The Cryptococcus neoformans Flc1 Homologue Controls Calcium Homeostasis and Confers Fungal Pathogenicity in the Infected Hosts.

Cryptococcus neoformans, an opportunistic yeast pathogen, relies on a complex network of stress response pathways that allow for proliferation in the host. In Saccharomyces cerevisiae, stress responses are regulated by integral membrane proteins containing a transient receptor potential (TRP) domain, including the flavin carrier protein 1 (Flc1), which regulates calcium homeostasis and flavin transport. Here, we report that deletion of C. neoformans FLC1 results in cytosolic calcium elevation and increased nuclear content of calcineurin-dependent transcription factor Crz1, which is associated with an aberrant cell wall chitin overaccumulation observed in the flc1Δ mutant. Absence of Flc1 or inhibition of calcineurin with cyclosporine A prevents vacuolar fusion under conditions of combined osmotic and temperature stress, which is reversed in the flc1Δ mutant by the inhibition of TORC1 kinase with rapamycin. Flc1-deficient yeasts exhibit compromised vacuolar fusion under starvation conditions, including conditions that stimulate formation of carbohydrate capsule. Consequently, the flc1Δ mutant fails to proliferate under low nutrient conditions and displays a defect in capsule formation. Consistent with the previously uncharacterized role of Flc1 in vacuolar biogenesis, we find that Flc1 localizes to the vacuole. The flc1Δ mutant presents a survival defect in J774A.1 macrophage cell-line and profound virulence attenuation in both the Galleria mellonella and mouse pulmonary infection models, demonstrating that Flc1 is essential for pathogenicity. Thus, cryptococcal Flc1 functions in calcium homeostasis and links calcineurin and TOR signaling with vacuolar biogenesis to promote survival under conditions associated with vacuolar fusion required for this pathogen's fitness and virulence. IMPORTANCE Cryptococcosis is a highly lethal infection with limited drug choices, most of which are highly toxic or complicated by emerging antifungal resistance. There is a great need for new drug targets that are unique to the fungus. Here, we identify such a potential target, the Flc1 protein, which we show is crucial for C. neoformans stress response and virulence. Importantly, homologues of Flc1 exist in other fungal pathogens, such as Candida albicans and Aspergillus fumigatus, and are poorly conserved in humans, which could translate into wider spectrum therapy associated with minimal toxicity. Thus, Flc1 could be an "Achille's heel" of C. neoformans to be leveraged therapeutically in cryptococcosis and possibly other fungal infections.

Establishing Minimal Conditions Sufficient for the Development of Titan-like Cells in Cryptococcus neoformans/gattii Species Complex.

Opportunistic pathogens of the anamorphic genus Cryptococcus are unique considering their virulence factors that in the context of pathogenesis allowed them to achieve evolutionary success. Morphological transformation into giant (Titan) cells is one of the factors contributing to cryptococcosis. Recently established in vitro protocols demonstrate that 5 or 10% fetal bovine serum (FBS) combined with 5% CO2, 37 °C, and sufficiently low cell density, triggers cellular enlargement (Serum protocols). However, the FBS components that promote this morphological transition remain incompletely characterized. In search of minimal conditions necessary for stimulating the formation of Titan cells, we performed a study where we eliminated serum from the protocol (Serum-free protocol) and instead systematically adjusted the amount of glucose, source of nitrogen (ammonium sulfate), and the pH. We found that exposing cells to PBS with adjusted pH to 7.3, and supplemented with 0.05% glucose, 0.025% ammonium sulfate, 0.004% K2HPO4, 0.0035% MgSO4, in the presence of 5% CO2 at 37 °C triggers Titan-like cell formation to the same degree as the previously established protocol that utilized 10% FBS as the sole nutrient source. Titan-like cells obtained according to this Serum-free protocol were characterized by cell body size over ten microns, a single enlarged vacuole, thick cell wall, extensive polysaccharide capsule, and changes in the level of cell ploidy, all currently known hallmarks of Titan cells found in vivo. Strikingly, we found that in both, Serum and Serum-free protocols, an optimal pH for Titan-like cell development is ~7.3 whereas relatively acidic pH (5.5) prevents this morphological transition and promotes robust proliferation, while alkaline pH (~8.0) has a profound growth inhibitory effect. Our study demonstrates a critical role of pH response to the formation of Titan cells and indicates that conditions that allow restricted proliferation in the presence of 5% CO2 are sufficient for this morphological transition to form enlarged cells in Cryptococcus neoformans and Cryptococcus gattii species complex.

Role of the anillin-like protein in growth of Cryptococcus neoformans at human host temperature.

Cryptococcus neoformans, a basidiomycete yeast, causes lethal meningitis in immunocompromised individuals. The ability of C. neoformans to proliferate at 37°C is essential for virulence. We identified anillin-like protein, CnBud4, as essential for proliferation of C. neoformans at 37°C and for virulence in a heterologous host Galleria mellonella at 25°C. C. neoformans cells lacking CnBud4 were inviable at 25°C in the absence of active calcineurin and were hypersensitive to membrane stress and an anti-fungal agent fluconazole, phenotypes previously described for C. neoformans mutants lacking septins. CnBud4 localized to the mother-bud neck during cytokinesis in a septin-dependent manner. In the absence of CnBud4, septin complex failed to transition from a collar-like single ring to the double ring during cytokinesis. In an ascomycete yeast, Saccharomyces cerevisiae, the anillin-like homologue ScBud4 participates in the organization of the septin ring at the mother-bud neck and plays an important role in specifying location for new bud emergence, known as axial budding pattern. In contrast to their role in S. cerevisiae, neither septins nor CnBud4 were needed to direct the position of the new bud in C. neoformans, suggesting that this function is not conserved in basidiomycetous yeasts. Our data suggest that the requirement of CnBud4 for growth at 37°C and pathogenicity in C. neoformans is based on its conserved role in septin complex organization.

Corrigendum: Advances in Plant-Derived Scaffold Proteins.

[This corrects the article DOI: 10.3389/fpls.2020.00122.].

Genetic contribution to high temperature tolerance in Cryptococcus neoformans.

The human fungal pathogen Cryptococcus neoformans relies on a complex signaling network for the adaptation and survival at the host temperature. Protein phosphatase calcineurin is central to proliferation at 37°C but its exact contributions remain ill-defined. To better define genetic contributions to the C. neoformans temperature tolerance, 4031 gene knockouts were screened for genes essential at 37°C and under conditions that keep calcineurin inactive. Identified 83 candidate strains, potentially sensitive to 37°C, were subsequently subject to technologically simple yet robust assay, in which cells are exposed to a temperature gradient. This has resulted in identification of 46 genes contributing to the maximum temperature at which C. neoformans can proliferate (Tmax). The 46 mutants, characterized by a range of Tmax on drug-free media, were further assessed for Tmax under conditions that inhibit calcineurin, which led to identification of several previously uncharacterized knockouts exhibiting synthetic interaction with the inhibition of calcineurin. A mutant that lacked septin Cdc11 was among those with the lowest Tmax and failed to proliferate in the absence of calcineurin activity. To further define connections with calcineurin and the role for septins in high temperature growth, the 46 mutants were tested for cell morphology at 37°C and growth in the presence of agents disrupting cell wall and cell membrane. Mutants sensitive to calcineurin inhibition were tested for synthetic lethal interaction with deletion of the septin-encoding CDC12 and the localization of the septin Cdc3-mCherry. The analysis described here pointed to previously uncharacterized genes that were missed in standard growth assays indicating that the temperature gradient assay is a valuable complementary tool for elucidating the genetic basis of temperature range at which microorganisms proliferate.

Registered report protocol: Quantitative analysis of septin Cdc10-associated proteome in Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast that primarily infects immunocompromised individuals. C. neoformans can thrive during infections due to its three main virulence-related characteristics: the ability to grow at host temperature (37°C), formation of carbohydrate capsule, and its ability to produce melanin. C. neoformans strains lacking septin proteins Cdc3 or Cdc12 are viable at 25°C; however, they fail to proliferate at 37°C and are avirulent in the murine model of infection. The basis of septin contribution to growth at host temperature remains unknown. Septins are a family of conserved filament-forming GTPases with roles in cytokinesis and morphogenesis. In the model organism Saccharomyces cerevisiae septins are essential. S. cerevisiae septins form a higher order complex at the mother-bud neck to scaffold over 80 proteins, including those involved in cell wall organization, cell polarity, and cell cycle control. In C. neoformans, septins also form a complex at the mother-bud neck but the septin interacting proteome in this species remains largely unknown. Moreover, it remains possible that septins play other roles important for high temperature stress that are independent of their established role in cytokinesis. Therefore, we propose to perform a global analysis of septin Cdc10 binding partners in C. neoformans, including those that are specific to high temperature stress. This analysis will shed light on the underlying mechanism of survival of this pathogenic yeast during infection and can potentially lead to the discovery of novel drug targets.

A case of anti- pityriasis versicolor therapy that preserves healthy mycobiome.

BACKGROUND: The impact of Malassezia yeasts on skin mycobiome and health has received considerable attention recently. Pityriasis versicolor (PV), a common dermatosis caused by Malassezia genus worldwide, is a manifestation of dysbiosis. PV can be associated with hyper- and/or hypopigmented skin lesions. This disease entity is characterized by high percentage of relapses, which demands a proper antifungal therapy that is based on unambiguous species identification and drug susceptibility testing. CASE PRESENTATION: Comprehensive analysis of PV case in man presenting simultaneously hyper- and hypopigmented skin lesions was performed. Conventional and molecular diagnostic procedures revealed Malassezia furfur and Malassezia sympodialis, respectively as etiological agents of skin lesions observed. Susceptibility tests showed significantly lowered sensitivity of M. furfur cells to fluconazole. Based on susceptibility profiles local antifungal therapy with drugs characterized by entirely different mechanism of action was included. CONCLUSIONS: Our study indicates that cases of PV represented by two types of skin lesions in one patient may be associated with distinct Malassezia species. Moreover, as observed in this case, each of the isolated etiological agents of PV may differ significantly in susceptibility to antifungals. This can significantly complicate the treatment of dermatosis, which by definition is associated with a significant percentage of relapses. In the presented case localized topical treatment was sufficient and successful while allowing maintaining the physiological mycobiome.

Titan cell formation is unique to Cryptococcus species complex.

Members of the Cryptococcus species complex stand out by unique virulence factors that allowed evolutionary transition to pathogenesis. Among the factors contributing to cryptococcosis is a morphological transformation into giant (Titan) cells. It remains unclear whether species outside of the C. neoformans/C. gattii species complex are capable of titanization. We utilized two recently developed protocols that allow obtaining Titan cells in vitro to test if titanization occurs in non-C. neoformans/C. gattii species. We find that none of the tested strains, representing 10 species of basidiomycetous yeasts and the ascomycetous yeast Saccharomyces cerevisiae, undergo significant titanization under conditions that promote robust Titan cell formation in C. neoformans/C. gattii species complex. C. terreus formed occasional enlarged cells through a mechanism potentially similar to that of titanization. Our findings suggest that titanization is a rare phenomenon among basidiomycetous yeasts that occurs mostly in members of the C. neoformans/C. gattii species complex.

Advances in Plant-Derived Scaffold Proteins.

Scaffold proteins form critical biomatrices that support cell adhesion and proliferation for regenerative medicine and drug screening. The increasing demand for such applications urges solutions for cost effective and sustainable supplies of hypoallergenic and biocompatible scaffold proteins. Here, we summarize recent efforts in obtaining plant-derived biosynthetic spider silk analogue and the extracellular matrix protein, collagen. Both proteins are composed of a large number of tandem block repeats, which makes production in bacterial hosts challenging. Furthermore, post-translational modification of collagen is essential for its function which requires co-transformation of multiple copies of human prolyl 4-hydroxylase. We discuss our perspectives on how the GAANTRY system could potentially assist the production of native-sized spider dragline silk proteins and prolyl hydroxylated collagen. The potential of recombinant scaffold proteins in drug delivery and drug discovery is also addressed.

Fluconazole induces ROS in Cryptococcus neoformans and contributes to DNA damage in vitro.

Pathogenic basidiomycetous yeast, Cryptococcus neoformans, causes fatal meningitis in immunocompromised individuals. Fluconazole (FLC) is a fungistatic drug commonly administered to treat cryptococcosis. Unfortunately, FLC-resistant strains characterized by various degree of chromosomal instability were isolated from clinical patients. Importantly, the underlying mechanisms that lead to chromosomal instability in FLC-treated C. neoformans remain elusive. Previous studies in fungal and mammalian cells link chromosomal instability to the reactive oxygen species (ROS). This study provides the evidence that exposure of C. neoformans to FLC induces accumulation of intracellular ROS, which correlates with plasma membrane damage. FLC caused transcription changes of oxidative stress related genes encoding superoxide dismutase (SOD1), catalase (CAT3), and thioredoxin reductase (TRR1). Strikingly, FLC contributed to an increase of the DNA damage in vitro, when complexed with iron or copper in the presence of hydrogen peroxide. Strains with isogenic deletion of copper response protein metallothionein were more susceptible to FLC. Addition of ascorbic acid (AA), an anti-oxidant at 10 mM, reduced the inhibitory effects of FLC. Consistent with potential effects of FLC on DNA integrity and chromosomal segregation, FLC treatment led to elevated transcription of RAD54 and repression of cohesin-encoding gene SCC1. We propose that FLC forms complexes with metals and contributes to elevated ROS, which may lead to chromosomal instability in C. neoformans.

Colony and Single Cell Level Analysis of the Heterogeneous Response of Cryptococcus neoformans to Fluconazole.

Cryptococcus neoformans is a human fungal pathogen that can cause fatal meningitis in immunocompromised individuals. Fluconazole (FLC) is a fungistatic drug administered to treat cryptococcosis. When exposed to the inhibitory concentration of FLC, C. neoformans exhibits heteroresistance where a small subpopulation of cells develops into FLC-resistant colonies. FLC-resistant cells are aneuploids with regard to specific beneficial chromosomal regions. Factors underlying the potential for only certain C. neoformans cells in a genetically isogenic population to become FLC-resistant are unknown. In this study, we systematically examine the heterogeneous response of C. neoformans to FLC at a colony and individual cell level. We find that the heterogeneity in response to FLC is reflected by variable diminishment of the ergosterol at the plasma membrane. A population of C. neoformans spread on a semi-solid medium displays two types of outcomes following FLC exposure. The first outcome is colonies consisting of non-resistant cells (survivors). The size of colonies consisting of survivors ranges from a few cells to visible colonies, which reflects intrinsic phenotypic heterogeneity of the C. neoformans population. The second outcome is FLC-resistant cells forming colonies of sizes significantly larger as compared to colonies made of survivors. We propose a model that describes how a distribution of these types of cellular responses within a population changes depending on FLC concentration and factors that influence the rate of cellular growth including temperature, media type, growth phase, and the age of cells. Our findings highlight a complex nature of the response to a fungistatic drug and provide insights that may help to optimize FLC therapy.

Continuous sheath-free separation of drug-treated human fungal pathogen Cryptococcus neoformans by morphology in biocompatible polymer solutions.

Cryptococcal meningitis caused by Cryptococcus neoformans is the leading cause of fungal central nervous system infections. Current antifungal treatments for cryptococcal infections are inadequate partly due to the occurrence of drug resistance. Recent studies indicate that the treatment of the azole drug fluconazole changes the morphology of C. neoformans to form enlarged "multimeras" that consist of three or more connected cells/buds. To analyze if these multimeric cells are a prerequisite for C. neoformans to acquire drug resistance, a tool capable of separating them from normal cells is critical. We extend our recently demonstrated sheath-free elasto-inertial particle separation technique to fractionate drug-treated C. neoformans cells by morphology in biocompatible polymer solutions. The separation performance is evaluated for both multimeric and normal cells in terms of three dimensionless metrics: efficiency, purity, and enrichment ratio. The effects of flow rate, polymer concentration, and microchannel height on cell separation are studied.

Yeast cell fractionation by morphology in dilute ferrofluids.

Morphology is an important particle (both biological and synthetic) property and potentially a useful marker for label-free particle separation. We present in this work a continuous-flow morphology-based fractionation of a heterogeneous mixture of drug-treated yeast cells in dilute ferrofluids. Such a diamagnetic cell separation technique utilizes the negative magnetophoretic motion to direct pre-focused yeast cells to morphology-dependent streamlines in a laminar flow. The separation performance is evaluated by comparing the exiting positions of the four classified groups of yeast cells: Singles, Doubles, Triples, and Others. We also develop a three-dimensional numerical model to simulate the separation process by the use of the experimentally determined correction factor for each group of non-spherical cells. The determining factors in this separation are studied both experimentally and numerically, the results of which show a reasonable agreement.

Mechanisms of Cytokinesis in Basidiomycetous Yeasts.

While mechanisms of cytokinesis exhibit considerable plasticity, it is difficult to precisely define the level of conservation of this essential part of cell division in fungi, as majority of our knowledge is based on ascomycetous yeasts. However, in the last decade more details have been uncovered regarding cytokinesis in the second largest fungal phylum, basidiomycetes, specifically in two yeasts, Cryptococcus neoformans and Ustilago maydis. Based on these findings, and current sequenced genomes, we summarize cytokinesis in basidiomycetous yeasts, indicating features that may be unique to this phylum, species-specific characteristics, as well as mechanisms that may be common to all eukaryotes.

Fluconazole-Induced Ploidy Change in Cryptococcus neoformans Results from the Uncoupling of Cell Growth and Nuclear Division.

Cryptococcus neoformans is a pathogenic yeast that causes lethal cryptococcal meningitis in immunocompromised patients. One of the challenges in treating cryptococcosis is the development of resistance to azole antifungals. Previous studies linked azole resistance to elevated numbers of copies of critical resistance genes in aneuploid cells. However, how aneuploidy is formed in the presence of azole drugs remains unclear. This study showed that treatment with inhibitory concentrations of an azole drug, fluconazole (FLC), resulted in a significant population of cells with increased DNA content, through the following defects: inhibition of budding, premature mitosis, and inhibition of cytokinesis followed by replication in the mother cell. Inhibition of and/or a delay in cytokinesis led to the formation of cells with two or more daughter cells attached (multimeric cells). To investigate which part of cytokinesis fails in the presence of FLC, the dynamics of the actomyosin ring (AMR), septins, and Cts1, a protein involved in cell separation, were analyzed with time-lapse microscopy. Following the constriction of the AMR, septins assembled and the septum was formed between the mother and daughter cells. However, final degradation of the septum was affected. Enlarged cells with aberrant morphology, including multimeric cells, exhibited an increased potential to proliferate in the presence of FLC. These findings suggest that pleiotropic effects of FLC on growth and mitotic division lead to an increase in DNA content, resulting in cells less sensitive to the drug. Cells with increased DNA content continue to proliferate and therefore increase the chance of forming resistant populations. IMPORTANCE Azoles are antifungals that are widely utilized due to relatively low toxicity and cost of treatment. One of their drawbacks, however, is that azoles are primarily cytostatic, leaving fungal cells capable of developing drug resistance. The human pathogen Cryptococcus neoformans acquires resistance to the azole drug fluconazole (FLC) through the development of aneuploidy, leading to elevated expression of key resistance genes, a mechanism that is also common for Candida albicans (K. J. Kwon-Chung and Y. C. Chang, PLoS Pathog 8:e1003022, 2012, https://doi.org/10.1371/journal.ppat.1003022; J. Morschhäuser, J Microbiol 54:192-201, 2016, https://doi.org/10.1007/s12275-016-5628-4). However, the exact ways in which FLC contributes to increased resistance in either of these important fungal pathogens remain unclear. Here we found that FLC treatment leads to an increase in DNA content in C. neoformans through multiple mechanisms, potentially increasing the size of a pool of cells from which aneuploids with increased resistance are selected. This study demonstrated the importance of FLC's inhibitory effects on growth and cytokinesis in the generation of cell populations with decreased sensitivity to the drug.

Relative Contributions of Prenylation and Postprenylation Processing in Cryptococcus neoformans Pathogenesis.

Prenyltransferase enzymes promote the membrane localization of their target proteins by directing the attachment of a hydrophobic lipid group at a conserved C-terminal CAAX motif. Subsequently, the prenylated protein is further modified by postprenylation processing enzymes that cleave the terminal 3 amino acids and carboxymethylate the prenylated cysteine residue. Many prenylated proteins, including Ras1 and Ras-like proteins, require this multistep membrane localization process in order to function properly. In the human fungal pathogen Cryptococcus neoformans, previous studies have demonstrated that two distinct forms of protein prenylation, farnesylation and geranylgeranylation, are both required for cellular adaptation to stress, as well as full virulence in animal infection models. Here, we establish that the C. neoformans RAM1 gene encoding the farnesyltransferase ß-subunit, though not strictly essential for growth under permissive in vitro conditions, is absolutely required for cryptococcal pathogenesis. We also identify and characterize postprenylation protease and carboxyl methyltransferase enzymes in C. neoformans. In contrast to the prenyltransferases, deletion of the genes encoding the Rce1 protease and Ste14 carboxyl methyltransferase results in subtle defects in stress response and only partial reductions in virulence. These postprenylation modifications, as well as the prenylation events themselves, do play important roles in mating and hyphal transitions, likely due to their regulation of peptide pheromones and other proteins involved in development. IMPORTANCE Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes through a posttranslational modification process known as prenylation. These studies investigate the roles of one of the prenylation enzymes, farnesyltransferase, as well as the postprenylation processing enzymes in C. neoformans. We demonstrate that the postprenylation processing steps are dispensable for the localization of certain substrate proteins. However, both protein farnesylation and the subsequent postprenylation processing steps are required for full pathogenesis of this fungus.

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.

Ordered kinetochore assembly in the human-pathogenic basidiomycetous yeast Cryptococcus neoformans.

UNLABELLED: Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes. IMPORTANCE: Successful propagation of genetic material in progeny is essential for the survival of any organism. A proper kinetochore-microtubule interaction is crucial for high-fidelity chromosome segregation. An error in this process can lead to loss or gain of chromosomes, a common feature of most solid cancers. Several proteins assemble on centromere DNA to form a kinetochore. However, significant differences in the process of kinetochore assembly exist between unicellular yeasts and multicellular metaozoa. Here, we examined the key events that lead to formation of a proper kinetochore in a basidiomycetous budding yeast, Cryptococcus neoformans. We found that, during the progression of the cell cycle, nonclustered centromeres gradually clustered and kinetochores assembled in an ordered manner concomitant with partial opening of the nuclear envelope in this organism. These events have higher similarity to mitotic events of metazoans than to those previously described in other yeasts.

Ras1 acts through duplicated Cdc42 and Rac proteins to regulate morphogenesis and pathogenesis in the human fungal pathogen Cryptococcus neoformans.

Proliferation and morphogenesis in eukaryotic cells depend on the concerted activity of Rho-type GTPases, including Ras, Cdc42, and Rac. The sexually dimorphic fungus Cryptococcus neoformans, which encodes paralogous, non-essential copies of all three, provides a unique model in which to examine the interactions of these conserved proteins. Previously, we demonstrated that RAS1 mediates C. neoformans virulence by acting as a central regulator of both thermotolerance and mating. We report here that ras1Δ mutants accumulate defects in polarized growth, cytokinesis, and cell cycle progression. We demonstrate that the ras1Δ defects in thermotolerance and mating can be largely explained by the compromised activity of four downstream Rho-GTPases: the Cdc42 paralogs, Cdc42 and Cdc420; and the Rac paralogs, Rac1 and Rac2. Further, we demonstrate that the separate GTPase classes play distinct Ras-dependent roles in C. neoformans morphogenesis and pathogenesis. Cdc42 paralogs primarily control septin localization and cytokinesis, while Rac paralogs play a primary role in polarized cell growth. Together, these duplicate, related signaling proteins provide a robust system to allow microbial proliferation in the presence of host-derived cell stresses.

Profiling a killer, the development of Cryptococcus neoformans.

The ability of fungi to transition between unicellular and multicellular growth has a profound impact on our health and the economy. Many important fungal pathogens of humans, animals, and plants are dimorphic, and the ability to switch between morphological states has been associated with their virulence. Cryptococcus neoformans is a human fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised and, in some cases, immunocompetent hosts. Cryptococcus neoformans grows vegetatively as a budding yeast and switches to hyphal growth during the sexual cycle, which is important in the study of cryptococcal pathogenicity because spores resulting from sexual development are infectious propagules and can colonize the lungs of a host. In addition, sexual reproduction contributes to the genotypic variability of Cryptococcus species, which may lead to increased fitness and virulence. Despite significant advances in our understanding of the mechanisms behind the development of C. neoformans, our knowledge is still incomplete. Recent studies have led to the emergence of many intriguing questions and hypotheses. In this review, we describe and discuss the most interesting aspects of C. neoformans development and address their impact on pathogenicity.