. neoformans var. neoformans). In addition, serotype AD strains, which are hybrids of these two varieties, are commonly isolated from clinical and environmental samples. While most isolates of serotype A and serotype D are haploid, AD strains are diploid or aneuploid, and contain two sets of chromosomes and two mating type alleles, MATa and MATalpha, one from each of the serotypes. The global population of serotype A is dominated by isolates with the MATalpha mating type (Aalpha); however, about half of the globally analyzed AD strains possess the extremely rare serotype A MATa allele (Aa). We previously described an unusual population of serotype A in Botswana, in which 25% of the strains contain the rare MATa allele. Here we utilized two methods, phylogenetic analysis of three genes and genotyping by scoring amplified fragment length polymorphisms, and discovered that AD hybrid strains possessing the rare serotype A MATa allele (genotype AaDalpha) cluster with isolates of serotype A from Botswana, whereas AD hybrids that possess the MATalpha serotype A allele (AalphaDa and AalphaDalpha) cluster with cosmopolitan isolates of serotype A. We also determined that AD hybrid strains are more resistant to UV irradiation than haploid serotype A strains from Botswana. These findings support two hypotheses: (i) AaDalpha strains originated in sub-Saharan Africa from a cross between strains of serotypes A and D; and (ii) this fusion produced hybrid strains with increased fitness, enabling the Botswanan serotype A MATa genome, which is otherwise geographically restricted, to survive, emigrate, and propagate throughout the world.

Figure 1. PCR Amplification of Portions of the STE20 Gene from the AD Hybrid Strains Using Mating Type– and Serotype-Specific Primers

(A) PCR primers specific to MATa serotype D allele (STE20Da); (B) PCR primers specific to MATa serotype A allele (STE20Aa); (C) PCR primers specific to MATα serotype A allele (STE20Aα); (D) PCR primers specific to MATα serotype A/D allele (STE20A/Dα). Lanes 1–12 represent the following strains (from left to right): CDC228, it755, ZG290, nc5–19, it756, it744, KW5, it752, CDC304, ZG287, nc34–21, and MMRL1365.

Figure 2. Computer-Generated AFLP Band Patterns of AD Hybrid Strains

Lanes 1–6 represent AaDα strains CDC228, CDC304, it752, nc34–21, and MMRL1365, respectively. Lanes 7–12 represent AαDa strains ZG287, KW5, it744, it755, it756, and nc5–19, respectively. Lane 12 represents the unusual nc5–19 isolate that carries both serotype A and serotype D MATα alleles (AαDα). Bands that are polymorphic between Aa and Aα groups are indicated with long arrows, and bands that are polymorphic among the individual strains are denoted with short arrows. AFLP bands that were consistent between independent DNA preparations and analyses (see Materials and Methods) are labeled with the asterisks.

Figure 3. Bayesian Consensus Tree for 12 AD Hybrid Strains and 45 Representative Strains of Serotype A Based on the Combined Data for the Three Loci

The tree is rooted with the sequences from the JEC21 strain of serotype D, and branch lengths leading to the outgroup are shortened to fit the figure. Thickened lines denote clades supported by Bayesian posterior probabilities ≥95%. VNI, VNII, and VNB refer to the subgroups (molecular types) identified within serotype A [22]. AaDα hybrid isolates are shown in blue, AαDa hybrid isolates are shown in green, and isolates from Botswana are shown in red. Strains of serotype A that possess MATa allele are marked with arrowheads.

Figure 4. Natural and Laboratory AaDα Hybrids Are More Resistant to UV Irradiation Compared to Serotype A and Serotype D Haploid Strains

(A) VNB strains are more sensitive to UV irradiation than isolates of serotype D, VNI strain of serotype A, and AD hybrid strains. Cells were grown overnight in liquid culture under the melanin-inducing conditions, and 10-fold serial dilutions of log-phase yeast cells of VNB strains, AD hybrid strains, and control serotype A and serotype D strains were plated in triplicates on YPD medium; plates were UV irradiated (∼48 mJ/cm²) for 24 (second panel) or 30 seconds (third panel). To evaluate the amount of melanin produced, strains were spotted on the plates containing low-glucose L-DOPA medium (last panel). (B) Quantitative assessment of UV resistance for each strain. The highest dilutions that yielded colonies after treatment with UV were identified, and the number of individual colonies was determined within each spot corresponding to the highest dilution that yielded viable colonies. From this number, the total number of CFU that survived the treatment was determined and plotted. The experiment was performed in triplicate, and error bars represent standard error of the mean based on three replicates. Black bars represent cells irradiated for 24 seconds and grey bars represent cells irradiated for 30 seconds. Some AD hybrids are aneuploid (>1n but <2n).