Serotype AD Strains of Cryptococcus neoformans Are Diploid or Aneuploid and Are Heterozygous at the Mating-Type Locus

Cryptococcus neoformans is a pathogenic basidiomycete with a defined sexual cycle involving mating between haploid yeast cells with a transient diploid state. C. neoformans occurs in four predominant serotypes (A, B, C, and D), which represent different varieties or species. Rare clinical and environmental isolates with an unusual AD serotype have been reported and suggested to be diploid. We found by fluorescence-activated cell sorter analysis that serotype AD strains are aneuploid or diploid. PCR analysis with primers specific for serotype A or D alleles of the CNA1, CLA4, and GPA1 genes revealed that both alleles are often present in serotype AD strains. PCR analysis with primers specific for genes in the MATa or MATα mating-type loci revealed that serotype AD strains are heterozygous for the mating-type locus. Interestingly, in several serotype AD strains, the MATα locus was derived from the serotype D parent and the MATa locus was inherited from a serotype A parent that has been thought to be extinct. Basidiospores from a self-fertile serotype AD strain bearing the putative serotype A MATa locus showed a very low viability (∼5%), and no fertile serotype A MATa strain could be recovered. Serotype AD strains were virulent in a murine model. Hybrid AD strains could readily be isolated following a laboratory cross between a serotype A strain and a serotype D strain. In summary, serotype AD strains of C. neoformans are unusual aneuploid or diploid strains that result from matings between serotype A and D strains. Self-fertile isolates fail to undergo normal meiosis because of genetic divergence. Our findings further suggest that serotype A MATa strains may exist in nature.

新型隐球菌（Cryptococcus neoformans）是一种致病性担子菌（basidiomycete），其拥有明确的有性生殖周期，该周期涉及单倍体酵母细胞之间的交配，并伴随短暂的二倍体状态。新型隐球菌存在四种主要血清型（A、B、C与D），分别对应不同的变种或物种。此前已有报道发现罕见的携带异常AD血清型的临床与环境分离株，并推测此类菌株为二倍体。我们通过荧光激活细胞分选分析发现，AD血清型菌株为非整倍体或二倍体。针对CNA1、CLA4与GPA1基因的血清型A或D等位基因设计特异性引物开展聚合酶链反应（PCR）分析，结果显示AD血清型菌株通常同时携带这两种等位基因。针对MATa与MATα交配型位点的基因设计特异性引物进行PCR分析，结果表明AD血清型菌株的交配型位点呈杂合状态。值得注意的是，在多株AD血清型菌株中，MATα位点源自血清型D亲本，而MATa位点则继承自一种此前被认为已灭绝的血清型A亲本。一株携带推定血清型A MATa位点的自育AD血清型菌株所产生的担孢子活力极低（约5%），且未分离得到可育的血清型A MATa菌株。AD血清型菌株在小鼠感染模型中具有致病性。通过实验室将血清型A菌株与血清型D菌株进行杂交，可轻松分离获得杂交AD菌株。综上，新型隐球菌的AD血清型菌株是一类由血清型A与D菌株交配产生的异常非整倍体或二倍体菌株。自育分离株因遗传分化无法完成正常减数分裂。我们的研究结果进一步提示，血清型A MATa菌株可能在自然界中存在。