A diverse population of Cryptococcus gattii molecular type VGIII in southern Californian HIV/AIDS patients

Abstract

Cryptococcus gattii infections in southern California have been reported in patients with HIV/AIDS. In this study, we examined the molecular epidemiology, population structure, and virulence attributes of isolates collected from HIV/AIDS patients in Los Angeles County, California. We show that these isolates consist almost exclusively of VGIII molecular type, in contrast to the VGII molecular type isolates causing the North American Pacific Northwest outbreak. The global VGIII population structure can be divided into two molecular groups, VGIIIa and VGIIIb. Isolates from the Californian patients are virulent in murine and macrophage models of infection, with VGIIIa significantly more virulent than VGIIIb. Several VGIII isolates are highly fertile and produce abundant sexual spores that may serve as infectious propagules. The a and α VGIII MAT locus alleles are largely syntenic with limited rearrangements compared to the known VGI (a/α) and VGII (α) MAT loci, but each has unique characteristics including a distinct deletion flanking the 5' VGIII MATa alleles and the α allele is more heterogeneous than the a allele. Our studies indicate that C. gattii VGIII is endemic in southern California, with other isolates originating from the neighboring regions of Mexico, and in rarer cases from Oregon and Washington state. Given that >1,000,000 cases of cryptococcal infection and >620,000 attributable mortalities occur annually in the context of the global AIDS pandemic, our findings suggest a significant burden of C. gattii may be unrecognized, with potential prognostic and therapeutic implications. These results signify the need to classify pathogenic Cryptococcus cases and highlight possible host differences among the C. gattii molecular types influencing infection of immunocompetent (VGI/VGII) vs. immunocompromised (VGIII/VGIV) hosts.

Figure 1. Molecular analysis of isolates from the Californian patient cohort reveals 93% are VGIII molecular type and genetically diverse.

Multilocus sequence typing analysis of eight loci for the 30 isolates of C. gattii from the southern California AIDS patient cohort. Unique alleles are colored differently for each marker for visual discrimination, and each number represents a GenBank accession number (Table S4). Orange coloration represents VGIIIa, green VGIIIb, and fuchsia shared alleles. In total, a single isolate is VGI, a single isolate is VGII, and 28 isolates are VGIII molecular type.

Figure 2. Global analysis of molecular markers illustrates high diversity and two distinct VGIII lineages: VGIIIa and VGIIIb.

Multilocus sequence typing analysis was conducted at eight loci for the global collection of 60 VGIII molecular type isolates. Geographic origins of isolates are indicated, with red indicating isolates from the southern California patient cohort. Unique alleles were assigned distinct colors for each marker for visual discrimination, and each number represents a GenBank accession number (Table S4). Orange coloration represents VGIIIa, green VGIIIb, and fuchsia shared alleles.

Figure 3. Clustering and phylogenetic analyses of global VGIII isolates reveals two well-supported lineages and one intermediate genotype.

A) A clustering analysis representation (ML) of the concatenated sequence data from global isolates, with the exclusion of MAT locus linked markers (SXI1α/SXI2 a). B) A phylogenetic representation (NJ) and supporting bootstrap values of the sequence data from global isolates, with the exclusion of MAT locus linked markers (SXI1α/SXI2 a). In total, 28 unique sequence types were observed. Details of isolates within each sequence type are presented in Table S5.

Figure 4. Haplotype mapping of markers harboring shared alleles between VGIIIa and VGIIIb shows evidence for both ancestral origins and introgression of shared alleles.

Alleles for each respective locus are indicated numerically. Orange coloration represents VGIIIa, green VGIIIb, and fuchsia shared alleles. Circles represent alleles extant in the population, and the smaller black circles represent alleles that have not been recovered, or which may no longer be extant in the population. Each line connected to an object represents one postulated evolutionary event, with the squared allele representing the posited ancestral allele. A–C) Haplotype networks for CAP10, TEF1, and PLB1, respectively.

Figure 5. Evidence for genetic recombination within the VGIII population, particularly within alleles unique to the VGIIIa lineage.

Informative paired allele graphs from VGIII global isolates. An hourglass shape indicates the presence of all four possible pairs of alleles and serves as evidence of recombination. A) An example of recombination evidence within VGIIIb (top) and VGIIIa (bottom). B) An example of recombination evidence within VGIIIb unique alleles (top) and an example involving a shared allele and three VGIIIa unique alleles (bottom). C) An example of recombination evidence involving both shared and VGIIIb unique alleles. All of the informative paired allele graphs constructed for this analysis can be found in Figure S3.

Figure 6. Scanning electron microscopy imaging of two VGIIIb isolates undergoing a-α mating to produce spores.

Mating images are of NIH312α×B4546a from a V8 media (pH = 5) mating assay. A) Imaging of yeast cells, hyphae, and a clamp cell (arrow) (7,500×). B) Representation of yeast cells and hyphae, with an emerging bud seen to the left of the panel and a blastospore forming off of the hyphae (arrow, 7,000×). C) Yeast, hyphae, basidia, and basidiospores. One detached spore is seen in the top left of the panel, and is characteristically elongated (6,000×). D) A high magnification image of a single basidium with four emerging basidiospores (10,000×). All scale bars are 3 µm.

Figure 7. The VGIII MAT locus of α and a strains share relative synteny with other molecular types but also show rearrangements in MATa.

A) The MAT a locus and flanking regions of molecular type VGIII compared to the locus of molecular type VGI. Fingerprint markers are shown with the order of strains from left to right as follows: B4546, CA1227, CA1232, CA1277, CA1508, CA1873, ICB108, and ATCC32608. B) The MATα locus and flanking regions of molecular type VGIII compared to the loci of molecular types VGI and VGII. Fingerprint markers are shown with the order of strains from left to right as follows: ICB88, DUMC140.97, CA1280, CA1093, NIH836 NIH312, CA1700, MMC08-897, WM161, MEX240, CA1514, 97/426, 97/433, and 97/428. The scale bar is equal to 10 kb and applies to both MAT locus alleles. Shading indicated the boundaries of the MAT locus alleles.

Figure 8. Isolates from the VGIIIa group exhibit increased virulence compared to VGIIIb in an in vivo murine model of infection.

A) Groups of five animals were each infected with an infectious inoculum of 1.0×10⁵ cells, and the survival of animals plotted against days post-infection. The legend is arranged from most virulent (top) to least virulent (bottom, based on survival). VGIIIa isolates are indicated by orange coloration and VGIIIb isolates are indicated by green coloration. B) C. gattii infections cause notable weight loss associated with the virulence level of strains. BALB/c mice were infected with 1.0×10⁵ yeast cells via the intranasal instillation route. The weight of each animal was monitored every other day after infection while asymptomatic. When the animals began to exhibit symptoms of infection, weight was measured daily and plotted.

Figure 9. Histopathology of lungs of mice infected with C. gattii.

Male, BALB/c mice were infected with 1×10⁵ yeast cells via intranasal instillation. The infected lungs were harvested at two weeks post-infection and processed as described above. Note that airway (arrow, A) and all the alveoli (double arrow, A) were packed with rapidly dividing yeasts with minimum tissue response (white arrow, B) in mice infected with highly virulent strain CA1499, while moderately virulent strain CA1292 showed a similar pattern except that several empty alveoli surrounding infected alveoli were observed (C, D). In contrast, the low virulene strain WM161 revealed less multiplication of C. gattii with a majority of alveoli containing one or two yeasts with an influx of inflammatory response (E, F). No yeast was seen in the lungs of mice infected with avirulent strain CA2339 and a vigorous inflammatory response was visible throughout the lung section (G, H). (Mucicarmine and H & E staining, ×100 magnification, scale bar 50 uM).

Figure 10. C. gattii organ load determination.

Male, BALB/c mice were infected intranasally with 1×10⁵ C. gattii yeast cells intranasally. After one and two weeks post-infection, lungs and brains from three infected mice were removed asceptically and fungal load was determined by CFU enumeration as described in materials and methods. The bar diagram represents mean ± SD of CFU per gram of lung tissues obtained from three mice each for different C. gattii strains.

Figure 11. In vitro analysis of intracellular proliferation rates (IPR) show increased proliferation levels in VGIIIa and IPR values correlate with in vivo virulence.

A) IPR values for the 12 isolates examined in mice; orange indicates VGIIIa and green indicates VGIIIb. B) Regression analysis illustrating a linear correlation of IPR and the time to 50% lethality in mice. C) IPR values for the isolates examined in mice, CA1089 excluded; orange indicates VGIIIa and green indicated VGIIIb. D) Regression analysis illustrating a linear correlation of IPR and the time to 50% lethality in mice with isolate CA1089 excluded.