Neoformans Species Complex Research Articles

Illuminating the Cryptococcus neoformans species complex: unveiling intracellular structures with fluorescent-protein-based markers.

Cryptococcus neoformans is a fungal pathogen of the top critical priority recognized by the World Health Organization. This clinically important fungus also serves as a eukaryotic model organism. A variety of resources have been generated to facilitate investigation of the C. neoformans species complex, including congenic pairs, well-annotated genomes, genetic editing tools, and gene deletion sets. Here, we generated a set of strains with all major organelles fluorescently marked. We tested short organelle-specific targeting sequences and successfully labeled the following organelles by fusing the targeting sequences with a fluorescence protein: the plasma membrane, the nucleus, the peroxisome, and the mitochondrion. We used native cryptococcal Golgi and late endosomal proteins fused with a fluorescent protein to label these two organelles. These fluorescence markers were verified via colocalization using organelle-specific dyes. All the constructs for the fluorescent protein tags were integrated in an intergenic safe haven region. These organelle-marked strains were examined for growth and various phenotypes. We demonstrated that these tagged strains could be employed to track cryptococcal interaction with the host in phagocytosis assays. These strains also allowed us to discover remarkable differences in the dynamics of proteins targeted to different organelles during sexual reproduction. Additionally, we revealed that "dormant" spores transcribed and synthesized their own proteins and trafficked the proteins to the appropriate subcellular compartments, demonstrating that spores are metabolically active. We anticipate that these newly generated fluorescent markers will greatly facilitate further investigation of cryptococcal biology and pathogenesis.

Multi-locus sequence typing and phylogenetics of Cryptococcus neoformans AD hybrids

Hybrid AD strains of the human pathogenic Cryptococcus neoformans species complex have been reported from many parts of the world. However, their origin, diversity, and evolution are incompletely understood. In this study, we analyzed 102 AD hybrid strains representing 21 countries on five continents. For each strain, we obtained its mating type and its allelic sequences at each of the seven loci that have been used for genotyping haploid serotypes A and D strains of the species complex by the Cryptococcus research community. Our results showed that most AD hybrids exhibited loss of heterozygosity at one or more of the seven analyzed loci. Phylogenetic and population genetic analyses of the allelic sequences revealed multiple origins of the hybrids within each continent, dating back to one million years ago in Africa and up to the present in other continents. We found evidence for clonal reproduction and long-distance dispersal of these hybrids in nature. Comparisons with the global haploid serotypes A and D strains identified new alleles and new haploid multi-locus genotypes in AD hybrids, consistent with the presence of yet-to-be discovered genetic diversity in haploid populations of this species complex in nature. Together, our results indicate that AD hybrids can be effectively genotyped using the same multi-locus sequencing type approach as that established for serotypes A and D strains. Our comparisons of the AD hybrids among each other as well as with the global haploid serotypes A and D strains revealed novel genetic diversity as well as evidence for multiple origins and dynamic evolution of these hybrids in nature.

Genotypic diversity and antifungal susceptibility of Cryptococcus neoformans species complex from China, including the diploid VNIII isolates from HIV-infected patients in Chongqing region.

Although previous studies on the genotypic diversity and antifungal susceptibility of the Cryptococcus neoformans species complex (CNSC) isolates from China revealed ST5 genotype isolates being dominant, the information of the CNSC isolates from Chinese HIV-infected patients is limited. In this study, 171 CNSC isolates from HIV-infected patients in the Chongqing region of Southwest China were genotyped using the International Society for Human and Animal Mycology-multilocus sequence typing (ISHAM-MLST) consensus scheme, and their antifungal drug susceptibilities were determined following CLSI M27-A3 guidelines. Among 171 isolates, six sequence types (STs) were identified, including the dominant ST5 isolates, the newly reported ST15, and four diploid VNIII isolates (ST632/ST636). Moreover, a total of 1019 CNSC isolates with STs and HIV-status information were collected and analyzed from mainland China in the present study. A minimum spanning analysis grouped these 1019 isolates into three main subgroups, which were dominated by the ST5 clonal complex (CC5), followed by the ST31 clonal complex (CC31) and ST93 clonal complex (CC93). The trend of resistance or decreasing susceptibility of clinical CNSC isolates to azole agents within HIV-infected patients from the Chongqing region is increasing, especially resistance to fluconazole (FLC).

Connecting Cryptococcal Meningitis and Gut Microbiome.

Fungal pathogens of the Cryptococcus neoformans species complex (C. neoformans SC) are a major cause of fungal meningitis in immunocompromised individuals. As with other melanotic microorganisms associated with human diseases, the cell-wall-associated melanin of C. neoformans SC is a major virulence factor that contributes to its ability to evade host immune responses. The levels of melanin substrate and the regulation of melanin formation could be influenced by the microbiota-gut-brain axis. Moreover, recent studies show that C. neoformans infections cause dysbiosis in the human gut microbiome. In this review, we discuss the potential association between cryptococcal meningitis and the gut microbiome. Additionally, the significant potential of targeting the gut microbiome in the diagnosis and treatment of this debilitating disease is emphasized.

Immunological Analysis of Cryptococcal Meningoencephalitis in a Murine Model.

Cryptococcal meningoencephalitis (CM), caused by the fungal pathogen Cryptococcus neoformans species complex, can lead to high mortality or severe neurological sequelae in survivors that are associated with excessive inflammation in the central nervous system (CNS), especially in those who develop immune reconstitution inflammatory syndrome (IRIS) or postinfectious immune response syndrome (PIIRS). While the means to establish a cause-and-effect relationship of a specific pathogenic immune pathway during CM by human studies are limited, mouse models allow dissection of the potential mechanistic links within the CNS immunological network. In particular, these models are useful for separating pathways contributing predominantly to immunopathology from those important for fungal clearance. In this protocol, we described methods to induce a robust, physiologically relevant murine model of C. neoformans CNS infection that reproduces multiple aspects of human cryptococcal disease immunopathology and subsequent detailed immunological analysis. Combined with tools including gene knockout mice, antibody blockade, cell adoptive transfer, as well as high throughput techniques such as single-cell RNA sequencing, studies using this model will provide new insights regarding the cellular and molecular processes that elucidate the pathogenesis of cryptococcal CNS diseases in order to develop more effective therapeutic strategies.

Antifungal susceptibility and molecular characteristics of Cryptococcus spp. based on whole-genome sequencing in Zhejiang Province, China.

Cryptococcus spp. is a complex species that often causes cryptococcosis, which is one of the most common opportunistic infections in adults living with HIV and has very high morbidity and mortality rates. This study aimed to investigate the antifungal susceptibility profiles and epidemiological characteristics of the Cryptococcus neoformans species complex (CNSC) and the Cryptococcus gattii species complex (CGSC) in Zhejiang Province, China. A total of 177 CNSC and 3 CGSC isolates were collected, and antifungal susceptibility was tested by FUNGUS 3 and verified with an E-test. Moreover, multiple classification methods and genomic analyses were performed. The majority of the isolates (96.11%) were C. neoformans (formerly C. neoformans var. grubii) (ST5-VNI-A-α). Our study highlights that most of the patients with cryptococcosis were non-HIV patients in China, and nearly half of them did not have underlying diseases that led to immune insufficiency. Most of the Cryptococcus spp. isolates in this study were sensitive to common antifungal drugs. Two 5-flucytosine (5-FC)-resistant strains were identified, and FUR1 mutation was detected in the 5-FC-resistant isolates. Typing based on whole-genome sequencing (WGS) showed better discrimination than that achieved with multilocus sequence typing (MLST) and indicated a clear population structure. A phylogenetic analysis based on WGS included more genomic information than traditional classification methods.

Analyses of the Global Multilocus Genotypes of the Human Pathogenic Yeast Cryptococcus neoformans Species Complex.

Cryptococcus neoformans species complex (CNSC) is a globally distributed human opportunistic yeast pathogen consisting of five major molecular types (VNI, VNII, VNB, VNIII and VNIV) belonging to two species, C. neoformans (VNI, VNII and VNB, collectively called serotype A) and C. deneoformans (VNIV, commonly called serotype D), and their hybrids (VNIII, serotype AD). Over the years, many studies have analyzed the geographical distribution and genetic diversity of CNSC. However, the global population structure and mode of reproduction remain incompletely described. In this study, we analyze the published multilocus sequence data at seven loci for CNSC. The combined sequences at the seven loci identified a total of 657 multilocus sequence types (STs), including 296 STs with known geographic information, representing 4200 non-redundant isolates from 31 countries and four continents. Among the 296 STs, 78 and 52 were shared among countries and continents, respectively, representing 3643 of the 4200 isolates. Except for the clone-corrected serotype D sample among countries, our analysis of the molecular variance of the 4200 isolates revealed significant genetic differentiations among countries and continents in populations of CNSC, serotype A, and serotype D. Phylogenetic analyses of the concatenated sequences of all 657 STs revealed several large clusters corresponding to the major molecular types. However, several rare but distinct STs were also found, representing potentially novel molecular types and/or hybrids of existing molecular types. Phylogenetic incompatibility analyses revealed evidence for recombination within all four major molecular types-VNI, VNII, VNIV and VNB-as well as within two VNB subclades, VNBI and VNBII, and two ST clusters around the most common STs, ST5 and ST93. However, linkage disequilibrium analyses rejected the hypothesis of random recombination across most samples. Together, our results suggest evidence for historical differentiation, frequent recent gene flow, clonal expansion and recombination within and between lineages of the global CNSC population.

S1.3c Diversity and hybridization in Malassezia furfur

S1.3 Malassezia: genetics, genomics, and biology, September 21, 2022, 11:00 AM - 12:30 PM The Basidiomycetous yeast Malassezia is the most abundant fungal genus on healthy human skin but may also cause various skin disorders such as seborrheic dermatitis, dandruff, and pityriasis versicolor. In recent years, Malassezia has increasingly been implicated in health and disease beyond the skin: as an underestimated cause of Malassezia bloodstream infections (BSIs) in immunocompromised patients and neonates, associated with Crohn's disease, promoting pancreatic oncogenesis, and exacerbating cystic fibrosis. Malassezia furfur is the number one Malassezia BSI cause and is also implicated in many skin disorders. With these new discoveries of Malassezia’s impact on human health, the need for a better understanding of its evolution and pathobiology also became more pressing. Hybridization has been suggested as a biological mechanism of adaptation to new hosts, and may lead to increased pathogenicity. Many examples of major hybrid yeast pathogens exist, such as Candida albicans, C. orthopsilosis, C. metapsilosis, and multiple examples in the Cryptococcus gattii/Cryptococcus neoformans species complex. Here the multiple hybridization events of the Malassezia furfur species complex will be discussed. Two distinct hybridization events occurred between the same parental lineages, and these parental strains were originally also hybrids. The identification of a pseudobipolar mating system and the analysis of the mating-type loci provide evidence that sexual liaisons of mating compatible cells from these parental lineages led to a diploid/aneuploid state in the hybrid lineages. Sequence similarity percentages suggest that both parental lineages in fact are two different species. The genetic diversity of ca 300 strains belonging to this species complex is evaluated in relationship to host background and phenotype.

Cryptococcus gattii Species Complex as an Opportunistic Pathogen: Underlying Medical Conditions Associated with the Infection.

ABSTRACTThe Cryptococcus gattii species complex has often been referred to as a primary pathogen due to its high infection frequency among apparently immunocompetent patients. In order to scrutinize the immune status of patients and the lineages of etiologic agents, we analyzed patient histories and the molecular types of etiologic agents from 135 global C. gattii cases. Eighty-six of 135 patients had been diagnosed as immunocompetent, although some of them had underlying medical issues, and 49 were diagnosed as immunocompromised with risk factors similar to those seen in Cryptococcus neoformans infection. We focused on the 86 apparently immunocompetent patients and were able to obtain plasma from 32 (37%) to analyze for the presence of autoantibodies against the granulocyte-macrophage colony-stimulating factor (GM-CSF) since these antibodies have been reported as a hidden risk factor for C. gattii infection. Among the 32 patients, 25 were free from any known other health issues, and 7 had various medical conditions at the time of diagnosis for cryptococcosis. Importantly, plasma from 19 (76%) of 25 patients with no recognized underlying medical condition showed the presence of GM-CSF autoantibodies, supporting this antibody as a major hidden risk factor for C. gattii infection. These data indicate that seemingly immunocompetent people with C. gattii infection warrant detailed evaluation for unrecognized immunologic risks. There was no relationship between molecular type and underlying conditions of patients. Frequency of each molecular type was related to its geographic origin exemplified by the overrepresentation of VGIV in HIV-positive (HIV+) patients due to its prevalence in Africa.

Phenotypic Plasticity in the Productions of Virulence Factors Within and Among Serotypes in the Cryptococcus neoformans Species Complex.

The Cryptococcus neoformans species complex (CNSC) is a common opportunistic human fungal pathogen and the most frequent cause of fungal meningitis. There are three major serotypes in CNSC: A, D, and their hybrids AD, and they have different geographic distributions and medical significance. Melanin pigment and a polysaccharide capsule are the two major virulence factors in CNSC. However, the relationships between serotype and virulence factor production and how environmental factors might impact their relationships are not known. This study investigated the expressions of melanin and capsular polysaccharide in a genetically diverse group of CNSC strains and how their phenotypic expressions were influenced by oxidative and nitrosative stress levels. We found significant differences in melanin and capsular polysaccharide productions among serotypes and across stress conditions. Under oxidative stress, the laboratory hybrids exhibited the highest phenotypic plasticity for melanin production while serotype A showed the highest for capsular polysaccharide production. In contrast, serotype D exhibited the highest phenotypic plasticity for capsular polysaccharide production and clinical serotype AD the highest phenotypic plasticity for melanin production under nitrosative stress. These results demonstrated that different serotypes have different environmental condition-specific mechanisms to modulate the expression of virulence factors.

Environmental Status of Cryptococcus neoformans and Cryptococcus gattii in Colombia.

The genus Cryptococcus comprises more than 80 species, including C. neoformans and C. gattii, which are pathogenic to humans, mainly affecting the central nervous system. The two species differ in geographic distribution and environmental niche. C. neoformans has a worldwide distribution and is often isolated from bird droppings. On the contrary, C. gattii is reported in tropical and subtropical regions and is associated with Eucalyptus species. This review aims to describe the distribution of environmental isolates of the Cryptococcus neoformans species complex and the Cryptococcus gattii species complex in Colombia. A systematic investigation was carried out using different databases, excluding studies of clinical isolates reported in the country. The complex of the species of C. gattii is recovered mainly from trees of the genus Eucalyptus spp., while the complex of the species of C. neoformans is recovered mainly from avian excrement, primarily Columba livia (pigeons) excrement. In addition, greater positivity was found at high levels of relative humidity. Likewise, an association was observed between the presence of the fungus in places with little insolation and cold or temperate temperatures compared to regions with high temperatures.

Cryptococcosis and Tuberculosis Coinfection in a Regional Reference Service of HIV-AIDS from Southern Brazil

Cryptococcosis (Cr) and tuberculosis (TB) are infectious diseases potentially fatal to HIV-AIDS patients, and coinfection with Cr/TB in endemic regions may represent a new challenge in the panorama of the HIV-AIDS pandemic. This retrospective study aims to describe the clinic-epidemiological data of cryptococcosis and tuberculosis (Cr/TB) coinfection in HIV-AIDS patients from Southern Brazil. A retrospective study including all cases of coinfection by the Cryptococcus neoformans species complex and Mycobacterium tuberculosis, diagnosed in HIV-AIDS patients at the Specialized Customer Service of University Hospital - Federal University of Rio Grande (SAE-HU-FURG), during the period January 2010 to December 2012 was performed. During the 3 years of study, ten cases of Cr/TB coinfection were detected. The mean age was 39.3 years and 80% were male. Cr meningitis with pulmonary TB occurred in 40% of the cases and pulmonary presentation in both diseases occurred in 30%. The mean TCD4 + rate was 118.6 cel/mm3 (SD = 135.36), with 80% of the patients maintaining TCD4 + < 100 cells/mm3. The viral load presented by 70% of the patients was equal or higher than 100,000 copies/mm3. Four patients (40%) died on an average, 29 days (SD = 8.75) after diagnosis of the coinfection. The study shows the necessity of a broad diagnostic investigation in the context of Cr/TB/HIV-AIDS coinfection.

Correlation between clinical outcome and tissue inflammatory response in kidney transplant recipients with cryptococcosis.

Cryptococcosis is the second most common invasive fungal infection reported in renal transplant recipients. Tissue granulomatous inflammation is necessary to contain Cryptococcus infection. This study aims to analyze the granuloma patterns and in situ expression of regulatory T (Treg) immune response in tissue samples from 12 renal transplant recipients with cryptococcosis. Fungal isolates were molecularly identified as Cryptococcus neoformans species complex. A detailed characterization of granulomas in tissue samples from 12 kidney transplant recipients with cryptococcosis was described by checking six lung and six skin biopsies by conventional histology and for immunohistochemical detection of CD4 and Treg markers: forkhead box P3 (FoxP3), interleukin (IL)-10 and transforming-growth factor (TGF)-β. Granulomas were classified as compact, loose or mixed. Patients with mixed (n=4) and compact (n=3) granulomatous inflammation patterns were associated with a better prognosis and presented a higher number of CD4+FoxP3+T cells compared to the group of patients with loose granulomas. In counterpart, three out of five patients with loose granulomas died with cryptococcosis. We suggest that Treg may have a protective role in the tissue response to Cryptococcus infection given its association with compact and mixed granulomas in patients with better clinical outcomes.

On the History and Applications of Congenic Strains in Cryptococcus Research.

Congenic strains have been utilized in numerous model organisms to determine the genetic underpinning of various phenotypic traits. Congenic strains are usually derived after 10 backcrosses to a recipient parent, at which point they are 99.95% genetically identical to the parental strain. In recent decades, congenic pairs have provided an invaluable tool for genetics and molecular biology research in the Cryptococcus neoformans species complex. Here, we summarize the history of Cryptococcus congenic pairs and their application in Cryptococcus research on topics including the impact of the mating type locus on unisexual reproduction, virulence, tissue tropism, uniparental mitochondrial inheritance, and the genetic underpinning of other various traits. We also discuss the limitations of these approaches and other biological questions, which could be explored by employing congenic pairs.

In vitro activity of isavuconazole against clinically isolated yeasts from Chile.

Isavuconazole is the last antifungal agent approved by the FDA and available for treatment of fungal infections. In the present study, the in vitro activity of isavuconazole against several yeasts was investigated. Two hundred forty-six isolates were included: 64 Candida albicans, 53 Candida parapsilosis sensu stricto, 48 Cryptococcus neoformans species complex, 27 C. glabrata sensu stricto, 17 C. lusitaniae, 17 C. tropicalis, 5 C. orthopsilosis, 4 C. krusei, 3 C. guilliermondii sensu stricto, 3 C. pelliculosa, 2 C. dubliniensis, 1 C. auris, 1 C. fermentati and 1 Trichosporon asahii. All isolates were recovered from clinical isolates from Chile, being 221 from hemoculture, 22 from cerebrospinal fluid, 1 pleural fluid, and 1 from tissue culture. The minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs) of isavuconazole were determined. Isavuconazole demonstrated good in vitro activity against all species tested. MIC90 values and MFC ranges of isavuconazole for Candida albicans were 0.03mg/L and 0.03- > 16mg/L respectively. Non-Candida albicans species isolates were inhibited by ≤ 1mg/L, with MFC ranges from < 0.03- > 16mg/L. Also, isavuconazole was active against the non-Candida yeasts, being inhibited with MIC values ≤ 0.06mg/L. Isavuconazole has exhibited potent in vitro activity against clinical isolates of Candida spp., Cryptococcus neoformans species complex, and other clinical yeast in Chile. Despite the results obtained in the present work, additional clinical studies are necessary to verify the level of efficacy of this azole.

New multilocus sequence typing primers to enable genotyping of AD hybrids within the Cryptococcus neoformans species complex.

We designed and tested new specific primers to amplify the two alleles of each of the seven MLST loci in C. neoformans species complex hybrids. The sequences obtained from hybrids can be compared with those present in the Cryptococcus global MLST database for future molecular epidemiology studies.

Cryptococcus Neoformans Species Complex Isolated for the First Time from the Environment in Croatia

Cryptococcus Neoformans Species Complex Isolated for the First Time from the Environment in Croatia

Cryptococcus neoformans species complex isolates living in a tree micro-ecosystem

Abstract An old oak tree (Quercus robur), colonized by two different lineages of the Cryptococcus neoformans species complex (VNI and VNIV), was investigated to determine the distribution of cryptococcal yeasts in the different micro-habitats of the tree. The yeasts were isolated from most of the tree micro-habitats such as bark, trunk cavities, soil beneath the tree, arthropods living on the tree, and air. Sunlight exposure and low temperatures decreased in vitro growth of VNI isolates but not that of VNIV ones. Both VNI and VNIV strains isolated from the tree were able to produce basidiospores by mating assays. The investigated tree represents an environmental reservoir of C. neoformans, where it is able to survive and probably to reproduce, and from where blastospores and basidiospores, exposed to wind, water and contact with arthropods, birds, and mammals, may be dispersed in the environment as potential infectious propagules.

Cryptococcus genetic diversity and mixed infections in Ivorian HIV patients: A follow up study.

Genetic diversity analyses were performed by sero-genotyping and multi-locus sequence typing on 252 cryptococcal isolates from 13 HIV-positive Ivorian patients followed-up for cryptococcal meningitis. Antifungal susceptibility analyses were performed according to the CLSI M27A3 method. The majority (67.8%) of the isolates belonged to the Cryptococcus neoformans (serotype A) species complex, with 93% being VNI and 7% being VNII. Cryptococcus deuterogattii VGII (serotype B) represented 16.7% of the strains, while C. neoformans/C. deneoformans VNIII (serotype AD) hybrids accounted for 15.1% of the strains. One strain (0.4%) was not identifiable. Nine different sequence types (STs 5, 6, 23, 40, 93, 207, 311, and a new ST; 555) were identified in the C. neoformans population, while the C. deuterogattii population comprised the single ST 173. The distribution of the strains showed that, while the majority of patients (9/13) harboured a single sequence type, 4 patients showed mixed infections. These patients experienced up to 4 shifts in strain content either at the species and/or ST level during their follow-up. This evolution of diversity over time led to the co-existence of up to 3 different Cryptococcus species and 4 different ST within the same individual during the course of infection. Susceptibility testing showed that all strains were susceptible to amphotericin B while 3.6% of them had a none-wild type phenotype to 5-flucytosine. Concerning fluconazole, 2.9% of C.neoformans serotype A strains and 2.4% of C. deuterogattii had also respectively a none-wild type phenotype to this molecule. All C. neoformans x C. deneoformans serotype AD hybrids had however a wild type phenotype to fluconazole. The present study showed that mixed infections exist and could be of particular importance for care outcomes. Indeed, (i) the different Cryptococcus species are known to exhibit different virulence and different susceptibility patterns to antifungal drugs and (ii) the strains genetic diversity within the samples may influence the susceptibility to antifungal treatment.

Production of a latex agglutination reagent for the rapid diagnosis of cryptococcal meningitis

Cryptococcosis is a fungal disease affecting more than one million people per year worldwide. Its main etiological agents are Cryptococcus neoformans species complex and Cryptococcus gattii species complex. Cryptococcal meningitis (CM) is considered an AIDS-defining condition. Rapid diagnosis by cryptococcal antigen assays, either the latex agglutination test (LA) or the lateral flow assay, is key to decreasing mortality due to cryptococcal disease. The aim of the study was to develop a latex agglutination reagent (LA-ANLIS) for the rapid and reliable diagnosis of cryptococcosis in Argentina. This reagent will be produced in order to supply the NMLN (National Mycology Laboratory Network). The evaluation of LA-ANLIS performance and its comparison with the Cryptococcus Antigen Latex Agglutination Test System (LA-IMMY) (Immuno-Mycologics, Inc., USA) were conducted in 94 samples of cerebrospinal fluid. LA-ANLIS and LA-IMMY compared exhibited 100% positive agreement and 97% negative agreement. LA-ANLIS showed 94% sensitivity and 97% specificity with the positive and negative predictive values of 94% and 97%, respectively. The LA-ANLIS is a reliable, reproducible and cost-effective reagent, especially useful in countries where the commercial kit is not generally available and must be obtained at a high cost. National production of reagents is the best choice for a reliable access to the rapid diagnosis of CM in Argentina.