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Species within the human pathogenic Cryptococcus species complex are major threats to public health, causing approximately 1 million annual infections globally. Cryptococcus amylolentus is the most closely known related species of the pathogenic Cryptococcus species complex, and it is non-pathogenic. Additionally, while pathogenic Cryptococcus species have bipolar mating systems with a single large mating type (MAT) locus that represents a derived state in Basidiomycetes, C. amylolentus has a tetrapolar mating system with 2 MAT loci (P/R and HD) located on different chromosomes. Thus, studying C. amylolentus will shed light on the transition from tetrapolar to bipolar mating systems in the pathogenic Cryptococcus species, as well as its possible link with the origin and evolution of pathogenesis. In this study, we sequenced, assembled, and annotated the genomes of 2 C. amylolentus isolates, CBS6039 and CBS6273, which are sexual and interfertile. Genome comparison between the 2 C. amylolentus isolates identified the boundaries and the complete gene contents of the P/R and HD MAT loci. Bioinformatic and chromatin immunoprecipitation sequencing (ChIP-seq) analyses revealed that, similar to those of the pathogenic Cryptococcus species, C. amylolentus has regional centromeres (CENs) that are enriched with species-specific transposable and repetitive DNA elements. Additionally, we found that while neither the P/R nor the HD locus is physically closely linked to its centromere in C. amylolentus, and the regions between the MAT loci and their respective centromeres show overall synteny between the 2 genomes, both MAT loci exhibit genetic linkage to their respective centromere during meiosis, suggesting the presence of recombinational suppressors and/or epistatic gene interactions in the MAT-CEN intervening regions. Furthermore, genomic comparisons between C. amylolentus and related pathogenic Cryptococcus species provide evidence that multiple chromosomal rearrangements mediated by intercentromeric recombination have occurred during descent of the 2 lineages from their common ancestor. Taken together, our findings support a model in which the evolution of the bipolar mating system was initiated by an ectopic recombination event mediated by similar repetitive centromeric DNA elements shared between chromosomes. This translocation brought the P/R and HD loci onto the same chromosome, and further chromosomal rearrangements then resulted in the 2 MAT loci becoming physically linked and eventually fusing to form the single contiguous MAT locus that is now extant in the pathogenic Cryptococcus species.

The pathogenic species of Cryptococcus are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of ERG11 at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of Cryptococcus species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis. IMPORTANCE Opportunistic infections caused by species of the pathogenic yeast Cryptococcus lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of Cryptococcus from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of Cryptococcus or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease. Opportunistic infections caused by species of the pathogenic yeast Cryptococcus lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of Cryptococcus from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of Cryptococcus or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease.

Cryptococcus spp., in particular Cryptococcus neoformans and Cryptococcus gattii, have an enormous impact on human health worldwide. The global burden of cryptococcal meningitis is almost a quarter of a million cases and 181,000 deaths annually, with mortality rates of 100% if infections remain untreated. Despite these alarming statistics, treatment options for cryptococcosis remain limited, with only three major classes of drugs approved for clinical use. Exacerbating the public health burden is the fact that the only new class of antifungal drugs developed in decades, the echinocandins, displays negligible antifungal activity against Cryptococcus spp., and the efficacy of the remaining therapeutics is hampered by host toxicity and pathogen resistance. Here, we describe the current arsenal of antifungal agents and the treatment strategies employed to manage cryptococcal disease. We further elaborate on the recent advances in our understanding of the intrinsic and adaptive resistance mechanisms that are utilized by Cryptococcus spp. to evade therapeutic treatments. Finally, we review potential therapeutic strategies, including combination therapy, the targeting of virulence traits, impairing stress response pathways and modulating host immunity, to effectively treat infections caused by Cryptococcus spp. Overall, understanding of the mechanisms that regulate anti-cryptococcal drug resistance, coupled with advances in genomics technologies and high-throughput screening methodologies, will catalyse innovation and accelerate antifungal drug discovery.Cryptococcosis is a serious fungal infection for which treatment options are limited. In this Review, Cowen and colleagues discuss the current antifungal treatments available for cryptococcal infections, the challenges in developing new treatments, and ongoing efforts to identify novel therapies.

Cryptococcosis has been reported to be mostly associated with non-HIV-related patients in China. However, little is known about the molecular characteristics of clinical isolates from the Cryptococcus neoformans species complex in this country. In this study, 115 clinical isolates were included. Molecular type VNI was the most representative (n=103), followed by VGI (n=8), VNIII (n=2), VNIV (n=1), and VGII (n=1). With the exception of a serotype D mating type a isolate, all possessed the MATα locus. Multilocus sequence typing (MLST) revealed that most Cryptococcus gattii isolates from China shared identical MLST profiles with the most common MLST genotype reported in the VGI group, and the only one VGII isolate resembled the Vancouver Island outbreak minor genotype. The C. gattii strains involved in this study were successfully grouped according to their molecular type and mating types by PCR-restriction fragment length polymorphism (RFLP) analysis of the GEF1 gene. Our results suggest that (1) in China, cryptococcosis is mostly caused by C. neoformans var. grubii (molecular type VNI), and mating type α; (2) The most common causative agents of C. gattii infection in China are closely related to a widely distributed MLST genotype; and (3) The PCR-RFLP analysis of the GEF1 gene has the potential to identify the molecular and mating types of C. gattii simultaneously.

Cryptococcosis is caused by the opportunistic pathogen Cryptococcus neoformans or by the primary pathogen Cryptococcus gattii . Epidemiological studies suggest that patients infected with C. gattii mainly present with pulmonary disease, while those infected with C. neoformans commonly manifest meningoencephalitis. We compared the pathogenesis of the two species using the C. neoformans H99 and C. gattii R265 strains in a murine inhalation model. C. neoformans grew faster in the brain and caused death by meningoencephalitis, while C. gattii grew faster in the lungs and caused death without producing fulminating meningoencephalitis. Despite the consistent failure to recover R265 cells from blood, a fraction of the R265 population was detected in the extrapulmonary organs, including the brain. Upon intravenous (i.v. ) inoculation of 10 4 cells via the tail vein, however, C. gattii produced severe meningoencephalitis, demonstrating that C. gattii cells can efficiently cross the blood-brain barrier. Interestingly, i.v. inoculation with five cells caused brain infection in only 10% of C. gattii -infected mice, compared to 60% of mice infected with C. neoformans . In mice that had been initially inoculated via the pulmonary route and subsequently challenged intravenously, a protective effect was observed only in mice infected with C. gattii . C. neoformans cells grew 10 to 100 times faster than C. gattii cells in blood or serum collected from naive mice. The paucity of meningoencephalitis upon inhalation of C. gattii , therefore, may be partly due to an unknown factor(s) in the host’s blood coupled with immune protection that reduces dissemination to the brain and fosters lung infection. IMPORTANCE While Cryptococcus neoformans is the most common cause of fatal meningoencephalitis, especially in HIV patients, Cryptococcus gattii causes disease mainly in non-HIV patients. Clinical studies revealed that most patients infected with C. gattii VGII strains have lung infections with minimal brain involvement. Despite extensive clinicopathological studies on cryptococcosis in animal models, only a few have included C. gattii . We compared the pathogenesis of the two species in mice using an inhalation model. Similar to infection in humans, even though C. gattii can cross the blood-brain barrier, it failed to cause fatal meningoencephalitis but caused fatal lung infection. We show that growth of C. gattii in mouse blood is significantly slower than that of C. neoformans and that a secondary protective phenomenon, though weak, manifests itself only in C. gattii infection. Our study provides a model for understanding the clinicopathological differences between these two closely genetically related pathogens. While Cryptococcus neoformans is the most common cause of fatal meningoencephalitis, especially in HIV patients, Cryptococcus gattii causes disease mainly in non-HIV patients. Clinical studies revealed that most patients infected with C. gattii VGII strains have lung infections with minimal brain involvement. Despite extensive clinicopathological studies on cryptococcosis in animal models, only a few have included C. gattii . We compared the pathogenesis of the two species in mice using an inhalation model. Similar to infection in humans, even though C. gattii can cross the blood-brain barrier, it failed to cause fatal meningoencephalitis but caused fatal lung infection. We show that growth of C. gattii in mouse blood is significantly slower than that of C. neoformans and that a secondary protective phenomenon, though weak, manifests itself only in C. gattii infection. Our study provides a model for understanding the clinicopathological differences between these two closely genetically related pathogens.

Cryptococcosis is a major fungal disease leading to morbidity and mortality worldwide. Cryptococcus neoformans is a major fungal species of public health concern, causing opportunistic systemic infections in immunocompromised patients. Cryptococcus gattii was traditionally a pathogenic fungus confined primarily to tropical regions, but in the 1990s, it emerged in the temperate climates of British Columbia, Canada and the Pacific Northwest of the United States. Outbreaks in these areas also led to the first host record of cryptococcosis in free-ranging cetaceans. C. gattii is particularly concerning as an emerging fungal pathogen due to its capacity to cause clinical disease in immunocompetent patients, its recent spread to a new ecological niche, and its higher resistance to antifungal therapies. Our research defines fungal characteristics that influence the transport of cryptococci through water and persistence of fungal cells near the water surface, improving our understanding of potential mechanisms for cryptococcal environmental transport.

Cryptococcus neoformans and C. gattii are pathogenic basidiomycetous yeasts and the commonest cause of fungal infection of the central nervous system. Cryptococci are typically haploid but several inter-species, inter-varietal and intra-varietal hybrids have been reported. It has a bipolar mating system with sexual reproduction occurring normally between two individuals with opposite mating types, α and a. This study set out to characterize hybrid isolates within the C. neoformans/C. gattii species complex: seven unisexual mating intra-varietal VNI/VNII (αAAα) and six novel inter-varietal VNII/VNIV (aADα). The URA5-RFLP pattern for VNII/VNIV (aADα) differs from the VNIII (αADa) hybrids. Analysis of the allelic patterns of selected genes for AD hybrids showed 79% or more heterozygosis for the studied loci except for CBS132 (VNIII), which showed 50% of heterozygosity. MALDI-TOF MS was applied to hybrids belonging to different sero/mating type allelic patterns. All hybrid isolates were identified as belonging to the same hybrid group with identification scores ranging between 2.101 to 2.634. All hybrids were virulent when tested in the Galleria mellonella (wax moth) model, except for VNII/VNIV (aADα) hybrids. VNI/VGII hybrids were the most virulent hybrids. Hybrids recovered from larvae manifested a significant increase in capsule and total cell size and produced a low proportion (5-10%) of giant cells compared with the haploid control strains. All strains expressed the major virulence factors-capsule, melanin and phospholipase B-and grew well at 37°C. The minimal inhibitory concentration of nine drugs was measured by micro-broth dilution and compared with published data on haploid strains. MICs were similar amongst hybrids and haploid parental strains. This is the first study reporting natural same sex αAAα intra-varietal VNI/VNII hybrids and aADα inter-varietal VNII/VNIV hybrids.

Cryptococcus neoformans causes life-threatening diseases mainly in immunosuppressed hosts such as AIDS patients; C. gattii causes disseminated infections even in healthy hosts. To identify the possible molecular mechanisms underlying this difference in virulence, we investigated the survival and histopathology of lung tissue in wild-type and CD4-depleted mice infected with C. neoformans H99 and C. gattii JP02 (the highly virulent strain isolated in Japan); we then compared dendritic cell (DC) cytokine release responses to different cell fractions from these two strains. JP02-infected mice exhibited shorter survival and fewer inflammatory cells in the lung than H99-infected control mice. Depletion of CD4-related cellular immunity reduced survival of H99-infected mice but had no effect on the survival or inflammatory cell infiltration in JP02-infected mice, suggesting that JP02 evades immune detection. To identify the molecule(s) conferring this difference, we measured cytokine production from murine DCs co-cultured with H99 and JP02 in vitro. The levels of inflammatory cytokines from DCs treated with intact JP02 cells, the extracted capsule, secreted extracellular polysaccharides, and purified glucuronoxylomannan (GXM) were markedly lower than those induced by intact H99 cells and corresponding H99 fractions. Structural analysis of GXM indicated that JP02 altered one of two O-acetyl groups detected in the H99 GXM. Deacetylated GXM lost the ability to induce inflammatory cytokine release from DCs, implicating these O-acetyl groups in immune recognition. We conclude that the highly virulent C. gattii processes a structural alteration in GXM that allows this pathogen to evade the immune response and therefore elimination.

Pathogenic microbes confront an evolutionary conflict between the pressure to maintain genome stability and the need to adapt to mounting external stresses. Bacteria often respond with elevated mutation rates, but little evidence exists of stable eukaryotic hypermutators in nature. Whole genome resequencing of the human fungal pathogen Cryptococcus deuterogattii identified an outbreak lineage characterized by a nonsense mutation in the mismatch repair component MSH2. This defect results in a moderate mutation rate increase in typical genes, and a larger increase in genes containing homopolymer runs. This allows facile inactivation of genes with coding homopolymer runs including FRR1, which encodes the target of the immunosuppresive antifungal drugs FK506 and rapamycin. Our study identifies a eukaryotic hypermutator lineage spread over two continents and suggests that pathogenic eukaryotic microbes may experience similar selection pressures on mutation rate as bacterial pathogens, particularly during long periods of clonal growth or while expanding into new environments. As humans, we often think of genetic mutations as being bad. Over the past several decades we have seen health warnings issued on a variety of environmental exposures, from cigarettes to tanning beds, and with good reason because they cause mutations. For multicellular organisms like humans, these mutations are strongly associated with cancer. But in bacteria, this is not true. In fact, the rate at which mutations occur sometimes increases to help bacteria cope with stressful environments. Unlike bacteria, humans are eukaryotes – the name given to organisms whose cells contain different compartments separated by membranes, such as the nucleus of the cell. For years, we have assumed that eukaryotic microbes, like fungi and parasites, act more like humans than like bacteria because work in budding yeast (another eukaryote) has suggested this to be the case. However, recent work in disease-causing fungi has shown that, much like bacteria, elevated mutation rates may help them to respond to stress. This could also enable fungi to become resistant to drugs used to treat fungal infections. Cryptococcus deuterogattii is a fungus that causes human diseases including meningoencephalitis and a lung infection called pulmonary cryptococcosis. An ongoing outbreak of the fungus began in the Pacific Northwest of Canada in the late 1990s and emerged in the United States in 2006/2007. Among isolates closely related to those fungi causing the outbreak, three were found that appear to have a specific mutation in their DNA mismatch repair pathway, meaning that they may also experience a higher mutation rate. These strains are also less able to cause disease than others. Billmyre et al. now demonstrate experimentally that all three isolates have a specific DNA mismatch repair defect, and show that these fungi experience elevated mutation rates, resulting in what is known as a hypermutator state. Furthermore, whole genome sequencing and phylogenetic analysis showed that these hypermutator strains are derived from the outbreak-causing fungi, and that their reduced ability to cause disease is likely a result of accumulating mutations and the loss of the ability to grow at the higher temperatures found in the human body. Fungal infections are difficult to treat, in part because there are a limited number of available drugs. Elevated mutation rates will likely increase how often and how rapidly fungi develop resistance to these drugs. Understanding how commonly fungi exhibit a hypermutator state that could impact the development of drug resistance will therefore be important for treating patients with fungal infections, which account for millions of infections and hundreds of thousands of deaths annually worldwide.

‘Same-sex’ mating game A virulent strain of the fungus Cryptococcus gattii emerged on Vancouver Island, Canada in 1999, causing an outbreak of meningoencephalitis, and it is still infecting humans and animals in the region. This was a surprise: C. gattii is normally restricted to the tropics where it grows on eucalyptus trees and only occasionally infects animals and humans. Geneticists now report that the outbreak was caused by genetic combination of the mildly pathogenic Australian incomer with another isolate of an unknown origin. Remarkably, these fungi combined via sexual reproduction to produce a hypervirulent strain, despite the fact that both were of the same ‘sex’. Both are classified ‘α’ rather than ‘a’ strains. It remains to be seen if such mating is common in the wild, and if it occurs in other parasites, such as Trypanosoma , Leishmania and Plasmodium falciparum , where similar α/a mating types are found. Genealogy can illuminate the evolutionary path of important human pathogens. In some microbes, strict clonal reproduction predominates, as with the worldwide dissemination of Mycobacterium leprae , the cause of leprosy 1 . In other pathogens, sexual reproduction yields clones with novel attributes, for example, enabling the efficient, oral transmission of the parasite Toxoplasma gondii 2 . However, the roles of clonal or sexual propagation in the origins of many other microbial pathogen outbreaks remain unknown, like the recent fungal meningoencephalitis outbreak on Vancouver Island, Canada, caused by Cryptococcus gattii 3 . Here we show that the C. gattii outbreak isolates comprise two distinct genotypes. The majority of isolates are hypervirulent and have an identical genotype that is unique to the Pacific Northwest. A minority of the isolates are significantly less virulent and share an identical genotype with fertile isolates from an Australian recombining population. Genotypic analysis reveals evidence of sexual reproduction, in which the majority genotype is the predicted offspring. However, instead of the classic a–α sexual cycle, the majority outbreak clone appears to have descended from two α mating-type parents. Analysis of nuclear content revealed a diploid environmental isolate homozygous for the major genotype, an intermediate produced during same-sex mating. These studies demonstrate how cryptic same-sex reproduction can enable expansion of a human pathogen to a new geographical niche and contribute to the ongoing production of infectious spores. This has implications for the emergence of other microbial pathogens and inbreeding in host range expansion in the fungal and other kingdoms.