Whole Genome Sequence Analysis of Cryptococcus gattii from the Pacific Northwest Reveals Unexpected Diversity

A recent emergence of Cryptococcus gattii in the Pacific Northwest involves strains that fall into three primarily clonal molecular subtypes: VGIIa, VGIIb and VGIIc. Multilocus sequence typing (MLST) and variable number tandem repeat analysis appear to identify little diversity within these molecular subtypes. Given the apparent expansion of these subtypes into new geographic areas and their ability to cause disease in immunocompetent individuals, differentiation of isolates belonging to these subtypes could be very important from a public health perspective. We used whole genome sequence typing (WGST) to perform fine-scale phylogenetic analysis on 20 C. gattii isolates, 18 of which are from the VGII molecular type largely responsible for the Pacific Northwest emergence. Analysis both including and excluding (289,586 SNPs and 56,845 SNPs, respectively) molecular types VGI and VGIII isolates resulted in phylogenetic reconstructions consistent, for the most part, with MLST analysis but with far greater resolution among isolates. The WGST analysis presented here resulted in identification of over 100 SNPs among eight VGIIc isolates as well as unique genotypes for each of the VGIIa, VGIIb and VGIIc isolates. Similar levels of genetic diversity were found within each of the molecular subtype isolates, despite the fact that the VGIIb clade is thought to have emerged much earlier. The analysis presented here is the first multi-genome WGST study to focus on the C. gattii molecular subtypes involved in the Pacific Northwest emergence and describes the tools that will further our understanding of this emerging pathogen

Division of labour in response to host oxidative burst drives a fatal Cryptococcus gattii outbreak

Cryptococcus gattii is an emerging intracellular pathogen and the cause of the largest primary outbreak of a life-threatening fungal disease in a healthy population. Outbreak strains share a unique mitochondrial gene expression profile and an increased ability to tubularize their mitochondria within host macrophages. However, the underlying mechanism that causes this lineage of C. gattii to be virulent in immunocompetent individuals remains unexplained. Here we show that a subpopulation of intracellular C. gattii adopts a tubular mitochondrial morphology in response to host reactive oxygen species. These fungal cells then facilitate the rapid growth of neighbouring C. gattii cells with non-tubular mitochondria, allowing for effective establishment of the pathogen within a macrophage intracellular niche. Thus, host reactive oxygen species, an essential component of the innate immune response, act as major signalling molecules to trigger a ‘division of labour’ in the intracellular fungal population, leading to increased pathogenesis within this outbreak lineage

Fatal Disseminated Cryptococcus gattii Infection in New Mexico

We report a case of fatal disseminated infection with Cryptococcus gattii in a patient from New Mexico. The patient had no history of recent travel to known C. gattii-endemic areas. Multilocus sequence typing revealed that the isolate belonged to the major molecular type VGIII. Virulence studies in a mouse pulmonary model of infection demonstrated that the strain was less virulent than other C. gattii strains. This represents the first documented case of C. gattii likely acquired in New Mexico

Gene expression in fungi

This contribution is based on the four presentations made at the Special Interest Group (SIG) meeting titled Gene Expression in Fungi held during IMC9 in Edinburgh. This overview is independent from other articles published or that will be published by each speaker. In the SIG meeting, basic principles of in vivo animal models for virulence studies were discussed. Infection associated genes of Candida albicans and fungal adaptation to the host was summarized. Azole susceptibility was evaluated as a combined result of several changes in expression of pertinent genes. Gene transfer in fungi, resulting in fungal evolution and gene adaptation to environmental factors, was reported

Automated Analysis of Cryptococcal Macrophage Parasitism Using GFP-Tagged Cryptococci

The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic immune effector cells, a phenomenon that correlates strongly with virulence in rodent models of infection. Despite the importance of phagocyte/Cryptococcus interactions to disease progression, current methods for assaying virulence in the acrophage system are both time consuming and low throughput. Here, we introduce the first stable and fully characterised GFP–expressing derivatives of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Both strains show unaltered responses to environmental and host stress conditions and no deficiency in virulence in the macrophage model system. In addition, we report the development of a method to effectively and rapidly investigate macrophage parasitism by flow cytometry, a technique that preserves the accuracy of current approaches but offers a four-fold improvement in speed

MALDI-TOF MS Enables the Rapid Identification of the Major Molecular Types within the Cryptococcus neoformans/C. gattii Species Complex

BACKGROUND: The Cryptococcus neoformans/C. gattii species complex comprises two sibling species that are divided into eight major molecular types, C. neoformans VNI to VNIV and C. gattii VGI to VGIV. These genotypes differ in host range, epidemiology, virulence, antifungal susceptibility and geographic distribution. The currently used phenotypic and molecular identification methods for the species/molecular types are time consuming and expensive. As Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) offers an effective alternative for the rapid identification of microorganisms, the objective of this study was to examine its potential for the identification of C. neoformans and C. gattii strains at the intra- and inter-species level. METHODOLOGY: Protein extracts obtained via the formic acid extraction method of 164 C. neoformans/C. gattii isolates, including four inter-species hybrids, were studied. RESULTS: The obtained mass spectra correctly identified 100% of all studied isolates, grouped each isolate according to the currently recognized species, C. neoformans and C. gattii, and detected potential hybrids. In addition, all isolates were clearly separated according to their major molecular type, generating greater spectral differences among the C. neoformans molecular types than the C. gattii molecular types, most likely reflecting a closer phylogenetic relationship between the latter. The number of colonies used and the incubation length did not affect the results. No spectra were obtained from intact yeast cells. An extended validated spectral library containing spectra of all eight major molecular types was established. CONCLUSIONS: MALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic Cryptococcus species and offers a simple method for the separation of the eight major molecular types and the detection of hybrid strains within this species complex in the clinical laboratory. The obtained mass spectra provide further evidence that the major molecular types warrant variety or even species status

Clonality and α-a Recombination in the Australian Cryptococcus gattii VGII Population - An Emerging Outbreak in Australia

BACKGROUND: Cryptococcus gattii is a basidiomycetous yeast that causes life-threatening disease in humans and animals. Within C. gattii, four molecular types are recognized (VGI to VGIV). The Australian VGII population has been in the spotlight since 2005, when it was suggested as the possible origin for the ongoing outbreak at Vancouver Island (British Columbia, Canada), with same-sex mating being suggested as the driving force behind the emergence of this outbreak, and is nowadays hypothesized as a widespread phenomenon in C. gattii. However, an in-depth characterization of the Australian VGII population is still lacking. The present work aimed to define the genetic variability within the Australian VGII population and determine processes shaping its population structure. METHODOLOGY/PRINCIPAL FINDINGS: A total of 54 clinical, veterinary and environmental VGII isolates from different parts of the Australian continent were studied. To place the Australian population in a global context, 17 isolates from North America, Europe, Asia and South America were included. Genetic variability was assessed using the newly adopted international consensus multi-locus sequence typing (MLST) scheme, including seven genetic loci: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1. Despite the overall clonality observed, the presence of MATa VGII isolates in Australia was demonstrated for the first time in association with recombination in MATα-MATa populations. Our results also support the hypothesis of a "smouldering" outbreak throughout the Australian continent, involving a limited number of VGII genotypes, which is possibly caused by a founder effect followed by a clonal expansion. CONCLUSIONS/SIGNIFICANCE: The detection of sexual recombination in MATα-MATa population in Australia is in accordance with the natural life cycle of C. gattii involving opposite mating types and presents an alternative to the same-sex mating strategy suggested elsewhere. The potential for an Australian wide outbreak highlights the crucial issue to develop active surveillance procedures.Fabian Carriconde, Félix Gilgado, Ian Arthur, David Ellis, Richard Malik, Nathalie van de Wiele, Vincent Robert, Bart J. Currie, Wieland Meye

Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence