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Cryptococcal meningitis has high mortality. Flucytosine is a key treatment but is expensive and rarely available. The anticancer agent tamoxifen has synergistic anti-cryptococcal activity with amphotericin in vitro. It is off-patent, cheap, and widely available. We performed a trial to determine its therapeutic potential. Open label randomized controlled trial. Participants received standard care - amphotericin combined with fluconazole for the first 2 weeks - or standard care plus tamoxifen 300 mg/day. The primary end point was Early Fungicidal Activity (EFA) - the rate of yeast clearance from cerebrospinal fluid (CSF). Trial registration https://clinicaltrials.gov/ct2/show/NCT03112031. Fifty patients were enrolled (median age 34 years, 35 male). Tamoxifen had no effect on EFA (-0.48log10 colony-forming units/mL/CSF control arm versus -0.49 tamoxifen arm, difference -0.005log10CFU/ml/day, 95% CI: -0.16, 0.15, p=0.95). Tamoxifen caused QTc prolongation. High-dose tamoxifen does not increase the clearance rate of from CSF. Novel, affordable therapies are needed. The trial was funded through the Wellcome Trust Asia Programme Vietnam Core Grant 106680 and a Wellcome Trust Intermediate Fellowship to JND grant number WT097147MA.

Background. The standard therapy for human immunodeficiency virus (HIV)–associated cryptococcal meningitis of amphotericin B (AmB; 0.7 mg/kg per day) plus flucytosine frequently takes >2 weeks to sterilize the cerebral spinal fluid, and acute mortality remains high. A dosage range for AmB of 0.7–1 mg/kg per day is noted in current guidelines, but there are no data comparing 0.7 mg/kg per day with 1 mg/kg per day. Methods. Sixty-four HIV-seropositive, antiretroviral therapy–naive patients in Cape Town, South Africa, who experienced their first episode of cryptococcal meningitis during the period May 2005–June 2006 were randomized to receive either (1) AmB, 0.7 mg/kg per day, plus flucytosine, 25 mg/kg 4 times per day (group 1; 30 patients); or (2) AmB, 1 mg/kg per day, plus flucytosine, 25 mg/kg 4 times per day (group 2; 34 patients). Regimens were given for 2 weeks, followed by treatment with oral fluconazole. The primary outcome measure was early fungicidal activity, as determined by results of serial, quantitative cerebral spinal fluid cryptococcal cultures. Secondary outcome measures were safety and mortality. The median duration of follow-up was 1 year. Results. Early fungicidal activity was significantly greater for group 2 than for group 1 (mean±SD, −0.56±0.24 vs. −0.45±0.16 log cfu/mL of cerebral spinal fluid per day; P=.02). The incidence of renal impairment did not significantly differ between the 2 groups. Anemia was associated with female sex and, less strongly, with membership in group 2. Renal impairment and anemia reversed after the regimen was switched to fluconazole. Two- and 10-week mortality rates were 6% and 24%, respectively, with no difference between groups. Conclusions. AmB, 1 mg/kg per day, plus flucytosine is more rapidly fungicidal than is standard-dose AmB plus flucytosine. Because of its size, this study provides limited data on any difference in toxicity between the regimens, but toxicities were manageable and reversible. Clinical trials registration number. ISRCTN68133435 (http://www.controlled-trials.com).

Background Cryptococcal meningitis (CM) is a major cause of mortality in HIV programmes in Africa despite increasing access to antiretroviral therapy (ART). Mortality is driven in part by limited availability of amphotericin-based treatment, drug-induced toxicities of amphotericin B deoxycholate and prolonged hospital admissions. A single, high-dose of liposomal amphotericin (L-AmB, Ambisome) on a fluconazole backbone has been reported as non-inferior to 14 days of standard dose L-AmB in reducing fungal burden. This trial examines whether single, high-dose L-AmB given with high-dose fluconazole and flucytosine is non-inferior to a seven-day course of amphotericin B deoxycholate plus flucytosine (the current World Health Organization [WHO] recommended treatment regimen). Methods An open-label phase III randomised controlled non-inferiority trial conducted in five countries in sub-Saharan Africa: Botswana, Malawi, South Africa, Uganda and Zimbabwe. The trial will compare CM induction therapy with (1) a single dose (10 mg/kg) of L-AmB given with 14 days of fluconazole (1200 mg/day) and flucytosine (100 mg/kg/day) to (2) seven days amphotericin B deoxycholate (1 mg/kg/day) given alongside seven days of flucytosine (100 mg/kg/day) followed by seven days of fluconazole (1200 mg/day). The primary endpoint is all-cause mortality at ten weeks with a non-inferiority margin of 10% and 90% power. Secondary endpoints are early fungicidal activity, proportion of grade III/IV adverse events, pharmacokinetic parameters and pharmacokinetic/pharmacodynamic associations, health service costs, all-cause mortality within the first two and four weeks, all-cause mortality within the first ten weeks (superiority analysis) and rates of CM relapse, immune reconstitution inflammatory syndrome and disability at ten weeks. A total of 850 patients aged ≥ 18 years with a first episode of HIV-associated CM will be enrolled (425 randomised to each arm). All patients will be followed for 16 weeks. All patients will receive consolidation therapy with fluconazole 800 mg/day to complete ten weeks of treatment, followed by fluconazole maintenance and ART as per local guidance. Discussion A safe, sustainable and easy to administer regimen of L-AmB that is non-inferior to seven days of daily amphotericin B deoxycholate therapy may reduce the number of adverse events seen in patients treated with amphotericin B deoxycholate and shorten hospital admissions, providing a highly favourable and implementable alternative to the current WHO recommended first-line treatment. Trial registration ISRCTN, ISRCTN72509687 . Registered on 13 July 2017.

Cryptococcus neoformans is an environmental pathogen that remodels its cellular physiology to survive within mammals and, in susceptible hosts, cause life-threatening meningoencephalitis. Of the many distinctions between the external environment and mammalian tissues, CO 2 concentration in the host is two orders of magnitude higher than in the environment and represents a critical stress for C. neoformans . C. neoformans strains that do not replicate at host CO 2 concentrations are less virulent in mouse models of infection, further supporting CO 2 tolerance as a virulence trait. To further understand the genetic determinants of C. neoformans CO 2 tolerance, we performed a near genome-wide screen for deletion mutants with altered CO 2 fitness using a competitive growth assay. A total of 301 of 4,692 deletion mutants showed altered CO 2 tolerance (245 reduced fitness; 56 increased fitness) demonstrating the global effect of host CO 2 on C. neoformans physiology. Based on this data set as well as a metabolomic analysis of C. neoformans adaptation to host CO 2 , we show that remodeling of central carbon metabolism, oxidative stress buffering, and membrane homeostasis represent an integrated response to CO 2 stress that is mediated in part by the TOR-Ypk1 signaling axis. We propose that CO 2 -induced capsule formation leads to reduced cellular glucose which, in turn, triggers remodeling of central carbon metabolism toward utilization of alternative carbon sources and increased mitochondrial respiration/reactive oxygen generation. Thus, these data provide a near genome-wide profile of the genetic determinants of C. neoformans CO 2 tolerance as well as a model for how this important environmental human fungal pathogen alters its physiology to proliferate in the host.

Cryptococcus neoformans is the leading cause of fungal meningoencephalitis. Cellular heterogeneity during cryptococcal infection contributes to host adaptation and fungal pathogenesis. C. neoformans titan cells and seed cells represent enlarged and small-sized morphotypes, respectively, which exhibit distinct transcriptional profiles and can be induced by environmental factors. In this work, we describe a distinct small morphotype of C. neoformans, referred to as ovoid cells. The formation of ovoid cells is promoted by host-related conditions such as nutrient limitation and elevated CO2 levels, which was observed during the late stage of cryptococcal infection. In addition to their smaller size compared to typical yeast cells, ovoid cells highly express OSP1, a marker distinguishes ovoid cells from other small morphotypes, including seed cells and titanides. These cells exhibit an increased budding and proliferation rate, which is consistent with transcriptome data that ovoid cells upregulate cell cycle related genes. We further demonstrate that the glucose repression signaling pathway and the cAMP/PKA pathway are involved in ovoid cell formation in C. neoformans. Ovoid cells show reduced fungal virulence but enhanced tolerance under long-term fluconazole treatment, indicating their important role in the balancing virulence and antifungal tolerance within C. neoformans populations.

Cryptococcus neoformans is a major life-threatening fungal pathogen. In response to the stress of the host environment, C. neoformans produces large polyploid titan cells. Titan cell production enhances the virulence of C. neoformans , yet whether the polyploid aspect of titan cells is specifically influential remains unknown. We show that titan cells were more likely to survive and produce offspring under multiple stress conditions than typical cells and that even their normally sized daughters maintained an advantage over typical cells in continued exposure to stress. Although polyploid titan cells generated haploid daughter cell progeny upon in vitro replication under nutrient-replete conditions, titan cells treated with the antifungal drug fluconazole produced fluconazole-resistant diploid and aneuploid daughter cells. Interestingly, a single titan mother cell was capable of generating multiple types of aneuploid daughter cells. The increased survival and genomic diversity of titan cell progeny promote rapid adaptation to new or high-stress conditions. IMPORTANCE The ability to adapt to stress is a key element for survival of pathogenic microbes in the host and thus plays an important role in pathogenesis. Here we investigated the predominantly haploid human fungal pathogen Cryptococcus neoformans , which is capable of ploidy and cell size increases during infection through production of titan cells. The enlarged polyploid titan cells are then able to rapidly undergo ploidy reduction to generate progeny with reduced ploidy and/or aneuploidy. Under stressful conditions, titan cell progeny have a growth and survival advantage over typical cell progeny. Understanding how titan cells enhance the rate of cryptococcal adaptation under stress conditions may assist in the development of novel drugs aimed at blocking ploidy transitions. The ability to adapt to stress is a key element for survival of pathogenic microbes in the host and thus plays an important role in pathogenesis. Here we investigated the predominantly haploid human fungal pathogen Cryptococcus neoformans , which is capable of ploidy and cell size increases during infection through production of titan cells. The enlarged polyploid titan cells are then able to rapidly undergo ploidy reduction to generate progeny with reduced ploidy and/or aneuploidy. Under stressful conditions, titan cell progeny have a growth and survival advantage over typical cell progeny. Understanding how titan cells enhance the rate of cryptococcal adaptation under stress conditions may assist in the development of novel drugs aimed at blocking ploidy transitions.

Fungi can cause devastating invasive infections, typically in immunocompromised patients. Treatment is complicated both by the evolutionary similarity between humans and fungi and by the frequent emergence of drug resistance. Studies in fungal pathogens have long been slowed by a lack of high-throughput tools and community resources that are common in model organisms. Here we demonstrate a high-throughput transposon mutagenesis and sequencing (TN-seq) system in Cryptococcus neoformans that enables genome-wide determination of gene essentiality. We employed a random forest machine learning approach to classify the C. neoformans genome as essential or nonessential, predicting 1,465 essential genes, including 302 that lack human orthologs. These genes are ideal targets for new antifungal drug development. TN-seq also enables genome-wide measurement of the fitness contribution of genes to phenotypes of interest. As proof of principle, we demonstrate the genome-wide contribution of genes to growth in fluconazole, a clinically used antifungal. We show a novel role for the well-studied RIM101 pathway in fluconazole susceptibility. We also show that insertions of transposons into the 5′ upstream region can drive sensitization of essential genes, enabling screenlike assays of both essential and nonessential components of the genome. Using this approach, we demonstrate a role for mitochondrial function in fluconazole sensitivity, such that tuning down many essential mitochondrial genes via 5′ insertions can drive resistance to fluconazole. Our assay system will be valuable in future studies of C. neoformans , particularly in examining the consequences of genotypic diversity.

Fungal diseases impact the lives of a millions of people across the globe, and with our current repertoire of therapeutic options dwindling, effective treatment strategies are urgently needed. Critically, the emergence of azole-resistant isolates in the clinic following prolonged treatment regimes, environmental fungicide exposure, and fungal evolution, threatens the outcome of current therapeutics, further endangering the survival of infected individuals. Here, we investigate the underpinnings of antifungal resistance using quantitative proteomics to discover protein-level signatures of fluconazole (FLC) resistance in the opportunistic human fungal pathogen, Cryptococcus neoformans . We explore ClpX, an ATP-dependent unfoldase, as a target to overcome FLC resistance and explore target efficacy through macrophage and murine models of cryptococcal infection. Here we show that disruption of ClpX, following gene deletion or targeted inhibition, re-introduces FLC susceptibility into resistant strains, rendering FLC treatment effective once again. Further, we identify and experimentally confirm mechanisms by which ClpX influences susceptibility to FLC, through association with both heme biosynthesis and ergosterol production. Overall, our results contribute to the understanding of mechanisms driving FLC resistance in a globally important fungal pathogen, and we provide avenues for targeting proteins as a therapeutic strategy to reverse antifungal resistance. Here, the authors reveal that the ATP-dependent unfoldase ClpX is a key driver of fluconazole resistance in Cryptococcus neoformans , and show that targeting ClpX restores drug susceptibility, offering a potential therapeutic strategy to reverse antifungal resistance and to render current drugs effective.

Copper is an essential micronutrient required for survival in all kingdoms of life, as it is used as a catalytic cofactor for many essential processes in the cell. In turn, this reactivity of copper ions makes elevated levels of free copper toxic to the cell. This dual nature of copper—essential for life but toxic at elevated levels—is used by our innate immune system in a process called nutritional immunity to combat and kill invading pathogens. In this work, we explore how the fungal human pathogen Cryptococcus neoformans senses high copper stress, a copper microenvironment encountered within the host lung. We identified a specific cysteine-rich motif within the copper-responsive transcription factor Cuf1 to be essential for high copper stress sensing. Mutation of this motif led to an impaired high copper stress adaptation, which did not affect the fitness of the yeast but did impact the containment and distribution of yeast cells inside the host lung.

Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, which is a major opportunistic infection in immunosuppressed individuals. Mammalian β-galactoside-binding protein Galectin-3 (Gal-3) modulates the host innate and adaptive immunity, and plays significant roles during microbial infections including some fungal diseases. Here we show that this protein plays a role also in C . neoformans infection. We find augmented Gal-3 serum levels in human and experimental infections, as well as in spleen, lung, and brain tissues of infected mice. Gal-3-deficient mice are more susceptible to cryptococcosis than WT animals, as demonstrated by the higher fungal burden and lower animal survival. In vitro experiments show that Gal-3 inhibits fungal growth and exerts a direct lytic effect on C . neoformans extracellular vesicles (EVs). Our results indicate a direct role for Gal-3 in antifungal immunity whereby this molecule affects the outcome of C . neoformans infection by inhibiting fungal growth and reducing EV stability, which in turn could benefit the host. The protein Galectin-3 modulates host immunity and plays roles during infections. Here, Almeida et al. show that this protein contributes to host defence against infection with the fungal pathogen Cryptococcus neoformans by inhibiting fungal growth and inducing lysis of fungal extracellular vesicles.