Evidence that the human pathogenic fungus Cryptococcus neoformans var. grubii may have evolved in Africa

Abstract

Most of the species of fungi that cause disease in mammals, including Cryptococcus neoformans var. grubii (serotype A), are exogenous and non-contagious. Cryptococcus neoformans var. grubii is associated worldwide with avian and arboreal habitats. This airborne, opportunistic pathogen is profoundly neurotropic and the leading cause of fungal meningitis. Patients with HIV/AIDS have been ravaged by cryptococcosis--an estimated one million new cases occur each year, and mortality approaches 50%. Using phylogenetic and population genetic analyses, we present evidence that C. neoformans var. grubii may have evolved from a diverse population in southern Africa. Our ecological studies support the hypothesis that a few of these strains acquired a new environmental reservoir, the excreta of feral pigeons (Columba livia), and were globally dispersed by the migration of birds and humans. This investigation also discovered a novel arboreal reservoir for highly diverse strains of C. neoformans var. grubii that are restricted to southern Africa, the mopane tree (Colophospermum mopane). This finding may have significant public health implications because these primal strains have optimal potential for evolution and because mopane trees contribute to the local economy as a source of timber, folkloric remedies and the edible mopane worm.

Figure 1. This unrooted dendrogram depicts the genetic relationships of MLST genotypes among isolates of C. neoformans var. grubii.

DNA sequences of eight loci were concatenated (totaling 4,443 base pairs) and analyzed with the neighbor joining method using uncorrected (“p”) genetic distances. The three major subpopulations or divergent clades of C. neoformans var. grubii, VNI, VNII, and VNB, are apparent. Isolates are clone-corrected (i.e., only one strain of each unique genotype is included). For each genotype, the country or countries of origin is/are shown in parentheses (RSA, Republic of South Africa; DCR, Democratic Republic of Congo). Strains that are unique to Africa are labeled in red, strains that are not found in Africa are labeled in blue, strains that are isolated from Africa and elsewhere are labeled in black. The ecological origin of each strain is indicated by colored ellipses. Green indicates that the strain was isolated from trees and/or soil at the base of trees, and an orange ellipse denotes that the strain was isolated from pigeon feces or soil contaminated with avian feces. Strains without an accompanying green or orange ellipse are clinical isolates that have not been yet been isolated from the environment. In addition, regardless of the ecological source of an isolate, most of the strains or their clones have been isolated from patients. Model strain H99, which is the subject of most molecular studies of C. neoformans, is labeled with arrow . Refer to Table S2 for details about each isolate.

Figure 2. The genetic relationships among 72 MLST genotypes are visualized by Principal Component analysis (PCA).

Each symbol represents a genotype with a unique eight-digit allelic profile (re Fig. 4). Red triangles represent genotypes of strains that are endemic to Africa, and blue circles represent genotypes of global strains. Genotypes associated with African trees are enclosed in green circles, and genotypes associated with pigeon excreta are enclosed in brown circles. Genotypes without circles represent clinical strains that to date have not been isolated from the environment.

Figure 3. Haplotype networks of the eight MLST loci.

Haplotypes of strains of C. neoformans var. grubii that have never been found outside Africa are shown in green: filled green circles designate haplotypes of strains that were obtained from trees (most were also found in patients), and empty green circles signify haplotypes that were obtained only from patients. Cosmopolitan haplotypes are shown in brown: filled brown circles designate haplotypes of strains from pigeon excreta (most were also found in patients), and empty brown circles signify haplotypes that were obtained only from patients. Circles that are half green and half brown, designated “1” and “2”, indicate haplotypes of strains found in trees and pigeon excreta, and they represent the ancestral haplotypes of global strains. Haplotypes from the global VNII subpopulation of C. neoformans var. grubii are used as an outgroup; they are shown in black and lightly encircled. Ancestral haplotypes are internal, and derived haplotypes occupy apical positions. Red dots on the lines connecting the haplotypes represent the most parsimonious number of mutational steps required to generate the allelic polymorphisms. Recombinant haplotypes identified by ARGs are excluded. The number of haplotypes per locus can also be observed. For example, 11 GPD1 haplotypes were detected in Africa (empty, solid or half-filled green circles), and only three GPD1 haplotypes were found among the global strains (empty, solid or half-filled brown circles). (One exception is the TEF1 locus, which has 16 African haplotypes, but only 15 are depicted because one haplotype was found in a pigeon strain [D16-16].) Overall, the non-recombinant African to global haplotype ratios varied from 6 to 3 for URA5 to 14 to 1 for SOD1 (Table 1).

Figure 4. Comparison of representative allelic profiles of isolates of C. neoformans var. grubii from (top panel) the global population (VNI), including seven clinical and seven pigeon isolates, (middle panel) the coprophilic African population associated with pigeons (VNI), and (bottom panel) the African population associated with trees (VNB and VNI).

Strain designations (see Table S2) are listed on the left. Under each locus, identical haplotypes are denoted with the same number and color. Haplotypes that are associated with both pigeons and trees are shown in pink (# 1) and yellow (# 2), respectively, and they represent ancestral haplotypes of the global population. The ancestral MLST genotype that might have emerged from Africa is marked with a red arrow. Emergence of any two strains comprised only of pink and yellow progenitor haplotypes can explain diversity in the contemporary global population of VNI.

Figure 5. Incongruence among four gene genealogies of C. neoformans var. grubii obtained by maximum parsimony.

For clarity, only representative stains are shown. The VNI and VNB subpopulations are indicated. Isolates that are inconsistently placed within the gene genealogies are marked with arrows.