Dating the Cryptococcus gattii Dispersal to the North American Pacific Northwest.

The emergence of Cryptococcus gattii, previously regarded as a predominantly tropical pathogen, in the temperate climate of the North American Pacific Northwest (PNW) in 1999 prompted several questions. The most prevalent among these was the timing of the introduction of this pathogen to this novel environment. Here, we infer tip-dated timing estimates for the three clonal C. gattii populations observed in the PNW, VGIIa, VGIIb, and VGIIc, based on whole-genome sequencing of 134 C. gattii isolates and using Bayesian evolutionary analysis by sampling trees (BEAST). We estimated the nucleotide substitution rate for each lineage (1.59 × 10-8, 1.59 × 10-8, and 2.70 × 10-8, respectively) to be an order of magnitude higher than common neutral fungal mutation rates (2.0 × 10-9), indicating a microevolutionary rate (e.g., successive clonal generations in a laboratory) in comparison to a species' slower, macroevolutionary rate (e.g., when using fossil records). The clonal nature of the PNW C. gattii emergence over a narrow number of years would therefore possibly explain our higher mutation rates. Our results suggest that the mean time to most recent common ancestor for all three sublineages occurred within the last 60 to 100 years. While the cause of C. gattii dispersal to the PNW is still unclear, our research estimates that the arrival is neither ancient nor very recent (i.e., <25 years ago), making a strong case for an anthropogenic introduction. IMPORTANCE The recent emergence of the pathogenic fungus Cryptococcus gattii in the Pacific Northwest (PNW) resulted in numerous investigations into the epidemiological and enzootic impacts, as well as multiple genomic explorations of the three primary molecular subtypes of the fungus that were discovered. These studies lead to the general conclusion that the subtypes identified likely emerged out of Brazil. Here, we conducted genomic dating analyses to determine the ages of the various lineages seen in the PNW and propose hypothetical causes for the dispersal events. Bayesian evolutionary analysis strongly suggests that these independent fungal populations in the PNW are all 60 to 100 years old, providing a timing that is subsequent to the opening of the Panama Canal, which allowed for more direct shipping between Brazil and the western North American coastline, a possible driving event for these fungal translocation events

Ancient dispersal of the human fungal pathogen Cryptococcus gattii from the Amazon rainforest.

Over the past two decades, several fungal outbreaks have occurred, including the high-profile 'Vancouver Island' and 'Pacific Northwest' outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals

Pathogenic diversity amongst serotype C VGIII and VGIV Cryptococcus gattii isolates

Cryptococcus gattii is one of the causative agents of human cryptococcosis. Highly virulent strains of serotype B C. gattii have been studied in detail, but little information is available on the pathogenic properties of serotype C isolates. In this study, we analyzed pathogenic determinants in three serotype C C. gattii isolates (106.97, ATCC 24066 and WM 779). Isolate ATCC 24066 (molecular type VGIII) differed from isolates WM 779 and 106.97 (both VGIV) in capsule dimensions, expression of CAP genes, chitooligomer distribution, and induction of host chitinase activity. Isolate WM 779 was more efficient than the others in producing pigments and all three isolates had distinct patterns of reactivity with antibodies to glucuronoxylomannan. This great phenotypic diversity reflected in differential pathogenicity. VGIV isolates WM 779 and 106.97 were similar in their ability to cause lethality and produced higher pulmonary fungal burden in a murine model of cryptococcosis, while isolate ATCC 24066 (VGIII) was unable to reach the brain and caused reduced lethality in intranasally infected mice. These results demonstrate a high diversity in the pathogenic potential of isolates of C. gattii belonging to the molecular types VGIII and VGIV

Sexual reproduction of human fungal pathogens

We review here recent advances in our understanding of sexual reproduction in fungal pathogens that commonly infect humans, including Candida albicans, Cryptococcus neoformans/gattii, and Aspergillus fumigatus. Where appropriate or relevant, we introduce findings on other species associated with human infections. In particular, we focus on rapid advances involving genetic, genomic, and population genetic approaches that have reshaped our view of how fungal pathogens evolve. Rather than being asexual, mitotic, and largely clonal, as was thought to be prevalent as recently as a decade ago, we now appreciate that the vast majority of pathogenic fungi have retained extant sexual, or parasexual, cycles. In some examples, sexual and parasexual unions of pathogenic fungi involve closely related individuals, generating diversity in the population but with more restricted recombination than expected from fertile, sexual, outcrossing and recombining populations. In other cases, species and isolates participate in global outcrossing populations with the capacity for considerable levels of gene flow. These findings illustrate general principles of eukaryotic pathogen emergence with relevance for other fungi, parasitic eukaryotic pathogens, and both unicellular and multicellular eukaryotic organisms

Cryptococcus gattii in North American Pacific Northwest: whole-population genome analysis provides insights into species evolution and dispersal

The emergence of distinct populations of Cryptococcus gattii in the temperate North American Pacific Northwest (PNW) was surprising, as this species was previously thought to be confined to tropical and semitropical regions. Beyond a new habitat niche, the dominant emergent population displayed increased virulence and caused primary pulmonary disease, as opposed to the predominantly neurologic disease seen previously elsewhere. Whole-genome sequencing was performed on 118 C. gattii isolates, including the PNW subtypes and the global diversity of molecular type VGII, to better ascertain the natural source and genomic adaptations leading to the emergence of infection in the PNW. Overall, the VGII population was highly diverse, demonstrating large numbers of mutational and recombinational events; however, the three dominant subtypes from the PNW were of low diversity and were completely clonal. Although strains of VGII were found on at least five continents, all genetic subpopulations were represented or were most closely related to strains from South America. The phylogenetic data are consistent with multiple dispersal events from South America to North America and elsewhere. Numerous gene content differences were identified between the emergent clones and other VGII lineages, including genes potentially related to habitat adaptation, virulence, and pathology. Evidence was also found for possible gene introgression from Cryptococcus neoformans var. grubii that is rarely seen in global C. gattii but that was present in all PNW populations. These findings provide greater understanding of C. gattii evolution in North America and support extensive evolution in, and dispersal from, South America. Importance: Cryptococcus gattii emerged in the temperate North American Pacific Northwest (PNW) in the late 1990s. Beyond a new environmental niche, these emergent populations displayed increased virulence and resulted in a different pattern of clinical disease. In particular, severe pulmonary infections predominated in contrast to presentation with neurologic disease as seen previously elsewhere. We employed population-level whole-genome sequencing and analysis to explore the genetic relationships and gene content of the PNW C. gattii populations. We provide evidence that the PNW strains originated from South America and identified numerous genes potentially related to habitat adaptation, virulence expression, and clinical presentation. Characterization of these genetic features may lead to improved diagnostics and therapies for such fungal infections. The data indicate that there were multiple recent introductions of C. gattii into the PNW. Public health vigilance is warranted for emergence in regions where C. gattii is not thought to be endemic

Cryptococcus gattii in North American Pacific Northwest: Whole-Population Genome Analysis Provides Insights into Species Evolution and Dispersal

The emergence of distinct populations of Cryptococcus gattii in the temperate North American Pacific Northwest (PNW) was surprising, as this species was previously thought to be confined to tropical and semitropical regions. Beyond a new habitat niche, the dominant emergent population displayed increased virulence and caused primary pulmonary disease, as opposed to the predominantly neurologic disease seen previously elsewhere. Whole-genome sequencing was performed on 118 C. gattii isolates, including the PNW subtypes and the global diversity of molecular type VGII, to better ascertain the natural source and genomic adaptations leading to the emergence of infection in the PNW. Overall, the VGII population was highly diverse, demonstrating large numbers of mutational and recombinational events; however, the three dominant subtypes from the PNW were of low diversity and were completely clonal. Although strains of VGII were found on at least five continents, all genetic subpopulations were represented or were most closely related to strains from South America. The phylogenetic data are consistent with multiple dispersal events from South America to North America and elsewhere. Numerous gene content differences were identified between the emergent clones and other VGII lineages, including genes potentially related to habitat adaptation, virulence, and pathology. Evidence was also found for possible gene introgression from Cryptococcus neoformans var. grubii that is rarely seen in global C. gattii but that was present in all PNW populations. These findings provide greater.IMPORTANCE Cryptococcus gattii emerged in the temperate North American Pacific Northwest (PNW) in the late 1990s. Beyond a new environmental niche, these emergent populations displayed increased virulence and resulted in a different pattern of clinical disease. In particular, severe pulmonary infections predominated in contrast to presentation with neurologic disease as seen previously elsewhere. We employed population-level whole-genome sequencing and analysis to explore the genetic relationships and gene content of the PNW C. gattii populations. We provide evidence that the PNW strains originated from South America and identified numerous genes potentially related to habitat adaptation, virulence expression, and clinical presentation. Characterization of these genetic features may lead to improved diagnostics and therapies for such fungal infections. The data indicate that there were multiple recent introductions of C. gattii into the PNW. Public health vigilance is warranted for emergence in regions where C. gattii is not thought to be endemic

Cryptococcus: from environmental saprophyte to global pathogen.

Cryptococcosis is a globally distributed invasive fungal infection that is caused by species within the genus Cryptococcus which presents substantial therapeutic challenges. Although natural human-to-human transmission has never been observed, recent work has identified multiple virulence mechanisms that enable cryptococci to infect, disseminate within and ultimately kill their human host. In this Review, we describe these recent discoveries that illustrate the intricacy of host-pathogen interactions and reveal new details about the host immune responses that either help to protect against disease or increase host susceptibility. In addition, we discuss how this improved understanding of both the host and the pathogen informs potential new avenues for therapeutic development

mBio

The emergence of distinct populations of Cryptococcus gattii in the temperate North American Pacific Northwest (PNW) was surprising, as this species was previously thought to be confined to tropical and semitropical regions. Beyond a new habitat niche, the dominant emergent population displayed increased virulence and caused primary pulmonary disease, as opposed to the predominantly neurologic disease seen previously elsewhere. Whole-genome sequencing was performed on 118 C. gattii isolates, including the PNW subtypes and the global diversity of molecular type VGII, to better ascertain the natural source and genomic adaptations leading to the emergence of infection in the PNW. Overall, the VGII population was highly diverse, demonstrating large numbers of mutational and recombinational events; however, the three dominant subtypes from the PNW were of low diversity and were completely clonal. Although strains of VGII were found on at least five continents, all genetic subpopulations were represented or were most closely related to strains from South America. The phylogenetic data are consistent with multiple dispersal events from South America to North America and elsewhere. Numerous gene content differences were identified between the emergent clones and other VGII lineages, including genes potentially related to habitat adaptation, virulence, and pathology. Evidence was also found for possible gene introgression from Cryptococcus neoformans var. grubii that is rarely seen in global C. gattii but that was present in all PNW populations. These findings provide greater understanding of C. gattii evolution in North America and support extensive evolution in, and dispersal from, South America. Importance: Cryptococcus gattii emerged in the temperate North American Pacific Northwest (PNW) in the late 1990s. Beyond a new environmental niche, these emergent populations displayed increased virulence and resulted in a different pattern of clinical disease. In particular, severe pulmonary infections predominated in contrast to presentation with neurologic disease as seen previously elsewhere. We employed population-level whole-genome sequencing and analysis to explore the genetic relationships and gene content of the PNW C. gattii populations. We provide evidence that the PNW strains originated from South America and identified numerous genes potentially related to habitat adaptation, virulence expression, and clinical presentation. Characterization of these genetic features may lead to improved diagnostics and therapies for such fungal infections. The data indicate that there were multiple recent introductions of C. gattii into the PNW. Public health vigilance is warranted for emergence in regions where C. gattii is not thought to be endemic.R21AI098059/AI/NIAID NIH HHS/United State

Genome Evolution and Innovation across the Four Major Lineages of Cryptococcus gattii

We acknowledge the Broad Institute Sequencing Platform and Imperial College London for generating the DNA sequence described here (and R265 Illumina sequences described previously [4]). We thank Sinéad Chapman for coordinating sequencing at the Broad Institute and Margaret Priest for assistance in submitting assemblies to NCBI. This project was supported by the National Human Genome Research Institute, grant no. U54HG003067. R.A.F. is supported by the Wellcome Trust. R.C.M. is supported by the Lister Institute for Preventive Medicine, the Medical Research Council UK, and the European Research Council.Peer reviewedPublisher PD