C5a-licensed phagocytes drive sterilizing immunity during systemic fungal infection.

Systemic candidiasis is a common, high-mortality, nosocomial fungal infection. Unexpectedly, it has emerged as a complication of anti-complement C5-targeted monoclonal antibody treatment, indicating a critical niche for C5 in antifungal immunity. We identified transcription of complement system genes as the top biological pathway induced in candidemic patients and as predictive of candidemia. Mechanistically, C5a-C5aR1 promoted fungal clearance and host survival in a mouse model of systemic candidiasis by stimulating phagocyte effector function and ERK- and AKT-dependent survival in infected tissues. C5ar1 ablation rewired macrophage metabolism downstream of mTOR, promoting their apoptosis and enhancing mortality through kidney injury. Besides hepatocyte-derived C5, local C5 produced intrinsically by phagocytes provided a key substrate for antifungal protection. Lower serum C5a concentrations or a C5 polymorphism that decreases leukocyte C5 expression correlated independently with poor patient outcomes. Thus, local, phagocyte-derived C5 production licenses phagocyte antimicrobial function and confers innate protection during systemic fungal infection.

Risk factors, management, and clinical outcomes of invasive Mycoplasma and Ureaplasma infections after lung transplantation.

Mollicute infections, caused by Mycoplasma and Ureaplasma species, are serious complications after lung transplantation; however, understanding of the epidemiology and outcomes of these infections remains limited. We conducted a single-center retrospective study of 1156 consecutive lung transplants performed from 2010-2019. We used log-binomial regression to identify risk factors for infection and analyzed clinical management and outcomes. In total, 27 (2.3%) recipients developed mollicute infection. Donor characteristics independently associated with recipient infection were age ≤40 years (prevalence rate ratio [PRR] 2.6, 95% CI 1.0-6.9), White race (PRR 3.1, 95% CI 1.1-8.8), and purulent secretions on donor bronchoscopy (PRR 2.3, 95% CI 1.1-5.0). Median time to diagnosis was 16 days posttransplant (IQR: 11-26 days). Mollicute-infected recipients were significantly more likely to require prolonged ventilatory support (66.7% vs 21.4%), undergo dialysis (44.4% vs 6.3%), and remain hospitalized ≥30 days (70.4% vs 27.4%) after transplant. One-year posttransplant mortality in mollicute-infected recipients was 12/27 (44%), compared to 148/1129 (13%) in those without infection (P <.0001). Hyperammonemia syndrome occurred in 5/27 (19%) mollicute-infected recipients, of whom 3 (60%) died within 10 weeks posttransplant. This study highlights the morbidity and mortality associated with mollicute infection after lung transplantation and the need for better screening and management protocols.

Mucorales: A systematic review to inform the World Health Organization priority list of fungal pathogens.

The World Health Organization, in response to the growing burden of fungal disease, established a process to develop a fungal priority pathogens list (FPPL). This systematic review aimed to evaluate the epidemiology and impact of invasive fungal disease due to Mucorales. PubMed and Web of Science were searched to identify studies published between January 1, 2011 and February 23, 2021. Studies reporting on mortality, inpatient care, complications and sequelae, antifungal susceptibility, risk factors, preventability, annual incidence, global distribution, and emergence during the study time frames were selected. Overall, 24 studies were included. Mortality rates of up to 80% were reported. Antifungal susceptibility varied across agents and species, with the minimum inhibitory concentrations lowest for amphotericin B and posaconazole. Diabetes mellitus was a common risk factor, detected in 65%-85% of patients with mucormycosis, particularly in those with rhino-orbital disease (86.9%). Break-through infection was detected in 13.6%-100% on azole or echinocandin antifungal prophylaxis. The reported prevalence rates were variable, with some studies reporting stable rates in the USA of 0.094-0.117/10 000 discharges between 2011 and 2014, whereas others reported an increase in Iran from 16.8% to 24% between 2011 and 2015. Carefully designed global surveillance studies, linking laboratory and clinical data, are required to develop clinical breakpoints to guide antifungal therapy and determine accurate estimates of complications and sequelae, annual incidence, trends, and global distribution. These data will provide robust estimates of disease burden to refine interventions and better inform future FPPL.

Aspergillus fumigatus-a systematic review to inform the World Health Organization priority list of fungal pathogens.

Recognizing the growing global burden of fungal infections, the World Health Organization established a process to develop a priority list of fungal pathogens (FPPL). In this systematic review, we aimed to evaluate the epidemiology and impact of invasive infections caused by Aspergillus fumigatus to inform the first FPPL. The pre-specified criteria of mortality, inpatient care, complications and sequelae, antifungal susceptibility, risk factors, preventability, annual incidence, global distribution, and emergence were used to search for relevant articles between 1 January 2016 and 10 June 2021. Overall, 49 studies were eligible for inclusion. Azole antifungal susceptibility varied according to geographical regions. Voriconazole susceptibility rates of 22.2% were reported from the Netherlands, whereas in Brazil, Korea, India, China, and the UK, voriconazole susceptibility rates were 76%, 94.7%, 96.9%, 98.6%, and 99.7%, respectively. Cross-resistance was common with 85%, 92.8%, and 100% of voriconazole-resistant A. fumigatus isolates also resistant to itraconazole, posaconazole, and isavuconazole, respectively. The incidence of invasive aspergillosis (IA) in patients with acute leukemia was estimated at 5.84/100 patients. Six-week mortality rates in IA cases ranged from 31% to 36%. Azole resistance and hematological malignancy were poor prognostic factors. Twelve-week mortality rates were significantly higher in voriconazole-resistant than in voriconazole-susceptible IA cases (12/22 [54.5%] vs. 27/88 [30.7%]; P = .035), and hematology patients with IA had significantly higher mortality rates compared with solid-malignancy cases who had IA (65/217 [30%] vs. 14/78 [18%]; P = .04). Carefully designed surveillance studies linking laboratory and clinical data are required to better inform future FPPL.

Susceptibility to Cryptococcus neoformans Infection with Bruton's Tyrosine Kinase Inhibition.

Patients receiving the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib have an increased likelihood of fungal infections. The objectives of this study were to determine if Cryptococcus neoformans infection severity was isolate dependent with BTK inhibition and whether blocking BTK impacted infection severity in a mouse model. We compared four clinical isolates from patients on ibrutinib to virulent (H99) and avirulent (A1-35-8) reference strains. BTK knockout (KO) and wild-type (WT) C57 mice and WT CD1 mice were infected by intranasal (i.n.), oropharyngeal aspiration (OPA), and intravenous (i.v.) routes. Infection severity was assessed by survival and fungal burden (CFU per gram of tissue). Ibrutinib (25 mg/kg) or vehicle was administered daily through intraperitoneal injections. In the BTK KO model, no isolate-dependent effect on fungal burden was observed, and infection severity was not significantly different from that of the WT with i.n., OPA, and i.v. routes. Ibrutinib treatment did not impact infection severity. However, when the four clinical isolates were compared to H99, two of these isolates were less virulent, with significantly longer survival and reduced rates of brain infection. In conclusion, C. neoformans infection severity in the BTK KO model does not appear to be isolate dependent. BTK KO and ibrutinib treatment did not result in significantly different infection severities. However, based on repeated clinical observations of increased susceptibility to fungal infections with BTK inhibitor therapy, further work is needed to optimize a mouse model with BTK inhibition to better understand the role that this pathway plays in susceptibility to C. neoformans infection.

Pharmacodynamics of ATI-2307 in a rabbit model of cryptococcal meningoencephalitis.

Cryptococcal meningoencephalitis (CM) is a devastating fungal disease with high morbidity and mortality. The current regimen that is standard-of-care involves a combination of three different drugs administered for up to one year. There is a critical need for new therapies due to both toxicity and inadequate fungicidal activity of the currently available antifungal drugs. ATI-2307 is a novel aryl amidine that disrupts the mitochondrial membrane potential and inhibits the respiratory chain complexes of fungi-it thus represents a new mechanism for direct antifungal action. Furthermore, ATI-2307 selectively targets fungal mitochondria via a fungal-specific transporter that is not present in mammalian cells. It has very potent in vitro anticryptococcal activity. In this study, the efficacy of ATI-2307 was tested in a rabbit model of CM. ATI-2307 demonstrated significant fungicidal activity at dosages between 1 and 2 mg/kg/d, and these results were superior to fluconazole and similar to amphotericin B treatment. When ATI-2307 was combined with fluconazole, the antifungal effect was greater than either therapy alone. While ATI-2307 has potent anticryptococcal activity in the subarachnoid space, its ability to reduce yeasts in the brain parenchyma was relatively less over the same study period. This new drug, with its unique mechanism of fungicidal action and ability to positively interact with an azole, has demonstrated sufficient anticryptococcal potential in this experimental setting to be further evaluated in clinical studies.

Genetic diversity and microevolution in clinical Cryptococcus isolates from Cameroon.

Cryptococcal meningitis is the second most common cause of death in people living with HIV/AIDS, yet we have a limited understanding of how cryptococcal isolates change over the course of infection. Cryptococcal infections are environmentally acquired, and the genetic diversity of these infecting isolates can also be geographically linked. Here, we employ whole genome sequences for 372 clinical Cryptococcus isolates from 341 patients with HIV-associated cryptococcal meningitis obtained via a large clinical trial, across both Malawi and Cameroon, to enable population genetic comparisons of isolates between countries. We see that isolates from Cameroon are highly clonal, when compared to those from Malawi, with differential rates of disruptive variants in genes with roles in DNA binding and energy use. For a subset of patients (22) from Cameroon, we leverage longitudinal sampling, with samples taken at days 7 and 14 post-enrollment, to interrogate the genetic changes that arise over the course of infection, and the genetic diversity of isolates within patients. We see disruptive variants arising over the course of infection in several genes, including the phagocytosis-regulating transcription factor GAT204. In addition, in 13% of patients sampled longitudinally, we see evidence for mixed infections. This approach identifies geographically linked genetic variation, signatures of microevolution, and evidence for mixed infections across a clinical cohort of patients affected by cryptococcal meningitis in Central Africa.

Cryptococcal meningitis, caused by Cryptococcus, results in approximately half a million deaths per year globally. We compare clinical Cryptococcus samples from Cameroon and Malawi to explore the genetic diversity of these isolates. We find instances of mixed-strain infections and identify genetic variants arising in Cryptococcus over disease.

Development and validation of a colorimetric antifungal susceptibility testing method for the dimorphic fungus Talaromyces marneffei.

Antifungal drug resistance is an emerging cause of treatment failure in invasive fungal infections, and antifungal susceptibility testing (AFST) may inform treatment decisions. Currently, there are no established AFST guidelines for Talaromyces marneffei (Tm) or other dimorphic fungi. We developed a colorimetric AFST method using a fluorescent redox indicator alamarBlue, which changes from blue to pink in proportion to cellular metabolic activity. We determined the optimal time for alamarBlue addition to be 24 h post-inoculation and for MIC reading to be 72 h post-inoculation. Our method allows three ways to determine minimum inhibitory concentration (MIC): visual inspection of color change, optical density, and fluorescence intensity. We validated the assay by determining the MICs for seven antifungals against 32 Tm clinical isolates and assessed the essential agreement (EA) and inter-rater reliability between our alamarBlue and the Clinical Laboratory Standard Institute (CLSI) broth microdilution methods. The MIC ranges (from low to high) were: 0.008-0.025 µg/ml for itraconazole, 0.004-0.13 µg/ml for voriconazole, 0.03-0.13 µg/ml for posaconazole, 0.06-0.5 µg/ml for flucytosine, 0.5-1 µg/ml for amphotericin B, 0.5-4 µg/ml for caspofungin, and 0.5-16 µg/ml for fluconazole. The EAs were 100% between all three MIC readouts of the alamarBlue method, and 94%-100% between the alamarBlue and CLSI methods. Our alamarBlue method had substantially higher inter-rater agreement and offers a more reliable method that can be standardized across laboratories in both high- and low-resource settings compared to the established CLSI methodology.

We developed a colorimetric alamarBlue method to determine the susceptibility of antifungal drugs against Talaromyces marneffei. We observed excellent agreement between the alamarBlue method and the Clinical Laboratory Standard Institute broth microdilution method, and the alamarBlue method had substantially higher inter-rater agreement.

The current state of Clinical Mycology in Eastern and South-Eastern Europe.

The ability of medical centers in Eastern and South-Eastern Europe to diagnose and treat fungal infections remains unknown. In order to investigate that, here we conducted a cross-sectional online survey, released at both The International Society for Human & Animal Mycology (ISHAM) and European Confederation of Medical Mycology (ECMM) websites. A total of 31 institutions responded to the questionnaire. Most centers (87.1%, n = 27) had access to Aspergillus spp. ELISA galactomannan testing as well as to Cryptococcus spp. antigen testing (83.9%, n = 26). Serological tests were mostly available for Aspergillus species (80.6%, n = 25); and most institutions reported access to mold-active antifungal drugs (83.9%; n = 26), but 5-flucytosine was available to only 29% (n = 9) of the participant centers. In conclusion, this study represents the first attempt to document the strengths and limitations of the Eastern and South-Eastern European region for diagnosing and treating fungal diseases. LAY SUMMARY: Our article is about the availability of diagnostic and treatments tools related to fungal infections in the countries of Eastern and South-Eastern region. Surveys like these are important to understand the gaps and point towards the fungal infections as a global health issue.

MSG07: An International Cohort Study Comparing Epidemiology and Outcomes of Patients With Cryptococcus neoformans or Cryptococcus gattii Infections.

BACKGROUND: Cryptococcosis due to Cryptococcus neoformans and Cryptococcus gattii varies with geographic region, populations affected, disease manifestations, and severity of infection, which impact treatment. METHODS: We developed a retrospective cohort of patients diagnosed with culture-proven cryptococcosis during 1995-2013 from 5 centers in North America and Australia. We compared underlying diseases, clinical manifestations, treatment, and outcomes in patients with C. gattii or C. neoformans infection. RESULTS: A total of 709 patients (452 C. neoformans; 257 C. gattii) were identified. Mean age was 50.2 years; 61.4% were male; and 52.3% were white. Time to diagnosis was prolonged in C. gattii patients compared with C. neoformans (mean, 52.2 vs 36.0 days; P < .003), and there was a higher proportion of C. gattii patients without underlying disease (40.5% vs 10.2%; P < .0001). Overall, 59% had central nervous system (CNS) infection, with lung (42.5%) and blood (24.5%) being common sites. Pulmonary infection was more common in patients with C. gattii than in those with C. neoformans (60.7% vs 32.1%; P < .0001). CNS or blood infections were more common in C. neoformans-infected patients (P ≤ .0001 for both). Treatment of CNS disease with induction therapy of amphotericin B and flucytosine occurred in 76.4% of patients. Crude 12-month mortality was higher in patients with C. neoformans (28.4% vs 20.2%; odds ratio, 1.56 [95% confidence interval, 1.08-2.26]). CONCLUSIONS: This study emphasizes differences in species-specific epidemiology and outcomes of patients with cryptococcosis, including underlying diseases, site of infection, and mortality. Species identification in patients with cryptococcosis is necessary to discern epidemiologic patterns, guide treatment regimens, and predict clinical progression and outcomes.

Correction: Cryptococcus neoformans resists to drastic conditions by switching to viable but non-culturable cell phenotype.

[This corrects the article DOI: 10.1371/journal.ppat.1007945.].

Cryptococcus neoformans resists to drastic conditions by switching to viable but non-culturable cell phenotype.

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce Cryptococcus neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro and then standardize the experimental conditions to consistently generate this dormancy in C. neoformans. We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30-40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and a precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial mass increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC. Altogether, these data show that we were able to generate for the first time VBNC phenotype in C. neoformans. This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast.

A link between urease and polyamine metabolism in Cryptococcus neoformans.

The urease enzyme of Cryptococcus neoformans is linked to different metabolic pathways within the yeast cell, several of which are involved in polyamine metabolism. Cryptococcal biogenic amine production is, however, largely unexplored and is yet to be investigated in relation to urease. The aim of this study was therefore to explore and compare polyamine metabolism in wild-type, urease-negative and urease-reconstituted strains of C. neoformans. Mass spectrometry analysis showed that agmatine and spermidine were the major extra- and intracellular polyamines of C. neoformans and significant differences were observed between 26 and 37 °C. In addition, compared to the wild-type, the relative percentages of extracellular putrescine and spermidine were found to be lower and agmatine higher in cultures of the urease-deficient mutant. The inverse was true for intracellular spermidine and agmatine. Cyclohexylamine was a more potent polyamine inhibitor compared to DL-α-difluoromethylornithine and inhibitory effects were more pronounced at 37 °C than at 26 °C. At both temperatures, the urease-deficient mutant was less susceptible to cyclohexylamine treatment compared to the wild-type. For both inhibitors, growth inhibition was alleviated with polyamine supplementation. This study has provided novel insight into the polyamine metabolism of C. neoformans, highlighting the involvement of urease in biogenic amine production.

Core Recommendations for Antifungal Stewardship: A Statement of the Mycoses Study Group Education and Research Consortium.

In recent years, the global public health community has increasingly recognized the importance of antimicrobial stewardship (AMS) in the fight to improve outcomes, decrease costs, and curb increases in antimicrobial resistance around the world. However, the subject of antifungal stewardship (AFS) has received less attention. While the principles of AMS guidelines likely apply to stewarding of antifungal agents, there are additional considerations unique to AFS and the complex field of fungal infections that require specific recommendations. In this article, we review the literature on AMS best practices and discuss AFS through the lens of the global core elements of AMS. We offer recommendations for best practices in AFS based on a synthesis of this evidence by an interdisciplinary expert panel of members of the Mycoses Study Group Education and Research Consortium. We also discuss research directions in this rapidly evolving field. AFS is an emerging and important component of AMS, yet requires special considerations in certain areas such as expertise, education, interventions to optimize utilization, therapeutic drug monitoring, and data analysis and reporting.

Invasive Fungal Infection After Lung Transplantation: Epidemiology in the Setting of Antifungal Prophylaxis.

BACKGROUND: Lung transplant recipients commonly develop invasive fungal infections (IFIs), but the most effective strategies to prevent IFIs following lung transplantation are not known. METHODS: We prospectively collected clinical data on all patients who underwent lung transplantation at a tertiary care academic hospital from January 2007-October 2014. Standard antifungal prophylaxis consisted of aerosolized amphotericin B lipid complex during the transplant hospitalization. For the first 180 days after transplant, we analyzed prevalence rates and timing of IFIs, risk factors for IFIs, and data from IFIs that broke through prophylaxis. RESULTS: In total, 156 of 815 lung transplant recipients developed IFIs (prevalence rate, 19.1 IFIs per 100 surgeries, 95% confidence interval [CI] 16.4-21.8%). The prevalence rate of invasive candidiasis (IC) was 11.4% (95% CI 9.2-13.6%), and the rate of non-Candida IFIs was 8.8% (95% CI 6.9-10.8%). First episodes of IC occurred a median of 31 days (interquartile range [IQR] 16-56 days) after transplant, while non-Candida IFIs occurred later, at a median of 86 days (IQR 40-121 days) after transplant. Of 169 IFI episodes, 121 (72%) occurred in the absence of recent antifungal prophylaxis; however, IC and non-Candida breakthrough IFIs were observed, most often representing failures of micafungin (n = 16) and aerosolized amphotericin B (n = 24) prophylaxis, respectively. CONCLUSIONS: Lung transplant recipients at our hospital had high rates of IFIs, despite receiving prophylaxis with aerosolized amphotericin B lipid complex during the transplant hospitalization. These data suggest benefit in providing systemic antifungal prophylaxis targeting Candida for up to 90 days after transplant and extending mold-active prophylaxis for up to 180 days after surgery.

Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium.

BACKGROUND: Invasive fungal diseases (IFDs) remain important causes of morbidity and mortality. The consensus definitions of the Infectious Diseases Group of the European Organization for Research and Treatment of Cancer and the Mycoses Study Group have been of immense value to researchers who conduct clinical trials of antifungals, assess diagnostic tests, and undertake epidemiologic studies. However, their utility has not extended beyond patients with cancer or recipients of stem cell or solid organ transplants. With newer diagnostic techniques available, it was clear that an update of these definitions was essential. METHODS: To achieve this, 10 working groups looked closely at imaging, laboratory diagnosis, and special populations at risk of IFD. A final version of the manuscript was agreed upon after the groups' findings were presented at a scientific symposium and after a 3-month period for public comment. There were several rounds of discussion before a final version of the manuscript was approved. RESULTS: There is no change in the classifications of "proven," "probable," and "possible" IFD, although the definition of "probable" has been expanded and the scope of the category "possible" has been diminished. The category of proven IFD can apply to any patient, regardless of whether the patient is immunocompromised. The probable and possible categories are proposed for immunocompromised patients only, except for endemic mycoses. CONCLUSIONS: These updated definitions of IFDs should prove applicable in clinical, diagnostic, and epidemiologic research of a broader range of patients at high-risk.

Population genomics and the evolution of virulence in the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes approximately 625,000 deaths per year from nervous system infections. Here, we leveraged a unique, genetically diverse population of C. neoformans from sub-Saharan Africa, commonly isolated from mopane trees, to determine how selective pressures in the environment coincidentally adapted C. neoformans for human virulence. Genome sequencing and phylogenetic analysis of 387 isolates, representing the global VNI and African VNB lineages, highlighted a deep, nonrecombining split in VNB (herein, VNBI and VNBII). VNBII was enriched for clinical samples relative to VNBI, while phenotypic profiling of 183 isolates demonstrated that VNBI isolates were significantly more resistant to oxidative stress and more heavily melanized than VNBII isolates. Lack of melanization in both lineages was associated with loss-of-function mutations in the BZP4 transcription factor. A genome-wide association study across all VNB isolates revealed sequence differences between clinical and environmental isolates in virulence factors and stress response genes. Inositol transporters and catabolism genes, which process sugars present in plants and the human nervous system, were identified as targets of selection in all three lineages. Further phylogenetic and population genomic analyses revealed extensive loss of genetic diversity in VNBI, suggestive of a history of population bottlenecks, along with unique evolutionary trajectories for mating type loci. These data highlight the complex evolutionary interplay between adaptation to natural environments and opportunistic infections, and that selection on specific pathways may predispose isolates to human virulence.

Titan cells formation in Cryptococcus neoformans is finely tuned by environmental conditions and modulated by positive and negative genetic regulators.

The pathogenic fungus Cryptococcus neoformans exhibits morphological changes in cell size during lung infection, producing both typical size 5 to 7 µm cells and large titan cells (> 10 µm and up to 100 µm). We found and optimized in vitro conditions that produce titan cells in order to identify the ancestry of titan cells, the environmental determinants, and the key gene regulators of titan cell formation. Titan cells generated in vitro harbor the main characteristics of titan cells produced in vivo including their large cell size (>10 µm), polyploidy with a single nucleus, large vacuole, dense capsule, and thick cell wall. Here we show titan cells derived from the enlargement of progenitor cells in the population independent of yeast growth rate. Change in the incubation medium, hypoxia, nutrient starvation and low pH were the main factors that trigger titan cell formation, while quorum sensing factors like the initial inoculum concentration, pantothenic acid, and the quorum sensing peptide Qsp1p also impacted titan cell formation. Inhibition of ergosterol, protein and nucleic acid biosynthesis altered titan cell formation, as did serum, phospholipids and anti-capsular antibodies in our settings. We explored genetic factors important for titan cell formation using three approaches. Using H99-derivative strains with natural genetic differences, we showed that titan cell formation was dependent on LMP1 and SGF29 genes. By screening a gene deletion collection, we also confirmed that GPR4/5-RIM101, and CAC1 genes were required to generate titan cells and that the PKR1, TSP2, USV101 genes negatively regulated titan cell formation. Furthermore, analysis of spontaneous Pkr1 loss-of-function clinical isolates confirmed the important role of the Pkr1 protein as a negative regulator of titan cell formation. Through development of a standardized and robust in vitro assay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways.

The virulence factor urease and its unexplored role in the metabolism of Cryptococcus neoformans.

Cryptococcal urease is believed to be important for the degradation of exogenous urea that the yeast encounters both in its natural environment and within the human host. Endogenous urea produced by the yeast's own metabolic reactions, however, may also serve as a substrate for the urease enzyme. Using wild-type, urease-deletion mutant and urease-reconstituted strains of Cryptococcus neoformans H99, we studied reactions located up- and downstream from endogenous urea. We demonstrated that urease is important for cryptococcal growth and that, compared to nutrient-rich conditions at 26°C, urease activity is higher under nutrient-limited conditions at 37°C. Compared to cells with a functional urease enzyme, urease-deficient cells had significantly higher intracellular urea levels and also showed more arginase activity, which may act as a potential source of endogenous urea. Metabolic reactions linked to arginase were also affected, since urease-positive and urease-negative cells differed with respect to agmatinase activity, polyamine synthesis, and intracellular levels of proline and reactive oxygen species. Lastly, urease-deficient cells showed higher melanin levels at 26°C than wild-type cells, while the inverse was observed at 37°C. These results suggest that cryptococcal urease is associated with the functioning of key metabolic pathways within the yeast cell.