Human Pathogen Cryptococcus Research Articles

Characterization of a methyltransferase for iterative N-methylation at the leucinostatin termini in Purpureocillium lilacinum

N-methyltransferase (NMT)-catalyzed methylation at the termini of nonribosomal peptides (NRPs) has rarely been reported. Here, we discover a fungal NMT LcsG for the iterative terminal N-methylation of a family of NRPs, leucinostatins. Gene deletion results suggest that LcsG is essential for leucinostatins methylation. Results from in vitro assays and HRESI-MS-MS analysis reveal the methylation sites as NH2, NHCH3 and N(CH3)2 in the C-terminus of various leucinostatins. LcsG catalysis yields new lipopeptides, some of which demonstrate effective antibiotic properties against the human pathogen Cryptococcus neoformans and the plant pathogen Phytophthora infestans. Multiple sequence alignments and site-directed mutagenesis of LcsG indicate the presence of a highly conserved SAM-binding pocket, along with two possible active site residues (D368 and D395). Molecular dynamics simulations show that the targeted N can dock between these two residues. Thus, this study suggests a method for increasing the variety of natural bioactivity of NPRs and a possible catalytic mechanism underlying the N-methylation of NRPs.

Studying Human Pathogenic Cryptococcus Gattii Lineages by Utilizing Simple Sequence Repeats to Create Diagnostic Markers and Analyzing Diversity.

In this study, we compared the occurrence, relative abundance (RA), and density (RD) of simple sequence repeats (SSRs) among the lineages of human pathogenicCryptococcus gattii using an in-silico approach to gain a deeper understanding of the structure and evolution of their genomes. C. gattii isolate MF34 showed the highest RA and RD of SSRs in both the genomic and transcriptomic sequences, followed by isolate WM276. In both the genomic (50%) and transcriptomic (65%) sequences, trinucleotide SSRs were the most common SSR class. A motif conservation study found that the isolates had stronger conservation (56.1%) of motifs, with isolate IND107 having the most (5.7%) unique motifs. We discovered the presence of SSRs in genes that are directly or indirectly associated with disease using gene enrichment analysis. Isolate-specific unique motifs identified in this study could be utilized as molecular probes for isolate identification. To improve genetic resources among C. gattii isolates, 6499 primers were developed. These genomic resources developed in this study could help with diversity analysis and the development of isolate-specific markers.

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.

Discovery of Novel Terpenoids from the Basidiomycete Pleurotus ostreatus through Genome Mining and Coculture Optimization.

In our previous work, postredienes A-C, three unusual linear sesterterpenes with high antifungal activities, were isolated from Pleurotus ostreatus SY10 when cocultured with Trametes robiniophila SY636. However, their titers were low, and exploration of newly biosynthesized trace analogues is required. Herein, genome mining analysis predicted that 17 gene clusters are involved in terpenoid biosynthesis in P. ostreatus. Thus, coculture conditions for strains SY10 and SY636 were optimized using a single-factor test and Box-Behnken design. As a result, the titers of postredienes A-C were increased by over 2.5-fold, reaching 1.28 to 8.40 mg/L. Moreover, five new terpenoids, named postredienes D-H (1-5), were successfully isolated. Compound 1 exhibited activities against the human pathogenic fungi Candida albicans and Cryptococcus neoformans comparable to those of amphotericin B. Compound 2 represents a novel sesterterpene with a five-membered ring at C-7. The absolute configurations of 1-5 were elucidated by making the methoxyphenylacetic acid esters and acetonide derivatives, combined with ECD and NMR calculation. Two potential gene clusters and relevant biosynthetic pathways for 1-5 were subsequently proposed based on real-time reverse transcription-quantitative PCR (RT-qPCR) analysis. The current study provides new insights into the research of terpenoid biosynthesis genes in P. ostreatus and other basidiomycetes.

A fungal lytic polysaccharide monooxygenase is required for cell wall integrity, thermotolerance, and virulence of the fungal human pathogen Cryptococcus neoformans.

Fungi often adapt to environmental stress by altering their size, shape, or rate of cell division. These morphological changes require reorganization of the cell wall, a structural feature external to the cell membrane composed of highly interconnected polysaccharides and glycoproteins. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that are typically secreted into the extracellular space to catalyze initial oxidative steps in the degradation of complex biopolymers such as chitin and cellulose. However, their roles in modifying endogenous microbial carbohydrates are poorly characterized. The CEL1 gene in the human fungal pathogen Cryptococcus neoformans (Cn) is predicted by sequence homology to encode an LPMO of the AA9 enzyme family. The CEL1 gene is induced by host physiological pH and temperature, and it is primarily localized to the fungal cell wall. Targeted mutation of the CEL1 gene revealed that it is required for the expression of stress response phenotypes, including thermotolerance, cell wall integrity, and efficient cell cycle progression. Accordingly, a cel1Δ deletion mutant was avirulent in two models of C. neoformans infection. Therefore, in contrast to LPMO activity in other microorganisms that primarily targets exogenous polysaccharides, these data suggest that CnCel1 promotes intrinsic fungal cell wall remodeling events required for efficient adaptation to the host environment.

Comprehensive genome-scale metabolic model of the human pathogen Cryptococcus neoformans: A platform for understanding pathogen metabolism and identifying new drug targets.

Introduction: The fungal priority pathogen Cryptococcus neoformans causes cryptococcal meningoencephalitis in immunocompromised individuals and leads to hundreds of thousands of deaths per year. The undesirable side effects of existing treatments, the need for long application times to prevent the disease from recurring, the lack of resources for these treatment methods to spread over all continents necessitate the search for new treatment methods. Methods: Genome-scale models have been shown to be valuable in studying the metabolism of many organisms. Here we present the first genome-scale metabolic model for C. neoformans, iCryptococcus. This comprehensive model consists of 1,270 reactions, 1,143 metabolites, 649 genes, and eight compartments. The model was validated, proving accurate when predicting the capability of utilizing different carbon and nitrogen sources and growth rate in comparison to experimental data. Results and Discussion: The compatibility of the in silico Cryptococcus metabolism under infection conditions was assessed. The steroid and amino acid metabolisms found in the essentiality analyses have the potential to be drug targets for the therapeutic strategies to be developed against Cryptococcus species. iCryptococcus model can be applied to explore new targets for antifungal drugs along with essential gene, metabolite and reaction analyses and provides a promising platform for elucidation of pathogen metabolism.

A fungal lytic polysaccharide monooxygenase is required for cell wall integrity, thermotolerance, and virulence of the fungal human pathogen Cryptococcus neoformans

A fungal lytic polysaccharide monooxygenase is required for cell wall integrity, thermotolerance, and virulence of the fungal human pathogen Cryptococcus neoformans

Terrien, a metabolite made by Aspergillus terreus, has activity against Cryptococcus neoformans.

Antimicrobial compounds, including antibiotics, have been a cornerstone of modern medicine being able to both treat infections and prevent infections in at-risk people, including those who are immune-compromised and those undergoing routine surgical procedures. Their intense use, including in people, animals, and plants, has led to an increase in the incidence of resistant bacteria and fungi, resulting in a desperate need for novel antimicrobial compounds with new mechanisms of action. Many antimicrobial compounds in current use originate from microbial sources, such as penicillin from the fungus Penicillium chrysogenum (renamed by some as P. rubens). Through a collaboration with Aotearoa New Zealand Crown Research Institute Manaaki Whenua–Landcare Research we have access to a collection of thousands of fungal cultures known as the International Collection of Microorganisms from Plants (ICMP). The ICMP contains both known and novel species which have not been extensively tested for their antimicrobial activity. Initial screening of ICMP isolates for activity against Escherichia coli and Staphylococcus aureus directed our interest towards ICMP 477, an isolate of the soil-inhabiting fungus, Aspergillus terreus. In our investigation of the secondary metabolites of A. terreus, through extraction, fractionation, and purification, we isolated nine known natural products. We evaluated the biological activity of selected compounds against various bacteria and fungi and discovered that terrein (1) has potent activity against the important human pathogen Cryptococcus neoformans.

Comparative miRNA transcriptomics of macaques and mice reveals MYOC is an inhibitor for Cryptococcus neoformans invasion into the brain

ABSTRACT Cryptococcal meningoencephalitis (CM) is emerging as an infection in HIV/AIDS patients shifted from primarily ART­naive to ART-experienced individuals, as well as patients with COVID-19 and immunocompetent hosts. This fungal infection is mainly caused by the opportunistic human pathogen Cryptococcus neoformans. Brain or central nervous system (CNS) dissemination is the deadliest process for this disease; however, mechanisms underlying this process have yet to be elucidated. Moreover, illustrations of clinically relevant responses in cryptococcosis are currently limited due to the low availability of clinical samples. In this study, to explore the clinically relevant responses during C. neoformans infection, macaque and mouse infection models were employed and miRNA-mRNA transcriptomes were performed and combined, which revealed cytoskeleton, a major feature of HIV/AIDS patients, was a centric pathway regulated in both infection models. Notably, assays of clinical immune cells confirmed an enhanced macrophage “Trojan Horse” in patients with HIV/AIDS, which could be shut down by cytoskeleton inhibitors. Furthermore, myocilin, encoded by MYOC, was found to be a novel enhancer for the macrophage “Trojan Horse,” and an enhanced fungal burden was achieved in the brains of MYOC-transgenic mice. Taken together, the findings from this study reveal fundamental roles of the cytoskeleton and MYOC in fungal CNS dissemination, which not only helps to understand the high prevalence of CM in HIV/AIDS but also facilitates the development of novel therapeutics for meningoencephalitis caused by C. neoformans and other pathogenic microorganisms.

Inositol Metabolism Regulates Capsule Structure and Virulence in the Human Pathogen Cryptococcus neoformans.

ABSTRACTThe environmental yeast Cryptococcus neoformans is the most common cause of deadly fungal meningitis in primarily immunocompromised populations. A number of factors contribute to cryptococcal pathogenesis. Among them, inositol utilization has been shown to promote C. neoformans development in nature and invasion of central nervous system during dissemination. The mechanisms of the inositol regulation of fungal virulence remain incompletely understood. In this study, we analyzed inositol-induced capsule growth and the contribution of a unique inositol catabolic pathway in fungal development and virulence. We found that genes involved in the inositol catabolic pathway are highly induced by inositol, and they are also highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. This pathway in C. neoformans contains three genes encoding myo-inositol oxygenases that convert myo-inositol into d-glucuronic acid, a substrate of the pentose phosphate cycle and a component of the polysaccharide capsule. Our mutagenesis analysis demonstrates that inositol catabolism is required for C. neoformans virulence and deletion mutants of myo-inositol oxygenases result in altered capsule growth as well as the polysaccharide structure, including O-acetylation. Our study indicates that the ability to utilize the abundant inositol in the brain may contribute to fungal pathogenesis in this neurotropic fungal pathogen.

Genetic and Phenotypic Diversities in Experimental Populations of Diploid Inter-Lineage Hybrids in the Human Pathogenic Cryptococcus.

To better understand the potential factors contributing to genome instability and phenotypic diversity, we conducted mutation accumulation (MA) experiments for 120 days for 7 diploid cryptococcal hybrids under fluconazole (10 MA lines each) and non-fluconazole conditions (10 MA lines each). The genomic DNA content, loss of heterozygosity (LOH) rate, growth ability, and fluconazole susceptibility were determined for all 140 evolved cultures. Compared to that of their ancestral clones, the evolved clones showed: (i) genomic DNA content changes ranging from ~22% less to ~27% more, and (ii) reduced, similar, and increased phenotypic values for each tested trait, with most evolved clones displaying increased growth at 40 °C and increased fluconazole resistance. Aside from the ancestral multi-locus genotypes (MLGs) and heterozygosity patterns (MHPs), 77 unique MLGs and 70 unique MPHs were identified among the 140 evolved cultures at day 120. The average LOH rates of the MA lines in the absence and presence of fluconazole were similar at 1.27 × 10−4 and 1.38 × 10−4 LOH events per MA line per mitotic division, respectively. While LOH rates varied among MA lines from different ancestors, there was no apparent correlation between the genetic divergence of the parental haploid genomes within ancestral clones and LOH rates. Together, our results suggest that hybrids between diverse lineages of the human pathogenic Cryptococcus can generate significant genotypic and phenotypic diversities during asexual reproduction.

Update on Pulmonary Cryptococcosis.

Pulmonary cryptococcosis is a common but underdiagnosed opportunistic fungal infection in both immunocompromised and immunocompetent patients. The causal agents include at least eight evolutionary distinct haploid lineages as well as their hybrids of the human pathogenic Cryptococcus complex. In this update, we review recent advances in epidemiology, mode of transmission, risk factors, diagnostic methods, and therapy of pulmonary cryptococcosis. Our review suggests significant challenges and opportunities for research, from bedside to benchside and back to bedside.

Molecular Markers Reveal Epidemiological Patterns and Evolutionary Histories of the Human Pathogenic Cryptococcus.

The human pathogenic Cryptococcus species are the main agents of fungal meningitis in humans and the causes of other diseases collectively called cryptococcosis. There are at least eight evolutionary divergent lineages among these agents, with different lineages showing different geographic and/or ecological distributions. In this review, we describe the main strain typing methods that have been used to analyze the human pathogenic Cryptococcus and discuss how molecular markers derived from the various strain typing methods have impacted our understanding of not only cryptococcal epidemiology but also its evolutionary histories. These methods include serotyping, multilocus enzyme electrophoresis, electrophoretic karyotyping, random amplified polymorphic DNA, restriction fragment length polymorphism, PCR-fingerprinting, amplified fragment length polymorphism, multilocus microsatellite typing, single locus and multilocus sequence typing, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and whole genome sequencing. The major findings and the advantages and disadvantages of each method are discussed. Together, while controversies remain, these strain typing methods have helped reveal (i) the broad phylogenetic pattern among these agents, (ii) the centers of origins for several lineages and their dispersal patterns, (iii) the distributions of genetic variation among geographic regions and ecological niches, (iv) recent hybridization among several lineages, and (v) specific mutations during infections within individual patients. However, significant challenges remain. Multilocus sequence typing and whole genome sequencing are emerging as the gold standards for continued strain typing and epidemiological investigations of cryptococcosis.

Production of Secreted Carbohydrates that Present Immunologic Similarities with the Cryptococcus Glucuronoxylomannan by Members of the Trichosporonaceae Family: A Comparative Study Among Species of Clinical Interest.

Glucuronoxylomannan (GXM) participates in several immunoregulatory mechanisms, which makes it an important Cryptococcus virulence factor that is essential for the disease. Trichosporon asahii and Trichosporon mucoides share with Cryptococcus species the ability to produce GXM. To check whether other opportunistic species in the Trichosporonaceae family produce GXM-like polysaccharides, extracts from 28 strains were produced from solid cultures and their carbohydrate content evaluated by the sulfuric acid / phenol method. Moreover, extracts were assessed for cryptococcal GXM cross-reactivity through latex agglutination and lateral flow assay methods. Cryptococcus neoformans and Saccharomyces cerevisiae were used as positive and negative controls, respectively. In addition to T. asahii, the species Trichosporon inkin, Apiotrichum montevideense, Trichosporon japonicum, Trichosporon faecale, Trichosporon ovoides, Cutaneotrichosporon debeurmannianum, and Cutaneotrichosporon arboriformis are also producers of a polysaccharide immunologically similar to the GXM produced by human pathogenic Cryptococcus species. The carbohydrate concentration of the extracts presented a positive correlation with the GXM contents determined by titration of both methodologies. These results add several species to the list of fungal pathogens that produce glycans of the GXM type and bring information about the origin of potential false-positive results on immunological tests for diagnosis of cryptococcosis based on GXM detection.

What Are the Best Parents for Hybrid Progeny? An Investigation into the Human Pathogenic Fungus Cryptococcus

Hybridization between more divergent organisms is likely to generate progeny with more novel genetic interactions and genetic variations. However, the relationship between parental genetic divergence and progeny phenotypic variation remains largely unknown. Here, using strains of the human pathogenic Cryptococcus, we investigated the patterns of such a relationship. Twenty-two strains with up to 15% sequence divergence were mated. Progeny were genotyped at 16 loci. Parental strains and their progeny were phenotyped for growth ability at two temperatures, melanin production at seven conditions, and susceptibility to the antifungal drug fluconazole. We observed three patterns of relationships between parents and progeny for each phenotypic trait, including (i) similar to one of the parents, (ii) intermediate between the parents, and (iii) outside the parental phenotypic range. We found that as genetic distance increases between parental strains, progeny showed increased fluconazole resistance and growth at 37 °C but decreased melanin production under various oxidative and nitrosative stresses. Our findings demonstrate that, depending on the traits, both evolutionarily more similar strains and more divergent strains may be better parents to generate progeny with hybrid vigor. Together, the results indicate the enormous potential of Cryptococcus hybrids in their evolution and adaptation to diverse conditions.

Bridgin connects the outer kinetochore to centromeric chromatin

The microtubule-binding outer kinetochore is coupled to centromeric chromatin through CENP-CMif2, CENP-TCnn1, and CENP-UAme1 linker pathways originating from the constitutive centromere associated network (CCAN) of the inner kinetochore. Here, we demonstrate the recurrent loss of most CCAN components, including certain kinetochore linkers during the evolution of the fungal phylum of Basidiomycota. By kinetochore interactome analyses in a model basidiomycete and human pathogen Cryptococcus neoformans, a forkhead-associated domain containing protein “bridgin” was identified as a kinetochore component along with other predicted kinetochore proteins. In vivo and in vitro functional analyses of bridgin reveal its ability to connect the outer kinetochore with centromeric chromatin to ensure accurate chromosome segregation. Unlike established CCAN-based linkers, bridgin is recruited at the outer kinetochore establishing its role as a distinct family of kinetochore proteins. Presence of bridgin homologs in non-fungal lineages suggests an ancient divergent strategy exists to bridge the outer kinetochore with centromeric chromatin.

Sphaerostilbellins, New Antimicrobial Aminolipopeptide Peptaibiotics from Sphaerostilbella toxica.

Sphaerostilbella toxica is a mycoparasitic fungus that can be found parasitizing wood-decay basidiomycetes in the southern USA. Organic solvent extracts of fermented strains of S. toxica exhibited potent antimicrobial activity, including potent growth inhibition of human pathogenic yeasts Candida albicans and Cryptococcus neoformans, the respiratory pathogenic fungus Aspergillus fumigatus, and the Gram-positive bacterium Staphylococcus aureus. Bioassay-guided separations led to the purification and structure elucidation of new peptaibiotics designated as sphaerostilbellins A and B. Their structures were established mainly by analysis of NMR and HRMS data, verification of amino acid composition by Marfey’s method, and by comparison with published data of known compounds. They incorporate intriguing structural features, including an N-terminal 2-methyl-3-oxo-tetradecanoyl (MOTDA) residue and a C-terminal putrescine residue. The minimal inhibitory concentrations for sphaerostilbellins A and B were measured as 2 μM each for C. neoformans, 1 μM each for A. fumigatus, and 4 and 2 μM, respectively, for C. albicans. Murine macrophage cells were unaffected at these concentrations.

In vitro interactions of amphotericin B and non-antifungal compounds against opportunistic human pathogen Cryptococcus neoformans

The incidence of opportunistic human pathogen Cryptococcus neoformans caused infections have been higher during the last few decades along with the increasing number of susceptible individuals around the world. Recommended treatment of cryptococcal meningoencephalitis is a combined therapy with amphotericin B deoxycholate and flucytosine. Despite of the efficiency of this drug combination, the mortality rate of the disease is high due to the limited accessibility and the high cost of these antifungals in the most severely affected areas. The broad-spectrum activity of non-antifungal drugs and their potential to enhance the efficiency of conventional antifungal agents have been recognised previously. In this study, the in vitro activity of amantadine, valproic acid, trifluoperazine and chlorpromazine was tested against five C. neoformans strains individually and in combination with amphotericin B. All the four compounds exerted slight antifungal activity against the studied C. neoformans strains. Their combination with amphotericin B revealed additive and synergistic interactions.

The contest of microbial pigeon neighbors: Interspecies competition between Serratia marcescens and the human pathogen Cryptococcus neoformans

In nature, microorganisms often exhibit competitive behavior for nutrients and limited space, allowing them to alter the virulence determinants of pathogens. The human pathogenic yeast Cryptococcus neoformans can be found organized in biofilms, a complex community composed of an extracellular matrix which confers protection against predation. The aim of this study was to evaluate and characterize antagonistic interactions between two cohabiting microorganisms: C. neoformans and the bacteria Serratia marcescens. The interaction of S. marcescens with C. neoformans expressed a negative effect on biofilm formation, polysaccharide capsule, production of urease, and melanization of the yeast. These findings evidence that competition in mixed communities can result in dominance by one species, with direct impact on the physiological modulation of virulence determinants. Such an approach is key for understating the response of communities to the presence of competitors and, ultimately, rationally designing communities to prevent and treat certain diseases.

Scanning electron microscopy and machine learning reveal heterogeneity in capsular morphotypes of the human pathogen Cryptococcus spp.

Phenotypic heterogeneity is an important trait for the development and survival of many microorganisms including the yeast Cryptococcus spp., a deadly pathogen spread worldwide. Here, we have applied scanning electron microscopy (SEM) to define four Cryptococcus spp. capsule morphotypes, namely Regular, Spiky, Bald, and Phantom. These morphotypes were persistently observed in varying proportions among yeast isolates. To assess the distribution of such morphotypes we implemented an automated pipeline capable of (1) identifying potentially cell-associated objects in the SEM-derived images; (2) computing object-level features; and (3) classifying these objects into their corresponding classes. The machine learning approach used a Random Forest (RF) classifier whose overall accuracy reached 85% on the test dataset, with per-class specificity above 90%, and sensitivity between 66 and 94%. Additionally, the RF model indicates that structural and texture features, e.g., object area, eccentricity, and contrast, are most relevant for classification. The RF results agree with the observed variation in these features, consistently also with visual inspection of SEM images. Finally, our work introduces morphological variants of Cryptococcus spp. capsule. These can be promptly identified and characterized using computational models so that future work may unveil morphological associations with yeast virulence.