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Sporothrix brasiliensis is the main agent of sporotrichosis in Brazil, with few therapeutic options. This study aimed to investigate the in vitro efficacy of photodynamic therapy using a diode laser (InGaAIP) in combination with the photosensitizer methylene blue against S. brasiliensis yeasts. Additionally, we evaluated the underexplored mitochondrial activity of S. brasiliensis and the impact of laser treatment on the fungal mitochondrial aspects post-treatment. Three strains of S. brasiliensis were used, including a non-wild-type strain to itraconazole. Yeast viability was determined by counting colony-forming units. For a comprehensive analysis of irradiated versus non-irradiated cells, we assessed combined therapy with itraconazole, scanning electron microscopy of cells, and mitochondrial activity. The latter included high-resolution respirometry, membrane potential analysis, and reactive oxygen species production. Methylene blue combined with photodynamic therapy inhibited the growth of the isolates, including the non-wild-type strain to itraconazole. Photodynamic therapy induced the production of reactive oxygen species, which negatively affected mitochondrial function, resulting in decreased membrane potential and cell death. Photodynamic therapy altered the ultrastructure and mitochondrial physiology of S. brasiliensis, suggesting a new therapeutic approach for sporotrichosis caused by this species., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Understanding Cryptococcus neoformans pathogenesis requires a detailed analysis of the various virulence factors that contribute to its ability to cause disease. Cyclosporine, calcineurin inhibitor, impairs C. neoformans production of a polysaccharide capsule and secretion of urease, which are critical for cryptococcal pathogenesis. Two particularly important virulence factors are the production of cell wall melanin and formation of biofilm. In this study, we investigated cyclosporine's effects on melanin production and biofilm formation in C. neoformans. Initially, we examined melanin production in planktonic cells treated with cyclosporine using an L-DOPA containing melanin-inducing medium. Visual inspection and optical microscopy revealed a notable reduction in the characteristic dark coloration of cultures treated with cyclosporine, which indicate decreased melanin production in daughter cells compared to mother cells. Spectrophotometric analysis also demonstrated a significantly altered ultraviolet-visible (UV/vis) absorption spectra in cyclosporine-treated yeast cells, indicative of structural changes in melanin. Additionally, cyclosporine-treated cells exhibited reduced conductance (P-value < 0.0001), suggesting altered cellular ionic properties. The impact of cyclosporine on biofilm formation and mature biofilm disruption was also assessed. Despite cyclosporine's efficacy in modifying virulence factors during planktonic growth, cyclosporine did not inhibit biofilm formation or melanization under biofilm growth conditions, nor did it disrupt mature biofilms in terms of biomass or metabolic activity. However, there was a significant reduction in extracellular matrix production in cyclosporine-treated non-melanized biofilms. Our findings underscore the complex interplay between cyclosporine and C. neoformans, highlighting its differential effects on melanization and biofilm dynamics, which provides new insights into the shortcomings of cyclosporin for combatting cryptococcosis and informs pathways for future therapeutic strategies against cryptococcosis., Competing Interests: Declaration of Competing Interest ☒ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Rodrigo Almeida-Paes reports financial support was provided by National Council for Scientific and Technological Development. Rodrigo Almeida-Paes and Susana Frases reports financial support was provided by Carlos Chagas Filho Foundation for Research Support of Rio de Janeiro State. Iara Bastos de Andrade reports financial support was provided by Coordination of Higher Education Personnel Improvement. Joshua Nosanchuk reports financial support was provided by National Institutes of Health. Joshua Nosanchuk reports financial support was provided by US Department of Defense. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Auranofin is an approved anti-rheumatic drug that has a broad-range inhibitory action against several microorganisms, including human pathogenic fungi. The auranofin activity against Histoplasma capsulatum, the dimorphic fungus that causes histoplasmosis, has not been properly addressed. Since there are few therapeutic options for this life-threatening systemic mycosis, this study evaluated the effects of auranofin on H. capsulatum growth and expression of virulence factors., Methodology/principal Findings: Minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) of auranofin against 15 H. capsulatum strains with distinct genetic backgrounds were determined using the yeast form of the fungus and a microdilution protocol. Auranofin activity was also assessed on a macrophage model of infection and on a Tenebrio molitor invertebrate animal model. Expression of virulence-related genes was compared between auranofin treated and untreated H. capsulatum yeast cells using a quantitative PCR assay. Auranofin affected the growth of different strains of H. capsulatum, with MIC and MFC values ranging from 1.25 to 5.0 μM and from 2.5 to >10 μM, respectively. Auranofin was able to kill intracellular H. capsulatum yeast cells and conferred protection against the fungus in the experimental animal model of infection. Moreover, the expression of catalase A, HSP70, superoxide dismutase, thioredoxin reductase, serine proteinase, cytochrome C peroxidase, histone 2B, formamidase, metallopeptidase, Y20 and YPS3 proteins were reduced after six hours of auranofin treatment. CONCLUSIONS/SIGNIFICANCE: Auranofin is fungicidal against H. capsulatum and reduces the expression of several virulence-related genes, which makes this anti-rheumatic drug a good candidate for new medicines against histoplasmosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 de Abreu Almeida et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Systemic candidiasis remains a significant public health concern worldwide, with high mortality rates despite available antifungal drugs. Drug-resistant strains add to the urgency for alternative therapies. In this context, vaccination has reemerged as a prominent immune-based strategy. Extracellular vesicles (EVs), nanosized lipid bilayer particles, carry a diverse array of native fungal antigens, including proteins, nucleic acids, lipids, and glycans. Previous studies from our laboratory demonstrated that Candida albicans EVs triggered the innate immune response, activating bone marrow-derived dendritic cells (BMDCs) and potentially acting as a bridge between innate and adaptive immunity. Vaccination with C. albicans EVs induced the production of specific antibodies, modulated cytokine production, and provided protection in immunosuppressed mice infected with lethal C. albicans inoculum. To elucidate the mechanisms underlying EV-induced immune activation, our study investigated pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) involved in EVs-phagocyte engagement. EVs from wild-type and mutant C. albicans strains with truncated mannoproteins were compared for their ability to stimulate BMDCs. Our findings revealed that EV decoration with O - and N -linked mannans and the presence of β -1,3-glucans and chitin oligomers may modulate the activation of specific PRRs, in particular Toll-like receptor 4 (TLR4) and dectin-1. The protective effect of vaccination with wild-type EVs was found to be dependent on TLR4. These results suggest that fungal EVs can be harnessed in vaccine formulations to selectively activate PRRs in phagocytes, offering potential avenues for combating or preventing candidiasis.IMPORTANCESystemic candidiasis is a serious global health concern with high mortality rates and growing drug resistance. Vaccination offers a promising solution. A unique approach involves using tiny lipid-coated particles called extracellular vesicles (EVs), which carry various fungal components. Previous studies found that Candida albicans EVs activate the immune response and may bridge the gap between innate and adaptive immunity. To understand this better, we investigated how these EVs activate immune cells. We demonstrated that specific components on EV surfaces, such as mannans and glucans, interact with receptors on immune cells, including Toll-like receptor 4 (TLR4) and dectin-1. Moreover, vaccinating with these EVs led to strong immune responses and full protection in mice infected with Candida . This work shows how harnessing fungal EVs might lead to effective vaccines against candidiasis., Competing Interests: The authors declare no conflict of interest.

The incidence of invasive fungal infections caused by Candida species is increasing, particularly in immunocompromised individuals. This increasing incidence poses a dual challenge, comprising escalating antifungal resistance and the necessity for accurate fungal identification. The Candida haemulonii complex further complicates these challenges due to limited identification tools. Like some other Candida species, infections involving this complex show resistance to multiple antifungals, requiring innovative therapeutic approaches. Rapamycin, known for its antifungal properties and immunosuppressive characteristics, was investigated against the C. haemulonii complex species. Results revealed a rapamycin minimal inhibitory concentration (MIC) range of 0.07 to >20 µM, with fungicidal effects in most strains. In vitro analyses using the rapamycin maximum plasma concentration (0.016 µM) showed reduced surface properties and decreased production of extracellular enzymes. Rapamycin also hindered biofilm formation by some strains. Even when treated at the human therapeutic dose, which is lower than the MIC, phenotypic variations in C. haemulonii were detected, hinting at the possible attenuation of some virulence factors when exposed to rapamycin., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Susana Frases reports financial support and equipment, drugs, or supplies were provided by National Council for Scientific and Technological Development. Susana Frases reports financial support and equipment, drugs, or supplies were provided by Carlos Chagas Filho Foundation for Research Support of Rio de Janeiro State. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The drugs available to treat sporotrichosis, an important yet neglected fungal infection, are limited. Some Sporothrix spp. strains present reduced susceptibility to these antifungals. Furthermore, some patients may not be indicated to use these drugs, while others may not respond to the therapy. The anthelmintic drug niclosamide is fungicidal against the Sporothrix brasiliensis type strain. This study aimed to evaluate whether niclosamide also has antifungal activity against Sporothrix globosa, Sporothrix schenckii and other S. brasiliensis strains with distinct genotypes and antifungal susceptibility status. Minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) were determined using the microdilution method according to the CLSI protocol. The checkerboard method was employed to evaluate niclosamide synergism with drugs used in sporotrichosis treatment. Metabolic activity of the strains under niclosamide treatment was evaluated using the resazurin dye. Niclosamide was active against all S. brasiliensis strains (n = 17), but it was ineffective (MIC > 20 µM) for some strains (n = 4) of other pathogenic Sporothrix species. Niclosamide MIC values for Sporothrix spp. were similar for mycelial and yeast-like forms of the strains (P = 0.6604). Niclosamide was fungicidal (MFC/MIC ratio ≤ 2) for most strains studied (89%). Niclosamide activity against S. brasiliensis is independent of the fungal genotype or non-wild-type phenotypes for amphotericin B, itraconazole, or terbinafine. These antifungal drugs presented indifferent interactions with niclosamide. Niclosamide has demonstrated potential for repurposing as a treatment for sporotrichosis, particularly in S. brasiliensis cases, instigating in vivo studies to validate the in vitro findings., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Mucormycosis is a severe fungal infection that demands immediate and decisive intervention upon suspicion. The causative agents of mucormycosis exhibit inherent resistance to echinocandins and voriconazole, and their in vitro susceptibility to terbinafine is highly variable and species-specific. Considering these factors and the limitations of currently available antifungal therapies, the identification of novel antifungals with potent activity against mucormycosis is of paramount importance. This study aims to identify compounds from the MMV Pathogen Box® presenting antifungal activity against selected mucormycosis agents and to evaluate their potential synergistic effects when combined with antifungal drugs. A screening of the Pathogen Box® compounds was conducted, isolated or in combination with sub-inhibitory concentrations of amphotericin B, isavuconazole or posaconazole, against a Rhizopus oryzae strain. Hits from the screenings were further evaluated against eight Mucoralean strains for minimal inhibitory and fungicidal concentration determinations and to confirm synergistic interactions using the checkerboard method. Ultrastructural studies were performed using scanning electron microscopy. MMV675968 exhibited fungicidal activity against a R. oryzae strain. All but one Rhizopus spp. strains presented MIC ≤ 1 μg/mL, with a geometric mean of 0.78 μg/mL observed across all isolates for this compound, which did not change significantly the cellular structure of this fungus. The combination screening with antifungal drugs revealed six additional compounds potentially active against the R. oryzae strain, two of them demonstrated proven synergism through the checkerboard assay. This first study with the MMV Pathogen Box® and Zigomycetes highlights promising new treatment options for mucormycosis in the future., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier B.V.)

In recent decades, the poultry farming industry has assumed a pivotal role in meeting the global demand for affordable animal proteins. While poultry farming makes a substantial contribution to food security and nutrition, it also presents environmental and public health challenges. The use of poultry litter as fertilizer for agricultural soils raises concerns about the transfer of pathogens and drug-resistant microorganisms from poultry farms to crop production areas. On the other hand, according to the Food and Agriculture Organization of the United Nations (FAO), fungicides represent the second most used chemical group in agricultural practices. In this context, agricultural soils receive the application of both poultry litter as a fertilizer and fungicides used in agricultural production. This practice can result in fungal contamination of the soil and the development of antifungal resistance. This article explores the necessity of monitoring antifungal resistance, particularly in food production areas with co-application of poultry litter and fungicides. It also highlights the role of fungi in ecosystems, decomposition, and mutualistic plant associations. We call for interdisciplinary research to comprehensively understand fungal resistance to fungicides in the environment. This approach seeks to promote sustainability in the realms of human health, agriculture, and the environment, aligning seamlessly with the One Health concept.

This chapter describes methodological details for preparing specimens of Cryptococcus neoformans (although it can be applied to any species of the genus) and their subsequent analysis by scanning and transmission electron microscopy. Adaptations to conventional protocols for better preservation of the sample, as well as to avoid artifacts, are presented. The protocols may be used to examine both the surface ultrastructure and the interior of this pathogenic fungus in detail., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Metal encapsulation delivers a straightforward strategy to improve miscellaneous nanoparticle properties and qualifies the resulting nanocomposite for exceptional application, including bioimaging, drug release, and theranostic development. Besides crucial applications, investigations associated with the nanocomposite impact on the biological media are highly relevant from a pharmacological viewpoint. Such studies can be conducted by exploring nanocomposite attributes and all aspects of their interaction with proteins existing in biofluids. Based on these aspects, the present work examines manganese-encapsulated carbonaceous nanocomposite (MnCQD) and their interaction with plasma proteins. On one side, the obtained nanocomposite has almost spherical shapes ( ≈ 12 nm in size), an appropriate composition and interesting optical properties for bioimaging applications. On another side, MnCQD quenches the fluorescence of two plasma proteins (BSA and HTF) following a static mechanism, confirming the formation of the MnCQD-BSA and MnCQD-HTF complexes. Although hydrophobic forces guide the stability of both formed complexes, MnCQD binds preferentially to BSA compared to HTF, with affinity constants differing by almost an order of magnitude. Furthermore, HTF and BSA underwent modifications in their secondary structure provoked due to contact with the nanocomposite, which also presented neglectable opsonization levels when exposed to appropriate biological media. These results highlight the MnCQD outstanding potential to be employed in diverse bioapplications.Communicated by Ramaswamy H. Sarma.

Fungal infections are a global public health challenge, especially among immunocompromised patients. Basidiomycetous yeasts, such as Rhodotorula mucilaginosa , have emerged as opportunistic pathogens, but have received less attention than Cryptococcus neoformans . This study aimed to characterize the polysaccharides of R. mucilaginosa and compare them with those of C. neoformans , analyzing their clinical implications. Comprehensive physicochemical, mechanical, and ultrastructural analyses of polysaccharides from both species were performed, revealing correlations with virulence and pathogenicity. R. mucilaginosa cells are surrounded by a capsule smaller than that produced by C. neoformans , but with similar polysaccharides. Those polysaccharides are also secreted by R. mucilaginosa . Cross-reactivity with R. mucilaginosa was observed in a diagnostic C. neoformans antigen test, using both in vitro and in vivo samples, highlighting the need for more reliable tests. Some R. mucilaginosa strains exhibited virulence comparable to that of C. neoformans in an invertebrate experimental model ( Tenebrio molitor ). This study contributes to a deeper understanding of yeast pathogenicity and virulence, highlighting the need for more accurate diagnostic tests to improve the differential diagnosis of infections caused by basidiomycetous yeasts.

Background: Sporothrix brasiliensis causes sporotrichosis, an important infection in some groups of patients. Aims: This work was designed to investigate the effects of isavuconazole against this species. Methods: An antifungal susceptibility test was performed to compare MIC values with other antifungal drugs used to treat sporotrichosis. A checkerboard assay was performed to understand isavuconazole interactions. Furthermore, isavuconazole growth inhibition on an itraconazole-resistant strain was tested. Results: Isavuconazole had similar MICs to other azoles against S. brasiliensis , presenting fungistatic activity. Isavuconazole did not interact in vitro with antifungals or immunosuppressive drugs and inhibited the growth of an itraconazole-resistant strain. Conclusion: Isavuconazole inhibits S. brasiliensis , its pharmacologic characteristics make it a candidate for patients with sporotrichosis and it may be useful to combat sporotrichosis caused by resistant isolates.

Background: Cryptococcus neoformans is an opportunistic fungal pathogen that causes infections mainly in immunosuppressed individuals, such as transplant recipients. Aims: This study investigated the effects of rapamycin, an immunosuppressant drug, on the cellular organization, biophysical characteristics, and main virulence factors of C. neoformans . Methods: Morphological, structural, physicochemical and biophysical analyses of cells and secreted polysaccharides of the reference H99 C. neoformans strain were investigated under the effect of subinhibitory concentrations of rapamycin. Results: Rapamycin at a minimum inhibitory concentration of 2.5 μM reduced C. neoformans cell viability by 53%, decreased capsule, increased cell size, chitin and lipid body formation, and changed peptidase and urease activity. Conclusion: Further studies are needed to assess how rapamycin affects the virulence factors and pathogenicity of C. neoformans.

Histoplasma capsulatum is the causative agent of histoplasmosis. Treating this fungal infection conventionally has significant limitations, prompting the search for alternative therapies. In this context, fungal extracellular vesicles (EVs) hold relevant potential as both therapeutic agents and targets for the treatment of fungal infections. To explore this further, we conducted a study using pharmacological inhibitors of chitinase (methylxanthines) to investigate their potential to reduce EV release and its subsequent impact on fungal virulence in an in vivo invertebrate model. Our findings revealed that a subinhibitory concentration of the methylxanthine, caffeine, effectively reduces EV release, leading to a modulation of H. capsulatum virulence. To the best of our knowledge, this is the first reported instance of a pharmacological inhibitor that reduces fungal EV release without any observed fungicidal effects.

Cryptococcosis is a systemic mycosis affecting immunosuppressed individuals, caused by various Cryptococcus species. The current treatment utilizes a combination of antifungal drugs, but issues such as nephrotoxicity, restricted or limited availability in certain countries, and resistance limit their effectiveness. Repurposing approved drugs presents a viable strategy for developing new antifungal options. This study investigates the potential of glatiramer acetate (Copaxone ® ) as a chemotherapy candidate for Cryptococcus neoformans infection. Various techniques are employed to evaluate the effects of glatiramer acetate on the fungus, including microdilution, XTT analysis, electron and light microscopy, and physicochemical measurements. The results demonstrate that glatiramer acetate exhibits antifungal properties, with an IC 50 of 0.470 mg/mL and a minimum inhibitory concentration (MIC) of 2.5 mg/mL. Furthermore, it promotes enhanced cell aggregation, facilitates biofilm formation, and increases the secretion of fungal polysaccharides. These findings indicate that glatiramer acetate not only shows an antifungal effect but also modulates the key virulence factor-the polysaccharide capsule. In summary, repurposing glatiramer acetate as a potential chemotherapy option offers new prospects for combating C. neoformans infection. It addresses the limitations associated with current antifungal therapies by providing an alternative treatment approach.

Sporothrix brasiliensis is the main agent of zoonotic sporotrichosis transmitted by domestic cats in South America. In humans, sporotrichosis commonly presents with cutaneous or lymphocutaneous lesions, and in cats, with multiple ulcerated skin lesions associated with enlarged lymph nodes and respiratory signs. Fungal virulence factors may affect the clinical presentation of the mycoses. Sporothrix spp. present some virulence factors. This study aims to compare 24 S. brasiliensis strains from 12 familiar outbreaks of cat-to-human transmitted sporotrichosis. Fungal growth in different substrates, thermotolerance, resistance to oxidative stress, and production of enzymes were evaluated. An invertebrate model of experimental infection was used to compare the virulence of the strains. The strains grew well on glucose and N -acetyl- D -glucosamine but poorly on lactate. Their thermotolerance was moderate to high. All strains were susceptible to hydrogen peroxide, and the majority produced hemolysins but not phospholipase and esterase. There was no significant difference in the putative virulence-associated factors studied among the different hosts. Moreover, strains isolated from a human and a cat from four familiar outbreaks presented a very similar profile of expression of these factors, reinforcing the zoonotic transmission of S. brasiliensis in Brazil and demonstrating the plasticity of this species in the production of virulence factors.

Sporotrichosis is a fungal infection caused by Sporothrix species, with Sporothrix brasiliensis as a prevalent pathogen in Latin America. Despite its clinical importance, the virulence factors of S. brasiliensis and their impact on the pathogenesis of sporotrichosis are still poorly understood. This study evaluated the morphostructural plasticity of S. brasiliensis , a fungus that causes sporotrichosis. Three cell surface characteristics, namely cell surface hydrophobicity, Zeta potential, and conductance, were assessed. Biofilm formation was also analyzed, with measurements taken for biomass, extracellular matrix, and metabolic activity. In addition, other potential and poorly studied characteristics correlated with virulence such as lipid bodies, chitin, and cell size were evaluated. The results revealed that the major phenotsypic features associated with fungal virulence in the studied S. brasiliensis strains were chitin, lipid bodies, and conductance. The dendrogram clustered the strains based on their overall similarity in the production of these factors. Correlation analyses showed that hydrophobicity was strongly linked to the production of biomass and extracellular matrix, while there was a weaker association between Zeta potential and size, and lipid bodies and chitin. This study provides valuable insights into the virulence factors of S. brasiliensis and their potential role in the pathogenesis of sporotrichosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Nannizzia gypsea is a geophylic agent of human and animal dermatophytosis. This study compares the metabolic and morphostructural plasticity of N. gypsea strains isolated from moss, sand, and a dog. The in vitro metabolic plasticity included the detection of extracellular enzymes, thermotolerance, resistance to oxidative stress, and assessment of fungal growth. Structural plasticity studies included cell surface hydrophobicity, electronegativity, and size of macroconidia. Virulence was assessed on Tenebrio mollitor model. The strains showed low thermotolerance, susceptibility to oxidative stress, and were producers of keratinase, lipase and catalase. N. gypsea strains were unable to produce hemolysin, esterase, and phospholipase although they were able to grow with different carbon sources. The electronegative properties of the surface did not vary between the strains under study. The knowledge about N. gypsea metabolic and morphostructural plasticity could be crucial for the development of therapeutic strategies and control of dermatophytosis., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Background: The Trichosporonaceae family comprises a large number of basidiomycetes widely distributed in nature. Some of its members, especially Trichosporon asahii, have the ability to cause human infections. This ability is related to a series of virulence factors, which include lytic enzymes production, biofilm formation, resistance to oxidising agents, melanin and glucuronoxylomannan in the cell wall, metabolic plasticity and phenotypic switching. The last two are poorly addressed within human pathogenic Trichosporonaceae., Objective: These factors were herein studied to contribute with the knowledge of these emerging pathogens and to uncover mechanisms that would explain the higher frequency of T. asahii in human infections., Methods: We included 79 clinical isolates phenotypically identified as Trichosporon spp. and performed their molecular identification. Lactate and N-acetyl glucosamine were the carbon sources of metabolic plasticity studies. Morphologically altered colonies after subcultures and incubation at 37°C indicated phenotypic switching., Results and Conclusion: The predominant species was T. asahii (n = 65), followed by Trichosporon inkin (n = 4), Apiotrichum montevideense (n = 3), Trichosporon japonicum (n = 2), Trichosporon faecale (n = 2), Cutaneotrichosporon debeurmannianum (n = 1), Trichosporon ovoides (n = 1) and Cutaneotrichosporon arboriforme (n = 1). T. asahii isolates had statistically higher growth on lactate and N-acetylglucosamine and on glucose during the first 72 h of culture. T. asahii, T. inkin and T. japonicum isolates were able to perform phenotypic switching. These results expand the virulence knowledge of Trichosporonaceae members and point for a role for metabolic plasticity and phenotypic switching on the trichosporonosis pathogenesis., (© 2022 Wiley-VCH GmbH.)

Cryptococcus neoformans ( Cn ) is an opportunistic, encapsulated, yeast-like fungus that causes severe meningoencephalitis, especially in countries with high HIV prevalence. In addition to its well-known polysaccharide capsule, Cn has other virulence factors such as phospholipases, a heterogeneous group of enzymes that hydrolyze ester linkages in glycerophospholipids. Phospholipase B (PLB1) has been demonstrated to play a key role in Cn pathogenicity. In this study, we used a PLB1 mutant ( plb1 ) and its reconstituted strain (Rec1) to assess the importance of this enzyme on Cn brain infection in vivo and in vitro . Mice infected with the plb1 strain survive significantly longer, have lower peripheral and central nervous system (CNS) fungal loads, and have fewer and smaller cryptococcomas or biofilm-like brain lesions compared to H99- and Rec1-infected animals. PLB1 causes extensive brain tissue damage and changes microglia morphology during cryptococcal disease, observations which can have important implications in patients with altered mental status or dementia as these manifestations are related to poorer survival outcomes. plb1 cryptococci are significantly more phagocytosed and killed by NR-9460 microglia-like cells. plb1 cells have altered capsular polysaccharide biophysical properties which impair their ability to stimulate glial cell responses or morphological changes. Here, we provide significant evidence demonstrating that Cn PLB1 is an important virulence factor for fungal colonization of and survival in the CNS as well as in the progression of cryptococcal meningoencephalitis. These findings may potentially help fill in a gap of knowledge in our understanding of cerebral cryptococcosis and provide novel research avenues in Cn pathogenesis. IMPORTANCE Cryptococcal meningoencephalitis (CME) is a serious disease caused by infection by the neurotropic fungal pathogen Cryptococcus neoformans. Due to the increasing number of cases in HIV-infected individuals, as well as the limited therapies available, investigation into potential targets for new therapeutics has become critical. Phospholipase B is an enzyme synthesized by Cn that confers virulence to the fungus through capsular enlargement, immunomodulation, and intracellular replication. In this study, we examined the properties of PLB1 by comparing infection of a Cn PLB1 mutant strain with both the wild-type and a PLB1-reconstituted strain. We show that PLB1 augments the survival and proliferation of the fungus in the CNS and strengthens virulence by modulating the immune response and enhancing specific biophysical properties of the fungus. PLB1 expression causes brain tissue damage and impacts glial cell functions, which may be responsible for the dementia observed in patients which may persist even after resolving from CME. The implications of PLB1 inhibition reveal its involvement in Cn infection and suggest that it may be a possible molecular target in the development of antifungal therapies. The results of this study support additional investigation into the mechanism of PLB1 to further understand the intricacies of cerebral Cn infection.

This study aimed to investigate the effects of cyclosporine on the morphology, cell wall structure, and secretion characteristics of Cryptococcus neoformans . The minimum inhibitory concentration (MIC) of cyclosporine was found to be 2 µM (2.4 µg/mL) for the H99 strain. Yeast cells treated with cyclosporine at half the MIC showed altered morphology, including irregular shapes and elongated projections, without an effect on cell metabolism. Cyclosporine treatment resulted in an 18-fold increase in chitin and an 8-fold increase in lipid bodies, demonstrating changes in the fungal cell wall structure. Cyclosporine also reduced cell body and polysaccharide capsule diameters, with a significant reduction in urease secretion in C. neoformans cultures. Additionally, the study showed that cyclosporine increased the viscosity of secreted polysaccharides and reduced the electronegativity and conductance of cells. The findings suggest that cyclosporine has significant effects on C. neoformans morphology, cell wall structure, and secretion, which could have implications for the development of new antifungal agents.

Histoplasmosis is a frequent mycosis in people living with HIV/AIDS and other immunocompromised hosts. Histoplasmosis has high rates of mortality in these patients if treatment is unsuccessful. Itraconazole and amphotericin B are used to treat histoplasmosis; however, both antifungals have potentially severe pharmacokinetic drug interactions and toxicity. The present study determined the minimal inhibitory and fungicidal concentrations of mebendazole, a drug present in the NIH Clinical Collection, to establish whether it has fungicidal or fungistatic activity against Histoplasma capsulatum . Protein extracts from H. capsulatum yeasts, treated or not with mebendazole, were analyzed by proteomics to understand the metabolic changes driven by this benzimidazole. Mebendazole inhibited the growth of 10 H. capsulatum strains, presenting minimal inhibitory concentrations ranging from 5.0 to 0.08 µM. Proteomics revealed 30 and 18 proteins exclusively detected in untreated and mebendazole-treated H. capsulatum yeast cells, respectively. Proteins related to the tricarboxylic acid cycle, cytoskeleton, and ribosomes were highly abundant in untreated cells. Proteins related to the nitrogen, sulfur, and pyrimidine metabolisms were enriched in mebendazole-treated cells. Furthermore, mebendazole was able to inhibit the oxidative metabolism, disrupt the cytoskeleton, and decrease ribosomal proteins in H. capsulatum . These results suggest mebendazole as a drug to be repurposed for histoplasmosis treatment., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Cryptococcosis is an opportunistic mycosis that mainly affects immunosuppressed patients. The treatment is a combination of three antifungal agents: amphotericin B, 5-flucytosine and fluconazole. However, these drugs have many disadvantages, such as high nephrotoxicity, marketing bans in some countries and fungal resistance. One of the solutions to find possible new drugs is pharmacological repositioning. This work presents repositioned drugs as an alternative for new antifungal therapies for cryptococcosis. All the studies here were performed in vitro or in animal models, except for sertraline, which reached phase III in humans. There is still no pharmacological repositioning approval for cryptococcosis in humans, though this review shows the potential of repurposing as a rapid approach to finding new agents to treat cryptococcosis.

The phenotypic plasticity of Cryptococcus neoformans is widely studied and demonstrated in vitro, but its influence on pathogenicity remains unclear. In this study, we investigated the dynamics of cryptococcal cell and transcriptional remodeling during pulmonary infection in a murine model. We showed that in Cryptococcus neoformans, cell size reduction (cell body ≤ 3 µm) is important for initial adaptation during infection. This change was associated with reproductive fitness and tissue invasion. Subsequently, the fungus develops mechanisms aimed at resistance to the host’s immune response, which is determinant for virulence. We investigated the transcriptional changes involved in this cellular remodeling and found an upregulation of transcripts related to ribosome biogenesis at the beginning (6 h) of infection and a later (10 days) upregulation of transcripts involved in the inositol pathway, energy production, and the proteasome. Consistent with a role for the proteasome, we found that its inhibition delayed cell remodeling during infection with the H99 strain. Altogether, these results further our understanding of the infection biology of C. neoformans and provide perspectives to support therapeutic and diagnostic targets for cryptococcosis.

During the geological eras, some fungi, through adaptation and/or environmental/ecological pressure, interacted directly and indirectly with humans, through occasionally harmful interaction interdependent on the individual's immunological condition. Infections caused by yeasts are underreported, subjugated, and underdiagnosed, and treatment is restricted to a few drugs, even after the significant progress of medicine and pharmacology. In the last centuries, antagonistically, there has been an exponential increase of immunocompromised individuals due to the use of immunosuppressive drugs such as corticosteroids, increased cases of transplants, chemotherapeutics, autoimmune diseases, neoplasms, and, more recently, coronavirus disease 2019 (COVID-19). This review aims to survey emerging and re-emerging yeast infections in the current clinical context. Currently, there is an immense clinical challenge for the rapid and correct diagnosis and treatment of systemic mycoses caused by yeasts due to the terrible increase in cases in the current context of COVID-19.

Aspergillus fumigatus causes invasive aspergillosis (IA) in immunocompromised patients, resulting in high mortality rates. Currently, no vaccine formulations to promote immune protection in at-risk individuals have been developed. In this work, we deleted the sterylglucosidase-encoding gene, sglA , in Aspergillus fumigatus and investigated its role in fungal virulence and host vaccine protection. The Δ sglA mutant accumulated sterylglucosides (SGs), newly studied immunomodulatory glycolipids, and exhibited reduced hyphal growth and altered compositions of cell wall polysaccharides. Interestingly, the Δ sglA mutant was avirulent in two murine models of IA and was fully eliminated from the lungs. Both corticosteroid-induced immunosuppressed and cyclophosphamide-induced leukopenic mice vaccinated with live or heat-killed Δ sglA conidia were fully protected against a lethal wild-type A. fumigatus challenge. These results highlight the potential of SG-accumulating strains as safe and promising vaccine formulations against invasive fungal infections. IMPORTANCE Infections by Aspergillus fumigatus occur by the inhalation of environmental fungal spores called conidia. We found that live mutant conidia accumulating glycolipids named sterylglucosides are not able to cause disease when injected into the lung. Interestingly, these animals are now protected against a secondary challenge with live wild-type conidia. Remarkably, protection against a secondary challenge persists even with vaccination with heat-killed mutant conidia. These results will significantly advance the field of the research and development of a safe fungal vaccine for protection against the environmental fungus A. fumigatus.

The overproduction of reactive oxygen species (ROS) induces oxidative stress, a well-known process associated with aging and several human pathologies, such as cancer and neurodegenerative diseases. A large number of synthetic compounds have been described as antioxidant enzyme mimics, capable of eliminating ROS and/or reducing oxidative damage. In this study, we investigated the antioxidant activity of a water-soluble 1,10-phenantroline-octanediaoate Mn 2+ -complex on cells under oxidative stress, and assessed its capacity to attenuate alpha-synuclein (aSyn) toxicity and aggregation, a process associated with increased oxidative stress. This Mn 2+ -complex exhibited a significant antioxidant potential, reducing intracelular oxidation and increasing oxidative stress resistance in S. cerevisiae cells and in vivo, in G. mellonella, increasing the activity of the intracellular antioxidant enzymes superoxide dismutase and catalase. Strikingly, the Mn 2+ -complex reduced both aSyn oligomerization and aggregation in human cell cultures and, using NMR and DFT/molecular docking we confirmed its interaction with the C-terminal region of aSyn. In conclusion, the Mn 2+ -complex appears as an excellent lead for the design of new phenanthroline derivatives as alternative compounds for preventing oxidative damages and oxidative stress - related diseases., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The increase in the prevalence and severity of fungal infections and the resistance to available antifungals highlights the imperative need for novel therapeutics and the search for new targets. High-content screening of libraries containing hundreds of compounds is a powerful strategy for searching for new drug candidates. In this study, we screened the Pandemic Response Box library (Medicines for Malaria Venture) of 400 diverse molecules against the Sporothrix pathogenic species. The initial screen identified twenty-four candidates that inhibited the growth of Sporothrix brasiliensis by more than 80%. Some of these compounds are known to display antifungal activity, including olorofim (MMV1782354), a new antifungal drug. Olorofim inhibited and killed the yeasts of S. brasiliensis , S. schenckii , and S. globosa at concentrations lower than itraconazole, and it also showed antibiofilm activity. According to the results obtained by fluorimetry, electron microscopy, and particle characterization analyses, we observed that olorofim induced profound alterations on the cell surface and cell cycle arrest in S. brasiliensis yeasts. We also verified that these morphophysiological alterations impaired their ability to adhere to keratinocytes in vitro. Our results indicate that olorofim is a promising new antifungal against sporotrichosis agents.

In this study, we investigated the influence of fungal extracellular vesicles (EVs) during biofilm formation and morphogenesis in Candida albicans. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated that C. albicans EVs inhibited biofilm formation in vitro . By time-lapse microscopy and SEM, we showed that C. albicans EV treatment stopped filamentation and promoted pseudohyphae formation with multiple budding sites. The ability of C. albicans EVs to regulate dimorphism was further compared to EVs isolated from different C. albicans strains, Saccharomyces cerevisiae, and Histoplasma capsulatum. C. albicans EVs from distinct strains inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 h. EVs from S. cerevisiae and H. capsulatum modestly reduced morphogenesis, and the effect was evident after 24 h of incubation. The inhibitory activity of C. albicans EVs on phase transition was promoted by a combination of lipid compounds, which were identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably, C. albicans EVs were also able to reverse filamentation. Finally, C. albicans cells treated with C. albicans EVs for 24 h lost their capacity to penetrate agar and were avirulent when inoculated into Galleria mellonella. Our results indicate that fungal EVs can regulate yeast-to-hypha differentiation, thereby inhibiting biofilm formation and attenuating virulence. IMPORTANCE The ability to undergo morphological changes during adaptation to distinct environments is exploited by Candida albicans and has a direct impact on biofilm formation and virulence. Morphogenesis is controlled by a diversity of stimuli, including osmotic stress, pH, starvation, presence of serum, and microbial components, among others. Apart from external inducers, C. albicans also produces autoregulatory substances. Farnesol and tyrosol are examples of quorum-sensing molecules (QSM) released by C. albicans to regulate yeast-to-hypha conversion. Here, we demonstrate that fungal EVs are messengers impacting biofilm formation, morphogenesis, and virulence in C. albicans. The major players exported in C. albicans EVs included sesquiterpenes, diterpenes, and fatty acids. The understanding of how C. albicans cells communicate to regulate physiology and pathogenesis can lead to novel therapeutic tools to combat candidiasis.

Melanin is one of the most studied virulence factors in pathogenic fungi. This pigment protects them from a series of both environmental and host stressors. Among basidiomycetes, Cryptococcus neoformans and Trichosporon asahii are known to produce melanin in the presence of phenolic precursors. Other species from the Trichosporonaceae family also produce this pigment, but the extent to this production among the clinically relevant species is unknown. For this reason, the aim of this study was to verify the production of melanin by different Trichosporonaceae species of clinical interest and to compare their pigments with the ones from C. neoformans and T. asahii , which are more prevalent in human infections. Melanin was produced in a minimal medium supplemented with 1 mM L-dihydroxyphenylalanine (L-DOPA). Pigment was evaluated using scanning electron microscopy, Zeta potential measurements, and energy-dispersive X-ray spectroscopy. It was found that, besides C. neoformans and T. asahii , Trichosporon japonicum , Apiotrichum montevideense , Trichosporon inkin , Trichosporon faecale , Cutaneotrichosporon debeurmannianum , and Cutaneotrichosporon arboriformis also produce melanin-like particles in the presence of L-DOPA. Melanin particles have negative charge and are smaller than original cells. Variations in color, fluorescence, and chemical composition was noticed between the studied strains. All melanins presented carbon, oxygen, sodium, and potassium in their composition. Melanins from the most pathogenic species also presented iron, zinc, and copper, which are important during parasitism. Biophysical properties of these melanins can confer to the Trichosporonaceae adaptive advantages to both parasitic and environmental conditions of fungal growth., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 de Andrade, Araújo, Brito-Santos, Figueiredo-Carvalho, Zancopé-Oliveira, Frases and Almeida-Paes.)

Competing Interests: The authors have declared that no competing interests exist.

Aim: Invasive human fungal infections have been a serious public health problem among immunocompromised patients. Wild bird species are related to the eco-epidemiology of some infectious diseases, mainly Cryptococcosis, Histoplasmosis, Aspergillosis, Chlamydiosis, Salmonellosis and allergic diseases. Falconry is the art of training predators for hunting. Nowadays, birds of prey are used as pets, which brings new sources of infections to humans. Materials & methods: We identified fungal pathogenic yeasts, Candida parapsilosis , Debaryomyces hansenii  and Rhodotorula mucilaginosa . Conclusion: Study new environmental niches of human pathogens is vitally important to establish preventive actions with the purpose of minimizing the risks of human contamination. Our work describes yeast microbiota from the excreta of Parabuteo unicinctus as a potential hazard for human disease.

Background: Treatment of mycoses is often ineffective, usually prolonged, and has some side effects. These facts highlight the importance of discovering new molecules to treat fungal infections., Objectives: To search the Medicines for Malaria Venture COVID Box for drugs with antifungal activity., Methods: Fourteen human pathogenic fungi were tested against the 160 drugs of this collection at 1.0 µM concentration. We evaluated the ability of the drugs to impair fungal growth, their fungicidal nature, and morphological changes caused to cells., Findings: Thirty-four molecules (21.25%) presented antifungal activity. Seven are antifungal drugs and one is the agricultural fungicide cycloheximide. The other drugs with antifungal activity included antibiotics (n = 3), antimalarials (n = 4), antivirals (n = 2), antiparasitcs (n = 3), antitumor agents (n = 5), nervous system agents (n = 3), immunosuppressants (n = 3), antivomiting (n = 1), antiasthmatic (n = 1), and a genetic disorder agent (n = 1). Several of these drugs inhibited Histoplasma capsulatum and Paracoccidioides brasiliensis growth (15 and 20, respectively), while Fusarium solani was not affected by the drugs tested. Most drugs were fungistatic, but niclosamide presented fungicidal activity against the three dimorphic fungi tested. Cyclosporine affected morphology of Cryptococcus neoformans., Main Conclusions: These drugs represent new alternatives to the development of more accessible and effective therapies to treat human fungal infections.

Background: Invasive fungal infections (IFI) are increasing in prevalence in recent years. In the last few months, the rise of COVID-19 patients has generated a new escalation in patients presenting opportunistic mycoses, mainly by Aspergillus. Candida infections are not being reported yet., Objectives: We aimed to determine the prevalence of systemic candidiasis in patients admitted to ICUs due to severe pneumonia secondary to SARS-CoV-2 infection and the existence of possible associated risk factors that led these patients to develop candidiasis., Patients/methods: We designed a study including patients with a confirmed diagnosis of COVID-19., Results: The prevalence of systemic candidiasis was 14.4%, and the main isolated species were C. albicans and C. parapsilosis. All patients that were tested positive for Candida spp. stayed longer in the ICU in comparison to patients who tested negative. Patients with candidiasis had higher MuLBSTA score and mortality rates and a worse radiological involvement. In our study, Candida spp. isolates were found in patients that were submitted to: tocilizumab, tocilizumab plus systemic steroids, interferon type 1β and Lopinavir-Ritonavir., Conclusions: Results suggested a high prevalence of systemic candidiasis in severe COVID-19-associated pneumonia patients. Patients with Candidiasis had the worst clinical outcomes. Treatment with tocilizumab could potentialize the risk to develop systemic candidiasis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Glucuronoxylomannan (GXM) participates in several immunoregulatory mechanisms, which makes it an important Cryptococcus virulence factor that is essential for the disease. Trichosporon asahii and Trichosporon mucoides share with Cryptococcus species the ability to produce GXM. To check whether other opportunistic species in the Trichosporonaceae family produce GXM-like polysaccharides, extracts from 28 strains were produced from solid cultures and their carbohydrate content evaluated by the sulfuric acid / phenol method. Moreover, extracts were assessed for cryptococcal GXM cross-reactivity through latex agglutination and lateral flow assay methods. Cryptococcus neoformans and Saccharomyces cerevisiae were used as positive and negative controls, respectively. In addition to T. asahii, the species Trichosporon inkin, Apiotrichum montevideense, Trichosporon japonicum, Trichosporon faecale, Trichosporon ovoides, Cutaneotrichosporon debeurmannianum, and Cutaneotrichosporon arboriformis are also producers of a polysaccharide immunologically similar to the GXM produced by human pathogenic Cryptococcus species. The carbohydrate concentration of the extracts presented a positive correlation with the GXM contents determined by titration of both methodologies. These results add several species to the list of fungal pathogens that produce glycans of the GXM type and bring information about the origin of potential false-positive results on immunological tests for diagnosis of cryptococcosis based on GXM detection.

Aim: Melanin has been linked to pathogenesis in several fungi. They often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in Candida glabrata . Methods & materials: C. glabrata was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment with denaturant and hot acid. Results: C. glabrata turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Biophysical properties demonstrated that the pigment was melanin. Melanized C. glabrata cells were effectively protected from azoles and amphotericin B, incubation at 42°C and macrophage killing. Conclusion: In the presence of L-DOPA, C. glabrata produces melanin, increases antifungal resistance and enhances host survival.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals. It is surrounded by three concentric structures that separate the cell from the extracellular space: the plasma membrane, the cell wall and the polysaccharide (PS) capsule. Although several studies have revealed the chemical composition of these structures, little is known about their ultrastructural organization and remodeling during C. neoformans budding events. Here, by combining the latest and most accurate light and electron microscopy techniques, we describe the morphological remodeling that occurs among the capsule, cell wall and plasma membrane during budding in C. neoformans . Our results show that the cell wall deforms to generate a specialized region at one of the cell's poles. This region subsequently begins to break into layers that are slightly separated from each other and with thick tips. We also observe a reorganization of the capsular PS around the specialized regions. While daughter cells present their PS fibers aligned in the direction of budding, mother cells show a similar pattern but in the opposite direction. Also, daughter cells form multilamellar membrane structures covering the continuous opening between both cells. Together, our findings provide compelling ultrastructural evidence for C. neoformans surface remodeling during budding, which may have important implications for future studies exploring these remodeled specialized regions as drug-targets against cryptococcosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Araújo, Alcantara, Rodrigues, de Souza, Pontes and Frases.)

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, who often have some inflammatory condition and, therefore, end up using glucocorticoids, such as dexamethasone and methylprednisolone. Although the effects of this class of molecules during cryptococcosis have been investigated, their consequences for the biology of C. neoformans is less explored. Here, we studied the effects of dexamethasone and methylprednisolone on the metabolism and on the induction of virulence factors in C. neoformans . Our results showed that both glucocorticoids increased fungal cell proliferation and surface electronegativity but reduced capsule and secreted polysaccharide sizes, as well as capsule compaction, by decreasing the density of polysaccharide fibers. We also tested whether glucocorticoids could affect the fungal virulence in Galleria mellonella and mice. Although the survival rate of Galleria larvae increased, those from mice showed a tendency to decrease, with infected animals dying earlier after glucocorticoid treatments. The pathogenesis of spread of cryptococcosis and the interleukin secretion pattern were also assessed for lungs and brains of infected mice. While increases in the spread of the fungus to lungs were observed after treatment with glucocorticoids, a significant difference in brain was observed only for methylprednisolone, although a trend toward increasing was also observed for dexamethasone. Moreover, increases in both pulmonary and cerebral IL-10 production, reduction of IL-6 production but no changes in IL-4, IL-17, and INF-γ were also observed after glucocorticoid treatments. Finally, histopathological analysis confirmed the increase in number of fungal cells in lung and brain tissues of mice previously subjected to dexamethasone or methylprednisolone treatments. Together, our results provide compelling evidence for the effects of dexamethasone and methylprednisolone on the biology of C. neoformans and may have important implications for future clinical treatments, calling attention to the risks of using these glucocorticoids against cryptococcosis or in immunocompromised individuals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Araújo, Alves, Martins-de-Souza, Guimarães, Honorato, Nimrichter, Takiya, Pontes and Frases.)

Background: As the global coronavirus pandemic (COVID-19) spreads across the world, new clinical challenges emerge in the hospital landscape. Among these challenges, the increased risk of coinfections is a major threat to the patients. Although still in a low number, due to the short time of the pandemic, studies that identified a significant number of hospitalised patients with COVID-19 who developed secondary fungal infections that led to serious complications and even death have been published., Objectives: In this scenario, we aim to determine the prevalence of invasive fungal infections (IFIs) and describe possible associated risk factors in patients admitted due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection., Patients/methods: We designed an open prospective observational study at the Rey Juan Carlos University Hospital (Mostoles, Spain), during the period from February 1 to April 30, 2020., Results: In this article, we reported seven patients with COVID-19-associated pulmonary aspergillosis (CAPA) who had a poor prognosis. Severely ill patients represent a high-risk group; therefore, we must actively investigate the possibility of aspergillosis in all of these patients. Larger cohort studies are needed to unravel the role of COVID-19 immunosuppressive therapy as a risk factor for aspergillosis., Conclusions: As the pandemic continues to spread across the world, further reports are needed to assess the frequency of emergent and highly resistant reemergent fungal infections during severe COVID-19. These coinfections are leading a significant number of patients with COVID-19 to death due to complications following the primary viral disease., (© 2020 Wiley-VCH GmbH.)

The cell wall is a ubiquitous structure in the fungal kingdom, with some features varying depending on the species. Additional external structures can be present, such as the capsule of Cryptococcus neoformans ( Cn ), its major virulence factor, mainly composed of glucuronoxylomannan (GXM), with anti-phagocytic and anti-inflammatory properties. The literature shows that other cryptococcal species and even more evolutionarily distant species, such as the Trichosporon asahii, T. mucoides , and Paracoccidioides brasiliensis can produce GXM-like polysaccharides displaying serological reactivity to GXM-specific monoclonal antibodies (mAbs), and these complex polysaccharides have similar composition and anti-phagocytic properties to cryptococcal GXM. Previously, we demonstrated that the fungus Histoplasma capsulatum (Hc) incorporates, surface/secreted GXM of Cn and the surface accumulation of the polysaccharide enhances Hc virulence in vitro and in vivo . In this work, we characterized the ability of Hc to produce cellular-attached (C-gly- Hc ) and secreted (E-gly) glycans with reactivity to GXM mAbs. These C-gly- Hc are readily incorporated on the surface of acapsular Cn cap59; however, in contrast to Cn GXM, C-gly- Hc had no xylose and glucuronic acid in its composition. Mapping of recognized Cn GXM synthesis/export proteins confirmed the presence of orthologs in the Hc database. Evaluation of C-gly and E-gly of Hc from strains of distinct monophyletic clades showed serological reactivity to GXM mAbs, despite slight differences in their molecular dimensions. These C-gly- Hc and E-gly- Hc also reacted with sera of cryptococcosis patients. In turn, sera from histoplasmosis patients recognized Cn glycans, suggesting immunogenicity and the presence of cross-reacting antibodies. Additionally, C-gly- Hc and E-gly- Hc coated Cn cap59 were more resistant to phagocytosis and macrophage killing. C-gly- Hc and E-gly- Hc coated Cn cap59 were also able to kill larvae of Galleria mellonella . These GXM-like Hc glycans, as well as those produced by other pathogenic fungi, may also be important during host-pathogen interactions, and factors associated with their regulation are potentially important targets for the management of histoplasmosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gonçalves, Rodriguez de La Noval, Ferreira, Honorato, Araújo, Frases, Pizzini, Nosanchuk, Cordero, Rodrigues, Peralta, Nimrichter and Guimarães.)

The increasing human population requires ongoing efforts in food production. This is frequently associated with an increased use of agrochemicals, leading to environmental contamination and altering microbial communities, including human fungal pathogens that reside in the environment. Cryptococcus gattii is an environmental yeast and is one of the etiological agents of cryptococcosis. Benomyl (BEN) is a broad-spectrum fungicide used on several crops. To study the effects of agrochemicals on fungal pathogens, we first evaluated the susceptibility of C. gattii to BEN and the interactions with clinical antifungals. Antagonistic interaction between BEN and fluconazole was seen and was strain- and concentration-dependent. We then induced BEN-resistance by culturing strains in increasing drug concentrations. One strain demonstrated to be more resistant and showed increased multidrug efflux pump gene (MDR1) expression and increased rhodamine 6G efflux, leading to cross-resistance between BEN and fluconazole. Morphologically, BEN-adapted cells had a reduced polysaccharide capsule; an increased surface/volume ratio; increased growth rate in vitro and inside macrophages and also higher ability in crossing an in vitro model of blood-brain-barrier. BEN-adapted strain demonstrated to be hypervirulent in mice, leading to severe symptoms of cryptococcosis, early mortality and higher fungal burden in the organs, particularly the brain. The parental strain was avirulent in murine model. In vivo cross-resistance between BEN and fluconazole was observed, with mice infected with the adapted strain unable to present any improvement in survival and behavior when treated with this antifungal. Furthermore, BEN-adapted cells cultured in drug-free media maintained the hypervirulent and cross-resistant phenotype, suggesting a persistent effect of BEN on C. gattii. In conclusion, exposure to BEN induces cross-resistance with fluconazole and increases the virulence of C. gattii. Altogether, our results indicate that agrochemicals may lead to unintended consequences on non-target species and this could result in severe healthy problems worldwide., Competing Interests: Declaration of competing interest All the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The emerging opportunistic pathogens comprising the Candida haemulonii complex (C. haemulonii [Ch], C. duobushaemulonii [Cd] and C. haemulonii var. vulnera[Chv]) are notable for their intrinsic antifungal resistance. Different clinical manifestations are associated with these fungal infections; however, little is known about their biology and potential virulence attributes. Herein, we evaluated some surface properties of 12 clinical isolates of Ch (n = 5), Cd (n = 4) and Chv (n = 3) as well as their virulence on murine macrophages and Galleria mellonella larvae. Scanning electron microscopy demonstrated the presence of homogeneous populations among the species of the C. haemulonii complex, represented by oval yeasts with surface irregularities able to form aggregates. Cell surface hydrophobicity was isolate-specific, exhibiting high (16.7%), moderate (25.0%) and low (58.3%) hydrophobicity. The isolates had negative surface charge, except for one. Mannose/glucose- and N-acetylglucosamine-containing glycoconjugates were evidenced in considerable amounts in all isolates; however, the surface expression of sialic acid was poorly detected. Cd isolates presented significantly higher amounts of chitin than Ch and Chv. Membrane sterol and lipid bodies, containing neutral lipids, were quite similar among all fungi studied. All isolates adhered to inert surfaces in the order: polystyrene > poly-L-lysine-coated glass > glass. Likewise, they interacted with murine macrophages in a quite similar way. Regarding in vivo virulence, the C. haemulonii species complex were able to kill at least 80% of the larvae after 120 hours. Our results evidenced the ability of C. haemulonii complex to produce potential surface-related virulence attributes, key components that actively participate in the infection process described in Candida spp., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)