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.

The Cyclin Cln1 Controls Polyploid Titan Cell Formation following a Stress-Induced G2 Arrest in Cryptococcus.

The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the in vivo and in vitro cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells. We identified unbudded 2C cells, referred to as a G2 arrest, produced both in vivo and in vitro in response to various stresses. Deletion of the nonessential cyclin Cln1 resulted in overproduction of titan cells in vivo and transient morphology defects upon release from stationary phase in vitro. Using a copper-repressible promoter PCTR4-CLN1 strain and a two-step in vitro titan cell formation assay, our in vitro studies revealed Cln1 functions after the G2 arrest. These studies highlight unique cell cycle alterations in C. neoformans that ultimately promote genomic diversity and virulence in this important fungal pathogen. IMPORTANCE Dysregulation of the cell cycle underlies many human genetic diseases and cancers, yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen Cryptococcus neoformans generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease. We analyzed the C. neoformans in vivo and in vitro cell cycles to identify changes required to generate the polyploid titan cells. C. neoformans paused cell cycle progression in response to various environmental stresses after DNA replication and before morphological changes associated with cell division, referred to as a G2 arrest. Release from this G2 arrest was coordinated by the cyclin Cln1. Reduced CLN1 expression after the G2 arrest was associated with polyploid titan cell production. These results demonstrate a mechanism to generate genomic diversity in eukaryotic cells through manipulation of the cell cycle that has broad disease implications.

Crowdsourced analysis of fungal growth and branching on microfluidic platforms.

Fungal hyphal growth and branching are essential traits that allow fungi to spread and proliferate in many environments. This sustained growth is essential for a myriad of applications in health, agriculture, and industry. However, comparisons between different fungi are difficult in the absence of standardized metrics. Here, we used a microfluidic device featuring four different maze patterns to compare the growth velocity and branching frequency of fourteen filamentous fungi. These measurements result from the collective work of several labs in the form of a competition named the "Fungus Olympics." The competing fungi included five ascomycete species (ten strains total), two basidiomycete species, and two zygomycete species. We found that growth velocity within a straight channel varied from 1 to 4 µm/min. We also found that the time to complete mazes when fungal hyphae branched or turned at various angles did not correlate with linear growth velocity. We discovered that fungi in our study used one of two distinct strategies to traverse mazes: high-frequency branching in which all possible paths were explored, and low-frequency branching in which only one or two paths were explored. While the high-frequency branching helped fungi escape mazes with sharp turns faster, the low-frequency turning had a significant advantage in mazes with shallower turns. Future work will more systematically examine these trends.

Docusate-Based Ionic Liquids of Anthelmintic Benzimidazoles Show Improved Pharmaceutical Processability, Lipid Solubility, and in Vitro Activity against Cryptococcus neoformans.

As the existing therapeutic modalities for the treatment of cryptococcal meningitis (CM) have suboptimal efficacy, repurposing existing drugs for the treatment of CM is of great interest. The FDA-approved anthelmintic benzimidazoles, albendazole, mebendazole, and flubendazole, have demonstrated potent but variable in vitro activity against Cryptococcus neoformans, the predominant fungal species responsible for CM. We performed molecular docking studies to ascertain the interaction of albendazole, mebendazole, and flubendazole with a C. neoformans ß-tubulin structure, which revealed differential binding interactions and explained the different in vitro efficacies reported previously and observed in this investigation. Despite their promising in vitro efficacy, the repurposing of anthelmintic benzimidazoles for oral CM therapy is significantly hampered due to their high crystallinity, poor pharmaceutical processability, low and pH-dependent solubility, and drug precipitation upon entering the intestine, all of which result in low and variable oral bioavailability. Here, we demonstrate that the anthelmintic benzimidazoles can be transformed into partially amorphous low-melting ionic liquids (ILs) with a simple metathesis reaction using amphiphilic sodium docusate as a counterion. In vitro efficacy studies on a laboratory reference and a clinical isolate of C. neoformans showed 2- to 4-fold lower IC90 values for docusate-based ILs compared to the pure anthelmintic benzimidazoles. Furthermore, using a C. neoformans strain with green fluorescent protein (GFP)-tagged ß-tubulin and albendazole and its docusate IL as model candidates, we showed that the benzimidazoles and their ILs reduce the viability of C. neoformans by interfering with its microtubule assembly. Unlike pure anthelmintic benzimidazoles, the docusate-based ILs showed excellent solubility in organic solvents and >30-fold higher solubility in bioavailability-enhancing lipid vehicles. Finally, the docusate ILs were successfully incorporated into SoluPlus, a self-assembling biodegradable polymer, which upon dilution with water formed polymeric micelles with a size of <100 nm. Thus, the development of docusate-based ILs represents an effective approach to improve the physicochemical properties and potency of anthelmintic benzimidazoles to facilitate their repurposing and preclinical development for CM therapy.

ATI-2307 Exhibits Equivalent Antifungal Activity in Cryptococcus neoformans Clinical Isolates With High and Low Fluconazole IC50.

Half maximal inhibitory concentrations (IC50) to the experimental drug ATI-2307 and complete inhibition (IC90) of the common clinically used antifungal drug amphotericin B were determined by microbroth dilution assay for a collection of 69 clinical isolates of Cryptococcus neoformans from Uganda that had high fluconazole IC50 values. The majority of the clinical isolates tested had fluconazole IC50 at or above 8 µg/mL, but were susceptible to both amphotericin B (IC90 ≤1 µg/mL) and ATI-2307 (IC50 ≤0.0312 µg/mL). No correlation between increased fluconazole minimum inhibitory concentration (MIC) and ATI-2307 or amphotericin B MIC was observed, suggesting that the cellular changes impacting fluconazole susceptibility did not impact the effectiveness of ATI-2307. Our results suggest that ATI-2307 is a promising new antifungal drug for use in the context of high fluconazole or other antifungal drug MICs and/or in combination drug therapy regimens.

The interplay of phenotype and genotype in Cryptococcus neoformans disease.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningitis primarily in immunocompromised individuals. In order to survive and proliferate during infection, C. neoformans must adapt to a variety of stresses it encounters within the host. Patient outcome depends on the interaction between the pathogen and the host. Understanding the mechanisms that C. neoformans uses to facilitate adaptation to the host and promote pathogenesis is necessary to better predict disease severity and establish proper treatment. Several virulence phenotypes have been characterized in C. neoformans, but the field still lacks a complete understanding of how genotype and phenotype contribute to clinical outcome. Furthermore, while it is known that C. neoformans genotype impacts patient outcome, the mechanisms remain unknown. This lack of understanding may be due to the genetic heterogeneity of C. neoformans and the extensive phenotypic variation observed between and within isolates during infection. In this review, we summarize the current understanding of how the various genotypes and phenotypes observed in C. neoformans correlate with human disease progression in the context of patient outcome and recurrence. We also postulate the mechanisms underlying the genetic and phenotypic changes that occur in vivo to promote rapid adaptation in the host.

A titanic drug resistance threat in Cryptococcus neoformans.

Increasing resistance to frontline antifungals is a growing threat to global health. In the face of high rates of relapse for patients with cryptococcal meningitis and frequent drug resistance in clinical isolates, recent insights into Cryptococcus neoformans morphogenesis and genome plasticity take on new and urgent meaning. Here we review the state of the understanding of mechanisms of drug resistance in the context of host-relevant changes in Cryptococcus morphology and cell ploidy.

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.

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.