MLST and Whole-Genome-Based Population Analysis of Cryptococcus gattii VGIII Links Clinical, Veterinary and Environmental Strains, and Reveals Divergent Serotype Specific Sub-populations and Distant Ancestors

Abstract

The emerging pathogen Cryptococcus gattii causes life-threatening disease in immunocompetent and immunocompromised hosts. Of the four major molecular types (VGI-VGIV), the molecular type VGIII has recently emerged as cause of disease in otherwise healthy individuals, prompting a need to investigate its population genetic structure to understand if there are potential genotype-dependent characteristics in its epidemiology, environmental niche(s), host range and clinical features of disease. Multilocus sequence typing (MLST) of 122 clinical, environmental and veterinary C. gattii VGIII isolates from Australia, Colombia, Guatemala, Mexico, New Zealand, Paraguay, USA and Venezuela, and whole genome sequencing (WGS) of 60 isolates representing all established MLST types identified four divergent sub-populations. The majority of the isolates belong to two main clades, corresponding either to serotype B or C, indicating an ongoing species evolution. Both major clades included clinical, environmental and veterinary isolates. The C. gattii VGIII population was genetically highly diverse, with minor differences between countries, isolation source, serotype and mating type. Little to no recombination was found between the two major groups, serotype B and C, at the whole and mitochondrial genome level. C. gattii VGIII is widespread in the Americas, with sporadic cases occurring elsewhere, WGS revealed Mexico and USA as a likely origin of the serotype B VGIII population and Colombia as a possible origin of the serotype C VGIII population. Serotype B isolates are more virulent than serotype C isolates in a murine model of infection, causing predominantly pulmonary cryptococcosis. No specific link between genotype and virulence was observed. Antifungal susceptibility testing against six antifungal drugs revealed that serotype B isolates are more susceptible to azoles than serotype C isolates, highlighting the importance of strain typing to guide effective treatment to improve the disease outcome.

Fig 1

Dendrogram showing the genetic relationships between 122 Cryptococcus gattii molecular type VGIII isolates based on the analysis of the seven concatenated MLST loci of the International Society for Human and Animal Mycology (ISHAM) consensus MLST scheme, using the program MEGA 6.0 [48] (A). Dendrogram showing the genetic relationships between 60 C. gattii isolates representing all published MLST sequence types for molecular type VGIII currently present in the MLST database based on the WGS SNP matrix (ii) (B). Numbers on the branches indicate bootstrap values above 50. The color of the branches indicates serotype B (blue) and C (red). Color of the font indicates clinical (black), veterinary (orange) and environmental (green) isolates recovered in AUS: Australia, COL: Colombia, GTM: Guatemala, MEX: Mexico, NZL: New Zealand, PRY: Paraguay, USA: USA, VEN: Venezuela and ZAF: South Africa (Country abbreviations according to the United Nation (UN) three letters ISO 3166–1 code). Asterisk (*) indicates mating type a isolates, all other isolates are mating type α.

Fig 2. Coalescence gene genealogy of the Cryptococcus gattii isolates evidencing the presence of two main clusters (Serotypes B and C) within the VGIII genotype that separated 0.96 to 2.7 million years ago.

Blue bars represent the 95% high posterior probability of the ages in the branches with a posterior probability limit of 0.5. The Bayesian posterior support values higher than 90% are described in the branches of the tree. The serotype C isolates are highlighted in red while serotype B isolates are highlighted in blue. The reference strains WM 179 (VGI, ST51), WM178 (VGII, ST21), and WM779 (VGIV, ST70) were included in the analysis as out-groups.

Fig 3. Haplotype network analysis by locus of the studied Cryptococcus gattii molecular type VGIII isolates.

Networks were constructed using the software Network 4.6.1.3. The size of the circles is proportional to the number of isolates. Color of the circles indicates serotype B (blue) and serotype C (red). Dual-coloration represents shared alleles between serotypes. Alleles for each locus are indicated numerically.

Fig 4. Minimum spanning tree of the studied Cryptococcus gattii molecular type VGIII isolates.

The tree was constructed using the goeBURST algorithm and shows the distribution of the STs according to the country of origin and the serotype B (blue) or serotype C (red) status of the isolates. STs are indicated numerically inside the circles. The size of the circles is proportional to the number of isolates per each ST. Circles surrounded by yellow lines represent the founder of each clonal complex. Numbers between STs represent one (black solid line), two (dark grey line), three (light grey line), and more than four (dashed line) allele differences.

Fig 5. Maximum parsimony phylogenetic analysis of the major molecular types of Cryptococcus gattii (VGI to VGIV) performed on WGST SNP data.

The analysis of the molecular type VGIII alone found 572,268 total SNPs, with 514,098 of them being parsimonious SNPs, with a consistency index (CI) of 0.7691. The tree shown is not rooted. Branch lengths represent numbers of SNPs between taxa, with the unit bar in the Figure. The taxa nomenclature include a unique strain identifier, the country and source of isolation and the ST. AUS: Australia, COL: Colombia, GTM: Guatemala, MEX: Mexico, NZL: New Zealand, PRY: Paraguay, USA: USA, VEN: Venezuela and ZAF: South Africa; Clin: clinical, Vet: veterinary, and Env: environmental.

Fig 6. Phylogenetic network of the main sub-populations of Cryptococcus gattii molecular type VGIII based on whole genome sequence analysis (A) and mitochondrial genome analysis (B).

The neighbor-joining phylogenetic splits tree represents the relationship between the VGIII STs and serotypes using the uncorrected P distance transformation. Each band of parallel edges indicates a split. The taxa nomenclature include a unique strain identifier, the country and source of isolation and the ST. AUS: Australia, COL: Colombia, GTM: Guatemala, MEX: Mexico, NZL: New Zealand, PRY: Paraguay, USA: USA, and VEN: Venezuela; Clin: clinical, Vet: veterinary, and Env: environmental. Isolates included in the virulence studies are in bold. High virulent strains are indicated with a red asterisk.

Fig 7. fineStructure analysis of the main sub-populations of Cryptococcus gattii molecular type VGIII.

To identify the population structure and possible recombination events between and within serotype B and C, fineStructure analysis was performed using a SNP matrix (iii). Whole-genome SNP data were reduced to a pairwise similarity matrix. The x-axis represents the strain as a “donor” and the y-axis represents the strain as a “recipient” of genomic regions. The scale bar represents the number of shared genome regions with blue being the greatest amount of sharing and yellow being the least. Blue and red boxes represent serotype B and serotype C isolates, respectively.

Fig 8. Survival curves of BALB/c mice inoculated with isolates of Cryptococcus gattii molecular type VGIII and the highly virulent VGIIa strain CDCR265 [69].

Blue lines indicates serotype B and red lines, serotype C. Asterisk (*) indicates mating type a isolates, all other isolates are mating type α. Clin: clinical and Vet: veterinary. The median survival time (in days) is indicated in brackets.

Fig 9. Pathological findings from BALB/c mice inoculated with a high virulent isolate of Cryptococcus gattii molecular type VGIII (WM 11.105).

A. Lungs with multiple gelatinous granulomata comprised mostly of masses of cryptococcal cells (cryptococcomas). B. India ink stained lung suspension examined microscopically displaying abundant cryptococcal cells. C. Histopathological section of infected lungs stained with haematoxylin and eosin showing alveoli containing large numbers of yeast cells and little or no inflammatory response.

Fig 10. Change in body weight (expressed as a percentage) of BALB/c mice inoculated with Cryptococcus gattii molecular types VGII and VGIII.

Blue lines indicates VGIII serotype B, red lines, VGIII serotype C and green lines, VGII isolates. Virulent VGIII isolates and isolates of subtypes VGIIa (CDCR265) and VGIIb (CDCR272) are labelled [69]. Code numbers of isolates that did not kill any mice at the end of the experiment are not included.