Comprehensive characterization of extracellular vesicles produced by environmental (Neff) and clinical (T4) strains of Acanthamoeba castellanii.

We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of A. castellanii EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for Acanthamoeba-related diseases.IMPORTANCEA comprehensive and fully comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains of distinct virulence, a Neff (environmental) and T4 (clinical), revealed striking differences in their morphology and protein, lipid, metabolites, and transcripts levels. Data integration highlighted the differences in enzyme profiles, metabolic processes, and potential distinct origin of EVs from both strains, shedding light on the diversity and complexity of A. castellanii EVs, with direct implications for understanding host-pathogen interactions, disease mechanisms, and developing new therapies for the clinical intervention of Acanthamoeba-related diseases.

Combination therapy of itraconazole and an acylhydrazone derivative (D13) for the treatment of sporotrichosis in cats.

Acylhydrazone (AH) derivatives represent a novel category of anti-fungal medications that exhibit potent activity against Sporothrix sp., both in vitro and in a murine model of sporotrichosis. In this study, we demonstrated the anti-fungal efficacy of the AH derivative D13 [4-bromo-N'-(3,5-dibromo-2-hydroxybenzylidene)-benzohydrazide] against both planktonic cells and biofilms formed by Sporothrix brasiliensis. In a clinical study, the effect of D13 was then tested in combination with itraconazole (ITC), with or without potassium iodide, in 10 cats with sporotrichosis refractory to the treatment of standard of care with ITC. Improvement or total clinical cure was achieved in five cases after 12 weeks of treatment. Minimal abnormal laboratory findings, e.g., elevation of alanine aminotransferase, were observed in four cats during the combination treatment and returned to normal level within a week after the treatment was ended. Although highly encouraging, a larger and randomized controlled study is required to evaluate the effectiveness and the safety of this new and exciting drug combination using ITC and D13 for the treatment of feline sporotrichosis. IMPORTANCE: This paper reports the first veterinary clinical study of an acylhydrazone anti-fungal (D13) combined with itraconazole against a dimorphic fungal infection, sporotrichosis, which is highly endemic in South America in animals and humans. Overall, the results show that the combination treatment was efficacious in ~50% of the infected animals. In addition, D13 was well tolerated during the course of the study. Thus, these results warrant the continuation of the research and development of this new class of anti-fungals.

Corrected and republished from: "Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal Virulence".

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants expected to lack NOP16 expression, we observed a reduced EV production. Whole-genome sequencing, RNA-Seq, and cellular proteomics revealed that, contrary to our initial findings, these mutants expressed Nop16 but exhibited altered expression of 14 genes potentially involved in sugar transport. Based on this observation, we designated these mutant strains as Past1 and Past2, representing potentially altered sugar transport. Analysis of the small molecule composition of EVs produced by wild-type cells and the Past1 and Past2 mutant strains revealed not only a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the Past1 and Past2 mutant strains were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the mutant cells in G. mellonella. These results connect EV biogenesis, cargo, and cryptococcal virulence.

ß-1,3-Glucan recognition by Acanthamoeba castellanii as a putative mechanism of amoeba-fungal interactions.

In this study, we conducted an in-depth analysis to characterize potential Acanthamoeba castellanii (Ac) proteins capable of recognizing fungal ß-1,3-glucans. Ac specifically anchors curdlan or laminarin, indicating the presence of surface ß-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to Ac was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, Histoplasma capsulatum (Hc) G217B, possessing a ß-1,3-glucan outer layer, showed significant adhesion to Ac compared to a Hc G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble ß-1,3-glucan substantially inhibited this adhesion, indicating the involvement of ß-1,3-glucan recognition. Biotinylated ß-1,3-glucan-binding proteins from Ac exhibited higher binding to Hc G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the ß-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identified several Ac proteins capable of binding ß-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identified proteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinity to ß-1,3-glucans. These findings underscore the complexity of binding via ß-1,3-glucan and suggest the existence of alternative fungal recognition pathways in Ac.IMPORTANCEAcanthamoeba castellanii (Ac) and macrophages both exhibit the remarkable ability to phagocytose various extracellular microorganisms in their respective environments. While substantial knowledge exists on this phenomenon for macrophages, the understanding of Ac's phagocytic mechanisms remains elusive. Recently, our group identified mannose-binding receptors on the surface of Ac that exhibit the capacity to bind/recognize fungi. However, the process was not entirely inhibited by soluble mannose, suggesting the possibility of other interactions. Herein, we describe the mechanism of ß-1,3-glucan binding by A. castellanii and its role in fungal phagocytosis and survival within trophozoites, also using macrophages as a model for comparison, as they possess a well-established mechanism involving the Dectin-1 receptor for ß-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.

Traversing the Cell Wall: The Chitinolytic Activity of Histoplasma capsulatum Extracellular Vesicles Facilitates Their Release.

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.

Unveiling the Morphostructural Plasticity of Zoonotic Sporotrichosis Fungal Strains: Possible Implications for Sporothrix brasiliensis Virulence and Pathogenicity.

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.

Coregulation of extracellular vesicle production and fluconazole susceptibility in Cryptococcus neoformans.

Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novel Cryptococcus neoformans regulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover, in vitro spontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen. IMPORTANCE Extracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation, but their functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogen Cryptococcus neoformans. Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated in C. neoformans clinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.

Cyclosporine Affects the Main Virulence Factors of Cryptococcus neoformans In Vitro.

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.

A tribute to a man of science: lessons from Professor Luiz R. Travassos (1938-2020).

Luiz Rodolpho Raja Gabaglia Travassos, MD, PhD was a world-class microbiologist and cell biologist whose contributions to science were remarkable at multiple levels and across diverse fields. Besides being responsible for the creation of a scientific school that contributed to the transmission of multidisciplinary knowledge through several generations in Brazil and abroad, Professor Travassos was a pioneer in the fields of Microbiology, Glycobiology, Mycology, Parasitology, and Cancer Biology. To fully measure his contribution to science is an impossible task. We, some of his former students, post-docs, and collaborators, will illustrate the joy of having Professor Travassos as a mentor and friend through highlighting some of his breakthroughs in the fields of microbial physiology, infection, and cancer biology immersed in backstage stories of how he influenced so many people in many aspects of life. We hope that our future scientific generations and all who are passionate about discovery will see Travassos as an inspiration and example of a love for science.

Professor Luiz R. Travassos and the study of surface structures of fungal pathogens.

Brazilian medical mycology considerably expanded in the last decades due to the efforts of several pioneers who started and expanded mycology during the twentieth century. In this manuscript, we highlight some of the contributions of one of these pioneers: Professor Luiz R. Travassos, who started his career in the field of microbiology in the 1960s. We will discuss his contributions to the areas of medical mycology and glycobiology, with a focus on glycosphingolipids, sialic acids, and surface enzymes.

Pathogenicity & virulence of Histoplasma capsulatum - A multifaceted organism adapted to intracellular environments.

Histoplasmosis is a systemic mycosis caused by the thermally dimorphic fungus Histoplasma capsulatum. Although healthy individuals can develop histoplasmosis, the disease is particularly life-threatening in immunocompromised patients, with a wide range of clinical manifestations depending on the inoculum and virulence of the infecting strain. In this review, we discuss the established virulence factors and pathogenesis traits that make H. capsulatum highly adapted to a wide variety of hosts, including mammals. Understanding and integrating these mechanisms is a key step toward devising new preventative and therapeutic interventions.

Melanization of Candida auris Is Associated with Alteration of Extracellular pH.

Candida auris is a recently emerged global fungal pathogen, which causes life-threatening infections, often in healthcare settings. C. auris infections are worrisome because the fungus is often resistant to multiple antifungal drug classes. Furthermore, C. auris forms durable and difficult to remove biofilms. Due to the relatively recent, resilient, and resistant nature of C. auris, we investigated whether it produces the common fungal virulence factor melanin. Melanin is a black-brown pigment typically produced following enzymatic oxidation of aromatic precursors, which promotes fungal virulence through oxidative stress resistance, mammalian immune response evasion, and antifungal peptide and pharmaceutical inactivation. We found that certain strains of C. auris oxidized L-DOPA and catecholamines into melanin. Melanization occurred extracellularly in a process mediated by alkalinization of the extracellular environment, resulting in granule-like structures that adhere to the fungus' external surface. C. auris had relatively high cell surface hydrophobicity, but there was no correlation between hydrophobicity and melanization. Melanin protected the fungus from oxidative damage, but we did not observe a protective role during infection of macrophages or Galleria mellonella larvae. In summary, C. auris alkalinizes the extracellular medium, which promotes the non-enzymatic oxidation of L-DOPA to melanin that attaches to its surface, thus illustrating a novel mechanism for fungal melanization.

Vaccination with Live or Heat-Killed Aspergillus fumigatus ΔsglA Conidia Fully Protects Immunocompromised Mice from Invasive Aspergillosis.

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 ΔsglA mutant accumulated sterylglucosides (SGs), newly studied immunomodulatory glycolipids, and exhibited reduced hyphal growth and altered compositions of cell wall polysaccharides. Interestingly, the ΔsglA 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 ΔsglA 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.

In vitro and in vivo efficacies of Dectin-1-Fc(IgG)(s) fusion proteins against invasive fungal infections.

Fungal infections have increased in the last years, particularly associated to an increment in the number of immunocompromised individuals and the emergence of known or new resistant species, despite the difficulties in the often time-consuming diagnosis. The controversial efficacy of the currently available strategies for their clinical management, apart from their high toxicity and severe side effects, has renewed the interest in the research and development of new broad antifungal alternatives. These encompass vaccines and passive immunization strategies with monoclonal antibodies (mAbs), recognizing ubiquitous fungal targets, such as fungal cell wall ß-1,3-glucan polysaccharides, which could be used in early therapeutic intervention without the need for the diagnosis at species level. As additional alternatives, based on the Dectin-1 great affinity to ß-1,3-glucan, our group developed broad antibody-like Dectin1-Fc(IgG)(s) from distinct subclasses (IgG2a and IgG2b) and compared their antifungal in vitro and passive immunizations in vivo performances. Dectin1-Fc(IgG2a) and Dectin1-Fc(IgG2b) demonstrated high affinity to laminarin and the fungal cell wall by ELISA, flow cytometry, and microscopy. Both Dectin-1-Fc(IgG)(s) inhibited Histoplasma capsulatum and Cryptococcus neoformans growth in a dose-dependent fashion. For Candida albicans, such inhibitory effect was observed with concentrations as low as 0.098 and 0.049 µg/ml, respectively, which correlated with the impairment of the kinetics and lengths of germ tubes in comparison to controls. Previous opsonization with Dectin-1-Fc(IgG)(s) enhanced considerably the macrophage antifungal effector functions, increasing the fungi macrophages interactions and significantly reducing the intraphagosome fungal survival, as lower CFUs were observed. The administration of both Dectin1-Fc(IgG)(s) reduced the fungal burden and mortality in murine histoplasmosis and candidiasis models, in accordance with previous evaluations in aspergillosis model. These results altogether strongly suggested that therapeutic interventions with Dectin-1-Fc(IgG)(s) fusion proteins could directly impact the innate immunity and disease outcome in favor of the host, by direct neutralization, opsonization, phagocytosis, and fungal elimination, providing interesting information on the potential of these new strategies for the control of invasive fungal infections. LAY SUMMARY: Mycoses have increased worldwide, and new efficient therapeutics are needed. Passive immunizations targeting universally the fungal cell would allow early interventions without the species-level diagnosis. Lectins with affinity to carbohydrates could be used to engineer 'antibody-like' strategies.

Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal Virulence and Requires the NOP16 Gene.

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants lacking NOP16 expression, we observed that this gene was required for EV production. Analysis of the small molecule composition of EVs produced by wild-type cells and two independent nop16Δ mutants revealed that the deletion of NOP16 resulted not only in a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the nop16Δ mutants were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were coinjected with the nop16Δ cells in G. mellonella. These results reveal a role for NOP16 in EV biogenesis and cargo, and also indicate that the composition of EVs is determinant for cryptococcal virulence.

Recognition of Cell Wall Mannosylated Components as a Conserved Feature for Fungal Entrance, Adaptation and Survival Within Trophozoites of Acanthamoeba castellanii and Murine Macrophages.

Acanthamoeba castellanii (Ac) is a species of free-living amoebae (FLAs) that has been widely applied as a model for the study of host-parasite interactions and characterization of environmental symbionts. The sharing of niches between Ac and potential pathogens, such as fungi, favors associations between these organisms. Through predatory behavior, Ac enhances fungal survival, dissemination, and virulence in their intracellular milieu, training these pathogens and granting subsequent success in events of infections to more evolved hosts. In recent studies, our group characterized the amoeboid mannose binding proteins (MBPs) as one of the main fungal recognition pathways. Similarly, mannose-binding lectins play a key role in activating antifungal responses by immune cells. Even in the face of similarities, the distinct impacts and degrees of affinity of fungal recognition for mannose receptors in amoeboid and animal hosts are poorly understood. In this work, we have identified high-affinity ligands for mannosylated fungal cell wall residues expressed on the surface of amoebas and macrophages and determined the relative importance of these pathways in the antifungal responses comparing both phagocytic models. Mannose-purified surface proteins (MPPs) from both phagocytes showed binding to isolated mannose/mannans and mannosylated fungal cell wall targets. Although macrophage MPPs had more intense binding when compared to the amoeba receptors, the inhibition of this pathway affects fungal internalization and survival in both phagocytes. Mass spectrometry identified several MPPs in both models, and in silico alignment showed highly conserved regions between spotted amoeboid receptors (MBP and MBP1) and immune receptors (Mrc1 and Mrc2) and potential molecular mimicry, pointing to a possible convergent evolution of pathogen recognition mechanisms.

Isolation of Extracellular Vesicles from Candida auris.

Extracellular vesicles (EVs) are structures released by a variety of cells from all kingdoms of life. EVs are typically involved in communication between tissues and organs, between distinct organisms, or inside microbial communities. The plasticity of these structures is reflected in the range of biological effects they are able to induce or inhibit. The study of fungal EVs is relatively new with the first report in 2007, but investigators have already demonstrated in several model systems that fungal EVs significantly modulate the host immune system and that the immunogenic materials in EV can be harnessed as vaccination platforms. This chapter describes the two main procedures used to isolate EVs from an emerging pathogenic fungus, Candida auris.