A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from an immunocompromised patient with cryptococcosis based on molecular analyses available in 2000. Here, we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MATα strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including eight nonsynonymous changes involving seven genes. To ascertain whether changes in these genes are selected for during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frameshift mutation in one of the seven genes altered in the human sample (LQVO5_000317), a gene predicted to encode an SWI–SNF chromatin-remodeling complex protein. In addition, both cockatoo and patient strains as well as mouse-passaged isolates obtained from brain tissue had a premature stop codon in a homologue of ZFC3 (LQVO5_004463), a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and reverted to a full-length protein in the mouse-passaged isolates obtained from lung tissue. The patient strain and mouse-passaged isolates show variability in virulence factors, with differences in capsule size, melanization, rates of nonlytic expulsion from macrophages, and amoeba predation resistance. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.

A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from the cerebral spinal fluid (CSF) of an immunocompromised patient with cryptococcosis based on the molecular analyses available in 2000. Here we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MATα strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including 8 nonsynonymous changes involving 7 genes. To ascertain whether changes in these genes are selected during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frame-shift mutation in one of the seven genes altered in the human sample, a gene predicted to encode a SWI-SNF chromatin-remodeling complex protein. Both cockatoo and patient strains as well as mouse passaged isolates obtained from brain tissue had a premature stop codon in a homolog of ZFC3, a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and appears to have reverted to a full-length protein in the mouse passaged isolates obtained from lung tissue. The patient strain and mouse passaged isolates show variability in the expression of virulence factors, with differences in capsule size, melanization, and rates on non-lytic expulsion from macrophages observed. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.

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Antibodies play critical roles in protection against infectious diseases and can be passively administered to protect patients. In the case of antibodies against glucuronoxylomannan (GXM), the major constituent of the Cryptococcus neoformans capsule, this results in protective, non-protective and disease enhancing outcomes. We sought to improve our basic understanding of these functionally different antibodies. Here we report a library of twenty-six synthetic GXM oligosaccharides, consisting of M2 (serotype A), M4 (serotype C), and M1 motifs (serotype D). These GXM glycans range in size from 1-mers to an 18-mer and were used to construct a microarray containing both O-acetylated and non-acetylated glycans. This allowed mapping of the binding preferences of sixteen functionally different monoclonal antibodies to the Cryptococcus capsule. This data is complemented by sequence comparison analysis of fragment antigen-binding (Fab) region which revealed how small changes (<13) in Fab sequence can affect antibody antigen specificity and function. While, changes in the constant region (isotype switching) altered glycan specificity. Furthermore, by combining immunofluorescence imaging of fungal cells and knowledge of antibody binding specificities, we gained direct evidence for the molecular complexity of cryptococcal capsules. Our results highlight the complex relationship between antibody epitope, affinity and isotype class, all of which act synergistically to contribute to a protective or non-protective immune response, meaning that binding specificity alone is not sufficient to predict an antibodys function. These results are relevant to the design of vaccines against Cryptococcosis.