Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the northwest United States

Abstract

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.

Figure 1. Geographic dispersal of pathogenic C. gattii genotypes in the United States.

Circles represent human cases and squares represent animal (non-human mammalian) cases. All cases shown have been reported from 2005 to 2009. Isolates are color coded by genotype, in which yellow and blue correspond to VGIIa/major genotype cases (yellow ST1, blue ST30), red corresponds to VGIIb/minor, green corresponds to the novel VGIIc genotype, and orange corresponds to two cases determined to be molecular type VGIII. In total, there were 39 cases (18 human, 21 animal) that have been confirmed by phenotypic and genotypic profiling.

Figure 2. Markers used in the study are dispersed in the genome.

A map of each chromosome is represented, illustrating the locations of each marker based on the genomic sequence of the C. gattii isolate WM276. MLST markers (n = 16) are indicated on the map by hexagons, with pink denoting the standard set used, blue the expanded set of loci, and red the MAT linked locus that is specific to α isolates. Green triangles represent the four VNTR loci that were examined.

Figure 3. Clustering analysis of global VGII isolates shows high global diversity.

This dendrogram, based on seven MLST loci and four VNTR loci, illustrates the global divergence seen in this molecular type. Major clusters are highlighted accordingly to illustrate the placements of the VGIIa/b/c super clusters as well as a unique NT cluster that has been found only in Australia thus far. Sequence types 1, 30, 19, and 20 are enlarged and represent the primary genotypes responsible for the Pacific NW outbreak. Boxed isolates represent those of the a mating type and all other sequence types represent the genotypes observed for mating type α isolates. Several genotypes are also combined with geographic information to illustrate the diversity surrounding several sequence types. Isolates from the VGI, VGIII, and VGIV molecular types serve as out-group sequence types.

Figure 4. Expanded molecular analysis reveals increased divergence in VGIIc.

A) Multilocus sequence typing analysis of 16 loci. Selected isolates from the outbreak in addition to global genotypes were selected for the expanded MLST analysis, including all nine of the VGIIc isolates available. Each unique allele is colored for each marker for visual discrimination, and each number represents a GenBank accession number (Table S2). B) A representation (ML) of the sequence data from panel A, with the exclusion of MAT locus linked markers (SXI1α/SXI2 a). C) A combination of the sequence data from panel B, with the addition of the four highly variable VNTR markers.

Figure 5. Haplotype networks define allele ancestry.

Allele placements are indicated numerically, with the VGIIa/major genotype also represented by blue coloration, the VGIIb/minor genotype by purple coloration, and the VGIIc/novel genotype by green coloration. Large circles represent alleles extant in the population, and the small circles represent alleles that have not been recovered, or which may no longer be extant in the population. Each connecting line represents one postulated evolutionary event, with the squared allele representing the posited ancestral allele (two possible ancestral alleles depicted for SXI1α). A–C) Haplotype networks of the unique VGIIc alleles, SXI1α, HOG1, and CRG1, respectively, with geographic origins indicated.

Figure 6. Evidence for recombination within the VGII molecular type.

Informative paired allele graphs from VGII global isolates. An hourglass shape indicates the presence of all four possible pairs of alleles and serves as evidence for recombination. A total of 56 graphs with at least one possible recombining allele pair were generated from a set of 25 representative genotypes within the VGII molecular type, including isolates of both mating type a and α (see also Figure S4).

Figure 7. In vitro analyses of intracellular proliferation and mitochondrial morphology provide evidence the VGIIc genotype is hypervirulent.

A) IPR rates of VGIIc isolates are similar to those from the VGIIa/major genotype and higher than those seen in the less-virulent VGIIb/minor genotype. Eight VGIIc isolates were tested individually, with the overall averages for the three primary outbreak genotypes presented. B) Percentage of cells with tubular mitochondrial morphology in DMEM. C) Percentage of cells with tubular mitochondrial morphology in macrophages. D) Linear correlation of IPR and percentage of tubular mitochondria after macrophage exposure.

Figure 8. Isolates from the United States outbreak are hypervirulent.

A) Groups of five animals were each infected with an infectious inoculum of 1.0×10⁵ cells of VGIIa isolates R265, CA1014, or NIH444, VGIIb isolate R272, or VGIIc isolates EJB15 or EJB18. B) Groups of nine or ten animals were each infected with an infectious inoculum of 5.0×10⁴ cells of VGIIa isolates R265, EJB51, CBS7750, or ICB107, VGIIb isolates R272 or EJB53, VGIIc isolates A6MR38, EJB12, EJB14, EJB15, or EJB18, or C. neoformans var. grubii isolate H99. C–D) Representative H&E stained histopathology slides from lung sections of severely morbid sacrificed animals from the VGIIa/major (R265) (C) and VGIIc (EJB18) (D) genotypes (sections from animals in panel B of this Figure).