Extracellular vesicles derived from Cryptococcus neoformans promoted neutrophil extracellular traps.

Investigation of the effects of polycations on Candida albicans membrane models and fungal cells. The effect of polymer structure and molecular weight.

Invasive fungal infections are a growing global health threat, driven by the limited availability of antifungal therapies and the rapid spread of multidrug-resistant yeasts. This study explored the effects of N-terminal lipidation on Trem-4, a decapeptide derived from the antimicrobial peptide Trematocine-HSK, to enhance its antifungal potential. Trem-4 was modified with caprylic (C8) and myristic (C14) fatty acid chains, generating two lipopeptides: Cap-T4 and Myr-T4. Biophysical analyses, including fluorescence-based assays and circular dichroism, showed that lipidation significantly improved peptide-membrane interactions compared to the non-lipidated form. The two derivatives displayed distinct behaviors, with differences in aggregation and membrane selectivity depending on acyl chain length. Antifungal activity was assessed against 60 clinically characterized yeast isolates, including Candida spp., Candidozyma haemuli complex, and Cryptococcus neoformans, under both planktonic and biofilm conditions. Both lipopeptides exhibited broad-spectrum activity, against clinically relevant species, with conserved activity against antifungal-resistant strains. Importantly, both compounds remained effective against mature biofilms, causing metabolic disruption and structural damage. Cap-T4 demonstrated consistent activity across species, while Myr-T4 showed enhanced potency against selected isolates, particularly C. auris. Biocompatibility evaluation revealed a concentration-dependent cytotoxicity and hemolysis, more pronounced for Myr-T4. However, both compounds were well tolerated in vivo in the Galleria mellonella model. Overall, Cap-T4 emerged as the most promising candidate for further preclinical development. These findings support N-terminal lipidation as an effective strategy to improve the antifungal efficacy of short antimicrobial peptides and highlight its potential for developing new treatments against drug-resistant fungal pathogens. KEY POINTS: N-terminal lipidation enhances Trem-4 membrane interaction and activity.Myr-T4 and Cap-T4 show broad activity against susceptible and resistant yeasts.Cap-T4 retains activity against yeasts and mature biofilms.

In Schizosaccharomyces pombe and Cryptococcus neoformans, the flavohemoglobins (flavoHbs) that protect these yeasts against nitrosative stress are Yhb1 and Fhb1, respectively. Although heme is an essential cofactor for flavoHb activity, the molecular circuitry governing the acquisition of exogenous heme by flavoHbs remains largely unknown. Because S. pombe is a powerful model organism for studying heme biology, Fhb1 was heterologously expressed in S. pombe mutant cells lacking Yhb1. Fhb1 expression functionally complements yhb1Δ cells that are defective in nitric oxide (˙NO) detoxification. Moreover, this Fhb1-mediated complementation requires the S. pombe heme transporter Str3 or the heterologous coexpression of two previously uncharacterized C. neoformans transporters, CnStr1 and CnStr3. Consistent with this finding, heterologous expression of CnStr1 and CnStr3 rescues the heme-dependent growth defect of S. pombe cells that are deficient in heme biosynthesis and lack endogenous heme uptake systems when hemin is provided as the sole heme source. Coimmunoprecipitation assays further demonstrate interactions between Fhb1 and S. pombe Str3 or C. neoformans Str3-like proteins when coexpressed in fission yeast cells. In C. neoformans, strains lacking CnStr1 and CnStr3 exhibit a heme-dependent growth defect in the absence of the cell-surface hemophore Cig1. Similar to the C. neoformans fhb1Δ mutant, a cig1Δ Cnstr1Δ Cnstr3Δ triple mutant fails to protect cells from nitrosative stress. Collectively, these findings reveal functional conservation between S. pombe Yhb1 and C. neoformans Fhb1 in their ability to acquire exogenous heme required for activation as defenders against nitrosative stress.

The incidence of invasive fungal infections is rising steadily, driven by the expanding immunocompromised patient population and the limited availability of effective antifungal interventions. Among medically important fungi, airborne Aspergillus conidia are especially relevant to environmental quality control because they can disseminate through air and contaminate healthcare settings. Here, we designed and synthesized a novel ultrafine sodium chloride powder (nanosalt) by anti-solvent precipitation coupled with high-energy nanonization and evaluated its in vitro antifungal activity in a controlled spray co-exposure system using Aspergillus spp. as the primary airborne fungal species. In addition, Candida albicans and Cryptococcus neoformans were also to be tested for the antifungal broad-spectrum by this system even though they do not belong to typical hospital airborne transmission. Nanosalt showed marked fungicidal activity against the tested human fungal pathogens. Further morphological observations in nanosalt-treated A. fumigatus conidia indicated severe cell wall and membrane damage, pore-like structural disruption, leakage-associated loss of integrity accompanied with a time-dependent increase of fungal intracellular reactive oxygen species. Findings in this study mainly identify nanosalt not only might be efficient anti-airborne fungal pathogens but also could be a potential broad-spectrum antifungal reagents. Although currently used aerosol chamber model still has limitation compared actual clinical environment, future practical air-disinfection applications in clinics and related biosafety evaluations are still required. KEY POINTS: • Novel nanosalt particles were generated by anti-solvent precipitation coupled with high-energy nanonization. • Nanosalts exhibit broad fungicidal activity by disrupting fungal cell walls and membranes. • Nanosalt is a candidate for dry environmental antifungal disinfectant.

Meiosis is a hallmark of sexual reproduction. Although the core meiotic machinery is evolutionarily conserved from unicellular yeasts to Metazoan, the key regulators responsible for initiating meiosis vary across species and remain largely unidentified in major eukaryotic lineages, including the major phylum Basidiomycota in the Kingdom Fungi. The basidiomycete fungus Cryptococcus neoformans is a critical pathogen listed by the World Health Organization that causes life-threatening meningoencephalitis worldwide. Meiosis not only indirectly facilitates cryptococcal pathogenicity through generating genetically diverse spores but also directly contributes to host adaptation and disease progression. Here, we identified a novel transcription factor Msd1 that is responsible for activating meiosis in C. neoformans. Deletion of MSD1 impaired multiple sexual development events and strikingly abolished meiosis and subsequent sporogenesis (gametogenesis). Conversely, overexpression of MSD1 alone is sufficient to drive meiosis and the formation of meiotic spores, even in the absence of the external mating-inducing cues or when the mating pathway is genetically inactivated. Mechanistically, we demonstrated that Msd1 initiates meiotic entry by targeting two interconnected pathways. First, Msd1 activates the transcription of multiple evolutionarily conserved core meiosis-specific genes such as meiotic recombinase Dmc1. Second, Msd1 activates two RNA-binding proteins, Csa1 and Csa2, which ensure spatiotemporal expression of the aforementioned meiosis-specific genes to drive meiosis and sporogenesis. Collectively, our findings demonstrate that meiosis and the sequential sexual development events are spatiotemporally orchestrated by the master regulator Msd1, and is the first reported regulator initiating meiotic entry in a basidiomycete fungus.

Domestic pigeons (Columba livia domestica) are potential reservoirs of opportunistic yeasts that may pose health risks to humans, particularly in urban environments. This study aimed to investigate the prevalence, seasonal distribution, and molecular identification of Candida species in pigeon feces across Riyadh, Saudi Arabia. Fecal samples were collected from 64 pigeons across four regions of Riyadh during all four seasons. Yeasts were isolated using selective Sabouraud dextrose agar and CHROMagar Candida, purified, and preserved. Species-level identification was performed via morphological analysis, MALDI-TOF mass spectrometry (MS), and molecular techniques targeting the ITS1-5.8S-ITS2 region. Phylogenetic analysis was conducted on confirmed Candida albicans isolates. Overall, 20.31% (n=13) of the samples were positive for fungal isolation. The predominant species were C. albicans, Pichia kudriavzevii, and Clavispora lusitaniae. Seasonal analysis revealed the highest prevalence in spring (7.81%), while the central region of Riyadh showed the highest regional prevalence (7.81%). MALDI-TOF MS successfully identified C. lusitaniae and P. kudriavzevii, whereas C. albicans was confirmed via PCR and ITS sequencing (GenBank: OR467467 and OR467468). Phylogenetic analysis placed C. albicans isolates in a well-supported monophyletic clade. Environmental factors such as temperature and humidity influenced fungal prevalence. Cryptococcus neoformans was not detected. Domestic pigeons in Riyadh serve as environmental reservoirs of pathogenic Candida species, highlighting potential public health risks, particularly for immunocompromised individuals. These findings emphasize the need for monitoring urban bird populations to mitigate opportunistic fungal transmission.

Cryptococcosis is a severe invasive fungal infection with limited therapeutic options beyond fluconazole-based regimens. Isavuconazole, a broad-spectrum triazole antifungal, has emerged as a potential alternative, although clinical data supporting its use remain scarce. We aimed to evaluate the real-world effectiveness and safety of isavuconazole in patients with different forms of cryptococcosis. A retrospective observational study was conducted at a tertiary-care hospital, including patients with cryptococcosis who received isavuconazole at any treatment phase. Standard microbiological methods were used for pathogen identification and susceptibility testing. Demographic, clinical, and microbiological data were collected. Clinical and microbiological responses and tolerability were assessed at end of treatment or until death. Eight patients with cryptococcosis received isavuconazole, most of whom were immunocompromised. Clinical presentations included pulmonary and disseminated disease, with Cryptococcus neoformans as the predominant species. Isavuconazole was primarily used during the consolidation and maintenance phases, after induction therapy with amphotericin B and flucytosine for 2weeks in most cases, and as salvage therapy in two patients. It was well tolerated during prolonged treatment (6-12months). In the two patients with isavuconazole therapeutic drug monitoring, plasma total trough concentrations were within the therapeutic range (5 and 3.5µg/mL, respectively), whereas cerebrospinal fluid total concentration levels were undetectable. A favorable clinical response was observed in four patients, while three remain on treatment with ongoing clinical improvement; one patient died early. Microbiological clearance was achieved in all culture-positive cases. Isavuconazole demonstrated clinical effectiveness in this cohort of patients across different presentations of cryptococcosis. Treatment was safe and well tolerated, supporting its role as an alternative antifungal option against Cryptococcus, particularly when fluconazole is limited by adverse effects or drug-drug interactions. However, data on central nervous system penetration were limited, and further studies are needed to better define its role in cryptococcal meningitis management.

Chitin deacetylase 3 of Cryptococcus neoformans produces chitosans with low degree and random pattern of acetylation.

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.

Phytochemical investigation of the tuberous roots of Paratinospora sagittata (Oliv.) Wei Wang led to the isolation of two new clerodane diterpenoids, tinosporoside E and tinosporane A (1 and 2), along with eight known compounds (3–10). The structures of the isolated compounds were determined using spectroscopic and spectrometric techniques. The isolates were screened for antimicrobial activity in vitro. Compounds 5, 9 and 10 showed moderate anti-cryptococcal activity against Cryptococcus neoformans with a minimum inhibitory concentration (MIC) of 64 μg/mL, while compounds 1, 3, 4, 7 and 8 exhibited weaker activity (MIC 128 μg/mL).

Systematic infectome\u2013phenome profiling reveals cryptococcal infection-associated proteins driving immune system remodeling and immunization potential

Cryptococcus neoformans is the leading cause of fungal meningitis in immunocompromised individuals. Understanding how this pathogen evades host immune mediated clearance is essential for developing new treatment strategies. Here, we demonstrated that Cdc50, the regulatory subunit of fungal lipid flippase complex, regulates membrane lipid homeostasis that governs extracellular vesicles (EV) biogenesis and macrophage immune responses. Loss of Cdc50 drives global membrane lipid remodeling, hyper-production of phospholipid enriched EVs that enhance macrophage phagocytosis, while the wild-type EV reduce macrophage phagocytosis. Contrary to the prevailing assumption that phosphatidylserine (PS) externalization on the fungal surfaces mimics the mammalian "eat-me signal", we show fungal PS does not engage canonical PS receptor MertK, revealing a fundamental difference between fungal and mammalian PS biology. Furthermore, cdc50 Δ cells are unable to resist phagosomal acidification, rendering them susceptible to macrophage killing. These findings establish how phospholipid homeostasis contributes to early host-pathogen interactions and serves as a compelling antifungal target in cryptococcosis.

The opportunistic pathogen Cryptococcus neoformans adapts to diverse host microenvironments during infection, yet the contribution of epigenetic mechanisms remains unclear. Comparative genomics across 50 basidiomycete species revealed that one chromatin protein, the linker histone H1, is uniquely duplicated across the entire Cryptococcus genus, generating paralogs H1.51 and H1.52 with divergent evolutionary trajectories. Molecular evolution analyses revealed that H1.51 experienced intensified selection with episodic positive selection in clinical isolates, while H1.52 evolved under sustained purifying selection, indicating adaptive neofunctionalization. H1.52 deletion triggered transcriptional reprogramming of 1561 genes (23% of the transcriptome), with downregulation of nucleolar function, ribosome biogenesis, and translation machinery, whereas H1.51 loss produced minimal transcriptional effects. We found that H1.52 governs chromatin architecture. Its deletion expanded H3K4me2 euchromatin coverage 1.54-fold (from 18.4% to 28.4% of the genome) into broader constitutive domains with loss of H3K9-marked heterochromatin islands. Following prolonged culture under host mimicking conditions (3-, 5-, and 7-day periods), H1.52 deletion resulted in enhanced metabolic activity and accelerated growth recovery compared to wild-type. These findings establish that Cryptococcus neoformans H1.52 coordinates chromatin compaction, maintains H3K9-marked heterochromatin islands and is a regulator of metabolic responses to host stress.

Cutaneous cryptococcosis, although rare, may arise as primary inoculation or as part of disseminated cryptococcal infection, particularly in immunocompromised hosts. We present the case of an 80-year-old man with type 2 diabetes mellitus receiving corticosteroid therapy for cryptogenic organizing pneumonia who developed cryptococcal cellulitis with a markedly elevated serum cryptococcal antigen (CrAg) titer but no discernible central nervous system (CNS) involvement. As high serum CrAg titers are strongly associated with CNS disease, we compared the patient isolate (designated MA-1) with the Cryptococcus neoformans reference strain KN99, using in vitro growth curves, a murine pulmonary infection model, and whole-genome sequencing. MA-1 grew more slowly than KN99 in tissue culture medium. Mice infected with MA-1 survived longer than those challenged with KN99, although brain dissemination of MA-1 still occurred by day 14, indicating that the strain was neurotropic. Genomic analysis revealed overall similarity to the C neoformans H99 strain but with distinctive structural features, including duplication of chromosome 12, a large deletion on chromosome 5, and multiple single-nucleotide polymorphisms. These alterations may have contributed to altered dissemination dynamics. This case underscores the importance of considering cryptococcosis in refractory cellulitis in immunocompromised patients. High CrAg titers, even without CNS findings, should prompt lumbar puncture and consideration of induction therapy per current guidelines. This integrated clinical and laboratory investigation suggests that pathogen-specific factors, as shown in vitro and in a murine model, may influence the tempo of dissemination in human disease.

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.

The Cryptococcus neoformans species complex (CNSC), responsible for cryptococcosis, is controlled by Th1-type immunity, in which IFN-γ-activated macrophages form granulomas that contain infection. Nevertheless, CNSC can evade host immunity and, after the primary infection phase, can shift to a latent infection similar to tuberculosis. Reactivation is thought to occur when immune control fails, but the underlying mechanisms remain poorly understood. FTY720, a functional antagonist of sphingosine-1-phosphate receptors, is primarily used in the treatment of multiple sclerosis. However, there have been reports linking its use to instances of cryptococcosis in patients undergoing treatment. To explore this, we established a novel mouse model using CnT-II mice, which express CD4+ T cell receptors specific for chitin deacetylase 2 (Cda2), a major T cell antigen of CNSC. Following infection with encapsulated CNSC, mice developed persistent pulmonary fungal burdens (10²-10³ CFU) accompanied by granulomatous responses, modeling latent infection. Upon FTY720 administration, mice exhibited increased pulmonary fungal burdens, disrupted granuloma integrity, and reduced IFN-γ and IL-12 production. FTY720 also markedly decreased CD4+ effector memory (Tem) and effector T cells (Teff) in the lungs, with a particularly profound loss of IFN-γ-producing Tem cells. These findings indicate that our model successfully recapitulates latent cryptococcal infection and reactivation, and demonstrate that FTY720 promotes relapse by depleting protective IFN-γ-producing CD4+ Tem cells and impairing Th1-mediated immunity. Given the rising use of FTY720, our study highlights its potential risk in patients with subclinical cryptococcosis and underscores the need for preventive strategies to avert disease progression.

Comparison of cationic beta-glucans with antifungal properties - Correlation between chemical structure and biological activity.

Primary cutaneous cryptococcosis (PCC) is a rare, localized fungal infection caused by Cryptococcus species, typically following direct inoculation of the yeast into the skin in immunocompetent individuals. Unlike disseminated cryptococcosis, which is more common in immunocompromised hosts, PCC remains confined to the skin and often arises after minor trauma or environmental exposure. Although traditionally associated with Cryptococcus neoformans (C. neoformans), Cryptococcus gattii (C. gattii) is also increasingly recognized in immunocompetent hosts. Dermatologists are crucial in the early identification of PCC, as its clinical presentation can resemble other dermatologic conditions such as bacterial cellulitis, pyoderma gangrenosum, or atypical mycobacterial infections. This review provides a comprehensive overview of the epidemiology, clinical features, diagnostic methods, and management strategies for PCC. It emphasizes the importance of differentiating PCC from disseminated cryptococcosis and other mimicking conditions. Treatment with oral azoles, such as fluconazole, is typically effective, with a favorable prognosis for most immunocompetent patients. The review also includes practical diagnostic algorithms to assist clinicians in the accurate and timely management of PCC, ultimately improving patient outcomes and reducing unnecessary interventions.

Cryptococcus neoformansis a global fungal pathogen that causes fatal cryptococcosis, underscoring the urgent need for novel therapeutics given the limitations of current antifungals. Here, we systematically investigate essential transcription factors (TFs) inC. neoformans, focusing on their roles in growth and their potential as drug targets. We developed experimental pipelines to assess growth requirement, essentiality, and function using conditional gene expression, constitutive overexpression, and meiotic spore analysis. Through these systematic analyses, we identify one quasi-essential (growth-required but non-essential) TF, Fhl1, and 13 essential TFs, including three (Ezt1, Ezt2 and Cbf1) that are highly divergent from counterparts in other eukaryotes. Notably, Ezt1 emerged as a central TF regulating over 1,200 genes, controlling growth, antifungal drug and stress responses, sexual development, and virulence. Collectively, our findings define the essential TF landscape ofC. neoformansand provide a framework for leveraging these regulators, particularly Ezt1, to develop targeted therapies for cryptococcosis.