Toll-like receptor 4 (TLR4) is the major pattern recognition receptor triggering the protective effect of a Candida albicans extracellular vesicle-based vaccine prototype in murine systemic candidiasis.

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 ß-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.

Phospholipase B Is Critical for Cryptococcus neoformans Survival in the Central Nervous System.

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.

Comparative Biophysical and Ultrastructural Analysis of Melanins Produced by Clinical Strains of Different Species From the Trichosporonaceae Family.

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.

Hypervirulence and cross-resistance to a clinical antifungal are induced by an environmental fungicide in Cryptococcus gattii.

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.

Effect of cell geometry in the evaluation of erythrocyte viscoelastic properties.

The red blood cell membrane-cytoskeleton is a complex structure mainly responsible for giving the cell rigidity and shape. It also provides the erythrocyte with the ability to pass through narrow capillaries of the vertebrate blood circulatory system. Although the red blood cell viscoelastic properties have been extensively studied, reported experimental data differ by up to three orders of magnitude. This could be attributed to the natural cell variability, to the different techniques employed, and also to the models used for the cell response, which are highly dependent on cell geometry. Here, we use two methodologies based on optical tweezers to investigate the viscoelastic behavior of healthy human red blood cells, one applying small cell deformations (microrheology) and another imposing large deformations (tether extraction). We also establish a defocusing microscopy-based method to characterize the cell geometry and thus the erythrocyte form factor, an essential parameter that allows comparisons among the viscoelastic properties at different conditions. Moreover, for small deformations, a soft glassy rheology model is used to discuss the results, while for large deformations two surface shear moduli and one surface viscosity are determined, together with the surface tension and bending modulus of the erythrocyte membrane lipid component. We also show that F-actin is not detected in tethers, although the erythrocyte membrane has physical properties like those of other adherent cells, known to have tethers containing F-actin inside. Altogether, our results show good agreement with the reported literature and we argue that, to properly compare the viscoelastic properties of red blood cells in different situations, the task of cell geometry characterization must be accomplished. This may be especially important when the influence of agents, like the malaria parasite, induces changes in both the geometry and chemical constituents of the erythrocyte membrane. Together, the new methodologies and procedures used in this study would allow the erythrocyte community to better explore the mechanical behavior of red blood cells and may be useful to characterize erythrocyte viscoelasticity changes in several blood diseases.

Histoplasma capsulatum Glycans From Distinct Genotypes Share Structural and Serological Similarities to Cryptococcus neoformans Glucuronoxylomannan.

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.

Atorvastatin as a promising anticryptococcal agent.

Cryptococcosis caused by Cryptococcus gattii leads to pneumonia and meningoencephalitis, and has a high mortality rate worldwide due to the inadequacy of available therapy and increasing drug resistance. There is a need to develop effective treatments, and drug repositioning is an interesting alternative to achieve new strategies to treat cryptococcosis. Atorvastatin (ATO), a statin currently used to treat hypercholesterolaemia, was tested in this study as an adjuvant to control infections caused by C. gattii. Several aspects of the effect of ATO on the host and the yeast were evaluated, with particular focus on the association of ATO with fluconazole (FLC), which (i) reduced ergosterol content in the cell membrane and altered properties of the polysaccharide capsule of C. gattii; (ii) increased the production of reactive oxygen species by macrophages; and (iii) reduced yeast phagocytosis and the intracellular proliferation rate. In an animal model, infected mice treated with ATO + FLC showed increased survival, improved clinical condition, and reduced fungal burden in the lungs and brain. This study is the first to perform in vivo tests with ATO + FLC for the treatment of cryptococcosis. The results suggest that ATO may be an important adjuvant for the treatment of cryptococcosis.