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Microbial pathogens generate extracellular vesicles (EVs) for intercellular communication and quorum sensing. Microbial EVs also induce inflammatory pathways within host innate immune cells. We previously demonstrated that EVs secreted by Candida albicans trigger type I interferon signaling in host cells specifically via the cGAS-STING innate immune signaling pathway. Here, we show that despite sharing similar properties of morphology and internal DNA content, the interactions between EVs and the innate immune system differ according to the parental fungal species. EVs secreted by C. albicans , Saccharomyces cerevisiae, Cryptococcus neoformans, and Aspergillus fumigatus are endocytosed at different rates by murine macrophages triggering varied cytokine responses, innate immune signaling, and subsequent immune cell recruitment. Notably, cell wall constituents that decorate C. neoformans and A. fumigatus EVs inhibit efficient internalization by macrophages and dampen innate immune activation. Our data uncover the transcriptional and functional consequences of the internalization of diverse fungal EVs by immune cells and reveal novel insights into the early innate immune response to distinct clinically significant fungal pathogens.

The urgency surrounding Candida auris as a public health threat is highlighted by both the Center for Disease Control (CDC) and World Health Organization (WHO) that categorized this species as a priority fungal pathogen. Given the current limitations of antifungal therapy for C. auris , particularly due to its multiple resistance to the current antifungals, the identification of new drugs is of paramount importance. Some alkaloids abundant in the venom of the red invasive fire ant ( Solenopsis invicta ), known as solenopsins, have garnered attention as potent inhibitors of bacterial biofilms, and there are no studies demonstrating such effects against fungal pathogens. Thus, we herein investigated the antibiotic efficacy of solenopsin alkaloids against C. auris biofilms and planktonic cells. Both natural and synthetic solenopsins inhibited the growth of C. auris strains from different clades, including fluconazole and amphotericin B-resistant isolates. Such alkaloids also inhibited matrix deposition and altered cellular metabolic activity of C. auris in biofilm conditions. Mechanistically, the alkaloids compromised membrane integrity as measured by propidium iodide uptake in exposed planktonic cells. Additionally, combining the alkaloids with AMB yielded an additive antifungal effect, even against AMB-resistant strains. Finally, both extracted solenopsins and the synthetic analogues demonstrated protective effect in vivo against C. auris infection in the invertebrate model Galleria mellonella . These findings underscore the potent antifungal activities of solenopsins against C. auris and suggest their inclusion in future drug development. Furthermore, exploring derivatives of solenopsins could reveal novel compounds with therapeutic promise.

The host type I interferon (IFN) pathway is a major signature of inflammation induced by the human fungal pathogen, Candida albicans. However, the molecular mechanism for activating this pathway in the host defence against C. albicans remains unknown. Here we reveal that mice lacking cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway components had improved survival following an intravenous challenge by C. albicans. Biofilm-associated C. albicans DNA packaged in extracellular vesicles triggers the cGAS-STING pathway as determined by induction of interferon-stimulated genes, IFNβ production, and phosphorylation of IFN regulatory factor 3 and TANK-binding kinase 1. Extracellular vesicle-induced activation of type I IFNs was independent of the Dectin-1/Card9 pathway and did not require toll-like receptor 9. Single nucleotide polymorphisms in cGAS and STING potently altered inflammatory cytokine production in human monocytes challenged by C. albicans. These studies provide insights into the early innate immune response induced by a clinically significant fungal pathogen., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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.

Fungal diseases are a leading threat to human health, especially in individuals with compromised immunity. Although there have been recent important advances in antifungal drug development, antifungal resistance, drug-drug interactions and difficulties in delivery remain major challenges. Among its pleiotropic actions, nitric oxide (NO) is a key molecule in host defense. We have developed a flexible nanoparticle platform that delivers sustained release of NO and have demonstrated the platform's efficacy against diverse bacteria as well as some fungal species. In this work, we investigate the effects of two NO-releasing particles against a panel of important human yeast. Our results demonstrate that the compounds are both effective against diverse yeast, including ascomycota and basidiomycota species, and that NO-releasing particles may be a potent addition to our armamentarium for the treatment of focal and disseminated mycoses., Competing Interests: Declaration of competing interest 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., (Published by Elsevier Ltd.)

Introduction: Fungal infections are caused by a broad range of pathogenic fungi that are found worldwide with different geographic distributions, incidences, and mortality rates. Considering that there are relatively few approved medications available for combating fungal diseases and no vaccine formulation commercially available, multiple groups are searching for new antifungal drugs, examining drugs for repurposing and developing antifungal vaccines, in order to control deaths, sequels, and the spread of these complex infections., Areas Covered: This review provides a summary of advances in fungal vaccine studies and the different approaches under development, such as subunit vaccines, whole organism vaccines, and DNA vaccines, as well as studies that optimize the use of adjuvants. We conducted a literature search of the PubMed with terms: fungal vaccines and genus of fungal pathogens (Cryptococcus spp. Candida spp. Coccidioides spp. Aspergillus spp. Sporothrix spp. Histoplasma spp. Paracoccidioides spp. Pneumocystis spp. and the Mucorales order), a total of 177 articles were collected from database., Expert Opinion: Problems regarding the immune response development in an immunocompromised organism, the similarity between fungal and mammalian cells, and the lack of attention by health organizations to fungal infections are closely related to the fact that, at present, there are no fungal vaccines available for clinical use.

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.

Antifungal resistance has become more frequent, either due to the emergence of naturally resistant species or the development of mechanisms that lead to resistance in previously susceptible species. Among these fungal species of global threat, Candida auris stands out for commonly being highly resistant to antifungal drugs, and some isolates are pan-resistant. The rate of mortality linked to C. auris infections varies from 28% to 78%. In this study, we characterized C. auris extracellular vesicles (EVs) in the presence of caspofungin, an echinocandin, which is the recommended first line antifungal for the treatment of infections due to this emerging pathogen. Furthermore, we also analyzed the protein and RNA content of EVs generated by C. auris cultivated with or without treatment with caspofungin. We observed that caspofungin led to the increased production of EVs, and treatment also altered the type and quantity of RNA molecules and proteins enclosed in the EVs. There were distinct classes of RNAs in the EVs with ncRNAs being the most identified molecules, and tRNA-fragments (tRFs) were abundant in each of the strains studied. We also identified anti-sense RNAs, varying from 21 to 55 nt in length. The differentially abundant mRNAs detected in EVs isolated from yeast subjected to caspofungin treatment were related to translation, nucleosome core and cell wall. The differentially regulated proteins identified in the EVs produced during caspofungin treatment were consistent with the results observed with the RNAs, with the enriched terms being related to translation and cell wall. Our study adds new information on how an echinocandin can affect the EV pathway, which is associated with the yeast cell being able to evade treatment and persist in the host. The ability of C. auris to efficiently alter the composition of EVs may represent a mechanism for the fungus to mitigate the effects of antifungal 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.

Methamphetamine (METH) is a major public health and safety problem in the United States. Chronic METH abuse is associated with a 2-fold-higher risk of HIV infection and, possibly, additional infections, particularly those that enter through the respiratory tract or skin. Cryptococcus neoformans is an encapsulated opportunistic yeast-like fungus that is a relatively frequent cause of meningoencephalitis in immunocompromised patients, especially in individuals with AIDS. C. neoformans melanizes during mammalian infection in a process that presumably uses host-supplied compounds such as catecholamines. l-3,4-Dihydroxyphenylalanine (l-Dopa) is a natural catecholamine that is frequently used to induce melanization in C. neoformans. l-Dopa-melanized cryptococci manifest resistance to radiation, phagocytosis, detergents, and heavy metals. Using a systemic mouse model of infection and in vitro assays to critically assess the impact of METH on C. neoformans melanization and pathogenesis, we demonstrated that METH-treated mice infected with melanized yeast cells showed increased fungal burdens in the blood and brain, exacerbating mortality. Interestingly, analyses of cultures of METH-exposed cryptococci supplemented with l-Dopa revealed that METH accelerates fungal melanization, an event of adaptation to external stimuli that can be advantageous to the fungus during pathogenesis. Our findings provide novel evidence of the impact of METH abuse on host homeostasis and increased permissiveness to opportunistic microorganisms.

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Emerging and reemerging pathogens are a worldwide concern, and it is predicted that these microbes will cause severe outbreaks. Candida auris affects people with weakened immune systems, particularly those who are hospitalized or are in health care facilities. Extracellular vesicles (EVs) are lipid bilayer structures released by organisms from all domains of life. EVs can deliver functional molecules to target cells, including proteins and nucleic acids, especially RNA molecules. EVs from several pathogenic fungi species play diverse biological roles related to cell-cell communication and pathogen-host interaction. In this study, we describe a data set which we produced by sequencing the RNA content of EVs from C. auris under normal growth conditions and in the presence of the antifungal caspofungin, a first-line drug to treat this fungus. To generate a more complete data set for future comparative studies, we also sequenced the RNA cellular content of EVs under the same conditions. This data set addresses a previously unexplored area of fungal biology regarding cellular small RNA and EV RNA. Our data will provide a molecular basis for the study of the aspects associated with antifungal treatment, gene expression response, and EV composition in C. auris. These data will also allow the exploration of small RNA content in the fungal kingdom and might serve as an informative basis for studies on the mechanisms by which molecules are directed to fungal EVs. IMPORTANCE Candida auris, a relevant emerging human-pathogenic yeast, is the first fungus to be called a global public health threat by the WHO. This is because of its rapid spread on all inhabited continents, together with its extremely high frequency of drug and multidrug resistance. In our study, we generated a large data set for 3 distinct strains of C. auris and obtained cellular small RNA fraction as well as extracellular vesicle RNA (EV-RNA) during normal growth conditions and after treatment with caspofungin, the first-line drug used to treat C. auris infection.

Lipids play a fundamental role in fungal cell biology, being essential cell membrane components and major targets of antifungal drugs. A deeper knowledge of lipid metabolism is key for developing new drugs and a better understanding of fungal pathogenesis. Here, we built a comprehensive map of the Histoplasma capsulatum lipid metabolic pathway by incorporating proteomic and lipidomic analyses. We performed genetic complementation and overexpression of H. capsulatum genes in Saccharomyces cerevisiae to validate reactions identified in the map and to determine enzymes responsible for catalyzing orphan reactions. The map led to the identification of both the fatty acid desaturation and the sphingolipid biosynthesis pathways as targets for drug development. We found that the sphingolipid biosynthesis inhibitor myriocin, the fatty acid desaturase inhibitor thiocarlide, and the fatty acid analog 10-thiastearic acid inhibit H. capsulatum growth in nanomolar to low-micromolar concentrations. These compounds also reduced the intracellular infection in an alveolar macrophage cell line. Overall, this lipid metabolic map revealed pathways that can be targeted for drug development. IMPORTANCE It is estimated that 150 people die per hour due to the insufficient therapeutic treatments to combat fungal infections. A major hurdle to developing antifungal therapies is the scarce knowledge on the fungal metabolic pathways and mechanisms of virulence. In this context, fungal lipid metabolism is an excellent candidate for developing drugs due to its essential roles in cellular scaffolds, energy storage, and signaling transductors. Here, we provide a detailed map of Histoplasma capsulatum lipid metabolism. The map revealed points of this fungus lipid metabolism that can be targeted for developing antifungal drugs.

Lipid microdomains or lipid rafts are dynamic and tightly ordered regions of the plasma membrane. In mammalian cells, they are enriched in cholesterol, glycosphingolipids, Glycosylphosphatidylinositol-anchored and signalling-related proteins. Several studies have suggested that mammalian pattern recognition receptors are concentrated or recruited to lipid domains during host-pathogen association to enhance the effectiveness of host effector processes. However, pathogens have also evolved strategies to exploit these domains to invade cells and survive. In fungal organisms, a complex cell wall network usually mediates the first contact with the host cells. This cell wall may contain virulence factors that interfere with the host membrane microdomains dynamics, potentially impacting the infection outcome. Indeed, the microdomain disruption can dampen fungus-host cell adhesion, phagocytosis and cellular immune responses. Here, we provide an overview of regulatory strategies employed by pathogenic fungi to engage with and potentially subvert the lipid microdomains of host cells. TAKE AWAY: Lipid microdomains are ordered regions of the plasma membrane enriched in cholesterol, glycosphingolipids (GSL), GPI-anchored and signalling-related proteins. Pathogen recognition by host immune cells can involve lipid microdomain participation. During this process, these domains can coalesce in larger complexes recruiting receptors and signalling proteins, significantly increasing their signalling abilities. The antifungal innate immune response is mediated by the engagement of pathogen-associated molecular patterns to pattern recognition receptors (PRRs) at the plasma membrane of innate immune cells. Lipid microdomains can concentrate or recruit PRRs during host cell-fungi association through a multi-interactive mechanism. This association can enhance the effectiveness of host effector processes. However, virulence factors at the fungal cell surface and extracellular vesicles can re-assembly these domains, compromising the downstream signalling and favouring the disease development. Lipid microdomains are therefore very attractive targets for novel drugs to combat fungal infections., (© 2021 John Wiley & Sons Ltd.)

Candida auris has emerged as a serious worldwide threat by causing opportunistic infections that are frequently resistant to one or more conventional antifungal medications resulting in high mortality rates. Against this backdrop, health warnings around the world have focused efforts on understanding C. auris fungal biology and effective prevention and treatment approaches to combat this fungus. To date, there is little information about the differentially expressed genes when this fungus is treated with conventional antifungals, and caspofungin is a standard echinocandin deployed in the therapy against C. auris . In this work, we treated two distinct strains of C. auris for 24 h with caspofungin, and the cellular responses were evaluated at the morphological, translational and transcriptional levels. We first observed that the echinocandin caused morphological alterations, aggregation of yeast cells, and modifications in the cell wall composition of C. auris . Transcriptomic analysis revealed an upregulation of genes related to the synthesis of the cell wall, ribosome, and cell cycle after exposure to caspofungin. Supporting these findings, the integrated proteomic analysis showed that caspofungin-treated cells were enriched in ribosome-related proteins and cell wall, especially mannoproteins. Altogether, these results provide further insights into the biology of C. auris and expands our understanding regarding the antifungal activity of caspofungin and reveal cellular targets, as the mannose metabolism, that can be further explored for the development of novel antifungals., 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., (© 2021 The Authors.)

Candida auris is a recently described multidrug-resistant pathogenic fungus that is increasingly responsible for health care-associated outbreaks across the world. Bloodstream infections of this fungus cause death in up to 70% of cases. Aggravating this scenario, the disease-promoting mechanisms of C. auris are poorly understood. Fungi release extracellular vesicles (EVs) that carry a broad range of molecules, including proteins, lipids, carbohydrates, pigments, and RNA, many of which are virulence factors. Here, we carried out a comparative molecular characterization of C. auris and Candida albicans EVs and evaluated their capacity to modulate effector mechanisms of host immune defense. Using proteomics, lipidomics, and transcriptomics, we found that C. auris released EVs with payloads that were significantly different from those of EVs released by C. albicans. EVs released by C. auris potentiated the adhesion of this yeast to an epithelial cell monolayer, while EVs from C. albicans had no effect. C. albicans EVs primed macrophages for enhanced intracellular yeast killing, whereas C. auris EVs promoted survival of the fungal cells. Moreover, EVs from both C. auris and C. albicans induced the activation of bone marrow-derived dendritic cells. Together, our findings show distinct profiles and properties of EVs released by C. auris and by C. albicans and highlight the potential contribution of C. auris EVs to the pathogenesis of this emerging pathogen. IMPORTANCE Candida auris is a recently described multidrug-resistant pathogenic fungus that is responsible for outbreaks across the globe, particularly in the context of nosocomial infections. Its virulence factors and pathogenesis are poorly understood. Here, we tested the hypothesis that extracellular vesicles (EVs) released by C. auris are a disease-promoting factor. We describe the production of EVs by C. auris and compare their biological activities against those of the better-characterized EVs from C. albicans. C. auris EVs have immunoregulatory properties, of which some are opposite those of C. albicans EVs. We also explored the cargo and structural components of those vesicles and found that they are remarkably distinct compared to EVs from C. auris's phylogenetic relative Candida albicans.

Extracellular vesicles (EVs) are lipid bilayer structures released by organisms from all kingdoms of life. The diverse biogenesis pathways of EVs result in a wide variety of physical properties and functions across different organisms. Fungal EVs were first described in 2007 and different omics approaches have been fundamental to understand their composition, biogenesis, and function. In this review, we discuss the role of omics in elucidating fungal EVs biology. Transcriptomics, proteomics, metabolomics, and lipidomics have each enabled the molecular characterization of fungal EVs, providing evidence that these structures serve a wide array of functions, ranging from key carriers of cell wall biosynthetic machinery to virulence factors. Omics in combination with genetic approaches have been instrumental in determining both biogenesis and cargo loading into EVs. We also discuss how omics technologies are being employed to elucidate the role of EVs in antifungal resistance, disease biomarkers, and their potential use as vaccines. Finally, we review recent advances in analytical technology and multi-omic integration tools, which will help to address key knowledge gaps in EVs biology and translate basic research information into urgently needed clinical applications such as diagnostics, and immuno- and chemotherapies to fungal infections., 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 Zamith-Miranda, Peres da Silva, Couvillion, Bredeweg, Burnet, Coelho, Camacho, Nimrichter, Puccia, Almeida, Casadevall, Rodrigues, Alves, Nosanchuk and Nakayasu.)

Melanins are ubiquitous complex polymers that are commonly known in humans to cause pigmentation of our skin. Melanins are also present in bacteria, fungi, and helminths. In this review, we will describe the diverse interactions of fungal melanin with the mammalian immune system. We will particularly focus on Cryptococcus neoformans and also discuss other major melanotic pathogenic fungi. Melanin interacts with the immune system through diverse pathways, reducing the effectiveness of phagocytic cells, binding effector molecules and antifungals, and modifying complement and antibody responses.

The prevalence of fungal infections has increased in immunocompromised patients, leading to millions of deaths annually. Arachidonic acid (AA) metabolites, such as eicosanoids, play important roles in regulating innate and adaptative immune function, particularly since they can function as virulence factors enhancing fungal colonization and are produced by mammalian and lower eukaryotes, such as yeasts and other fungi ( Candida albicans , Histoplasma capsulatum and Cryptococcus neoformans ). C. albicans produces prostaglandins (PG), Leukotrienes (LT) and Resolvins (Rvs), whereas the first two have been well documented in Cryptococcus sp. and H. capsulatum . In this review, we cover the eicosanoids produced by the host and fungi during fungal infections. These fungal-derived PGs have immunomodulatory functions analogous to their mammalian counterparts. Prostaglandin E 2 (PGE 2 ) protects C. albicans and C. parapsilosis cells from the phagocytic and killing activity of macrophages. H. capsulatum PGs augment the fungal burden and host mortality rates in histoplasmosis. However, PGD 2 potentiates the effects and production of LTB 4 , which is a very potent neutrophil chemoattractant that enhances host responses. Altogether, these data suggest that eicosanoids, mainly PGE 2 , may serve as a new potential target to combat diverse fungal infections.

Replicative aging is an underexplored field of research in medical mycology. Cryptococcus neoformans ( Cn ) and Candida glabrata ( Cg ) are dreaded fungal pathogens that cause fatal invasive infections. The fungal cell wall is essential for yeast viability and pathogenesis. In this study, we provide data characterizing age-associated modifications to the cell wall of Cn and Cg . Here, we report that old yeast cells upregulate genes of cell wall biosynthesis, leading to cell wall reorganization and increased levels of all major components, including glucan, chitin, and its derivatives, as well as mannan. This results in a significant thickening of the cell wall in aged cells. Old-generation yeast cells exhibited drastic ultrastructural changes, including the presence of abundant vesicle-like particles in the cytoplasm, and enlarged vacuoles with altered pH homeostasis. Our findings suggest that the cell wall modifications could be enabled by augmented intracellular trafficking. This work furthers our understanding of the cell phenotype that emerges during aging. It highlights differences in these two fungal pathogens and elucidates mechanisms that explain the enhanced resistance of old cells to antifungals and phagocytic attacks. IMPORTANCE Cryptococcus neoformans and Candida glabrata are two opportunistic human fungal pathogens that cause life-threatening diseases. During infection, both microorganisms have the ability to persist for long periods, and treatment failure can occur even if standard testing identifies the yeasts to be sensitive to antifungals. Replicative life span is a trait that is measured by the number of divisions a cell undergoes before death. Aging in fungi is associated with enhanced tolerance to antifungals and resistance to phagocytosis, and characterization of old cells may help identify novel antifungal targets. The cell wall remains an attractive target for new therapies because it is essential for fungi and is not present in humans. This study shows that the organization of the fungal cell wall changes remarkably during aging and becomes thicker and is associated with increased intracellular trafficking as well as the alteration of vacuole morphology and pH homeostasis.

Histoplasma capsulatum is a major endemic mycosis. Our laboratories have demonstrated that H. capsulatum produces extracellular vesicles (EV) that are loaded with diverse compounds that influence virulence. We have further shown that H. capsulatum dynamically regulates the loading and release of fungal EV in response to stimuli and growth conditions. This chapter details the current knowledge of EV biology in H. capsulatum and the impact of this information on our understanding of this important process that is closely linked to pathogenesis., (© 2021. Springer Nature Switzerland AG.)

Cryptococcus neoformans is a human pathogenic fungus that mainly afflicts immunocompromised patients. One of its virulence strategies is the production of extracellular vesicles (EVs), containing cargo with immunomodulatory properties. We evaluated EV's characteristics produced by capsular and acapsular strains of C. neoformans (B3501 and ΔCap67, respectively) growing in nutritionally poor or rich media and co-cultures with bone marrow-derived macrophages or dendritic cells from C57BL/6 mice. EVs produced under a poor nutritional condition displayed a larger hydrodynamic size, contained more virulence compounds, and induced a more robust inflammatory pattern than those produced in a rich nutritional medium, independently of strain. We treated infected mice with EVs produced in the rich medium, and the EVs inhibited more genes related to the inflammasome than untreated infected mice. These findings suggest that the EVs participate in the pathogenic processes that result in the dissemination of C. neoformans . Thus, these results highlight the versatility of EVs' properties during infection by C. neoformans in different tissues and support ongoing efforts to harness EVs to prevent and treat cryptococcosis.

Antibodies play a central role in host immunity by directly inactivating or recognizing an invading pathogen to enhance different immune responses to combat the invader. However, the cellular responses of pathogens to the presence of antibodies are not well-characterized. Here, we used different mass spectrometry techniques to study the cellular responses of the pathogenic fungus Histoplasma capsulatum to monoclonal antibodies (mAb) against HSP60, the surface protein involved in infection. A proteomic analysis of H. capsulatum yeast cells revealed that mAb binding regulates a variety of metabolic and signaling pathways, including fatty acid metabolism, sterol metabolism, MAPK signaling and ubiquitin-mediated proteolysis. The regulation of the fatty acid metabolism was accompanied by increases in the level of polyunsaturated fatty acids, which further augmented the degree of unsaturated lipids in H. capsulatum 's membranes and energy storage lipids, such as triacylglycerols, phosphatidylcholines, phosphatidylethanolamines and phosphatidylinositols. MAb treatment also regulated sterol metabolism by increasing the levels of cholesterol and ergosterol in the cells. We also showed that global changes in the lipid profiles resulted in an increased susceptibility of H. capsulatum to the ergosterol-targeting drug amphotericin B. Overall, our data showed that mAb induction of global changes in the composition of H. capsulatum membranes can potentially impact antifungal treatment during histoplasmosis.

Candida auris is a recently described pathogenic fungus that is causing invasive outbreaks on all continents. The fungus is of high concern given the numbers of multidrug-resistant strains that have been isolated in distinct sites across the globe. The fact that its diagnosis is still problematic suggests that the spreading of the pathogen remains underestimated. Notably, the molecular mechanisms of virulence and antifungal resistance employed by this new species are largely unknown. In the present work, we compared two clinical isolates of C. auris with distinct drug susceptibility profiles and a Candida albicans reference strain using a multi-omics approach. Our results show that, despite the distinct drug resistance profile, both C. auris isolates appear to be very similar, albeit with a few notable differences. However, compared to C. albicans both C. auris isolates have major differences regarding their carbon utilization and downstream lipid and protein content, suggesting a multifactorial mechanism of drug resistance. The molecular profile displayed by C. auris helps to explain the antifungal resistance and virulence phenotypes of this new emerging pathogen. IMPORTANCE Candida auris was first described in Japan in 2009 and has now been the cause of significant outbreaks across the globe. The high number of isolates that are resistant to one or more antifungals, as well as the high mortality rates from patients with bloodstream infections, has attracted the attention of the medical mycology, infectious disease, and public health communities to this pathogenic fungus. In the current work, we performed a broad multi-omics approach on two clinical isolates isolated in New York, the most affected area in the United States and found that the omic profile of C. auris differs significantly from C. albicans In addition to our insights into C. auris carbon utilization and lipid and protein content, we believe that the availability of these data will enhance our ability to combat this rapidly emerging pathogenic yeast., (Copyright © 2019 Zamith-Miranda et al.)

Histoplasmosis is an infection caused by the dimorphic fungus Histoplasma capsulatum . Histoplasmosis is typically self-limited and presents asymptomatically in most people. Nevertheless, histoplasmosis can cause severe pulmonary disease and death. Histoplasmosis is increasingly found worldwide; however, it is best documented in the endemic region of the Mississippi river valley system in the Eastern part of the United States (US). Epidemiological studies from the US detailing the morbidity, mortality, and cost associated with histoplasmosis underscore the need to develop a vaccine., Purpose of Review: This review will detail some of the major developments in potential vaccines against histoplasmosis, with particular emphasis on those that could be used to immunize immunocompromised hosts. Additionally, this review will highlight some non-traditional vaccine-like ideas for the prevention of diverse mycoses., Recent Findings: Historically, immunization strategies against histoplasmosis have largely focused on identifying immunogenic proteins that confer protection in animal models. More recently, novel active, therapeutic, and immunomodulatory strategies have been explored as potential alternatives for those with various immune-deficiencies., Summary: The studies summarized in this review demonstrate that more research is needed to clarify the immunobiology, clinical role and efficacy of each candidate vaccine in the ever-expanding potential armamentarium against histoplasmosis., Competing Interests: Conflict of Interest Maxwell T. Roth, Daniel Zamith-Miranda, and Joshua D. Nosanchuk declare no conflict of interest.

Eukaryotic cells, including fungi, release extracellular vesicles (EVs). These lipid bilayered compartments play essential roles in cellular communication and pathogenesis. EV composition is complex and includes proteins, glycans, pigments, and RNA. RNAs with putative roles in pathogenesis have been described in EVs produced by fungi. Here we describe the RNA content in EVs produced by the G186AR and G217B strains of Histoplasma capsulatum , an important human-pathogenic fungal pathogen. A total of 124 mRNAs were identified in both strains. In this set of RNA classes, 93 transcripts were enriched in EVs from the G217B strain, whereas 31 were enriched in EVs produced by the G186AR strain. This result suggests that there are important strain-specific properties in the mRNA composition of fungal EVs. We also identified short fragments (25 to 40 nucleotides in length) that were strain specific, with a greater number identified in EVs produced by the G217B strain. Remarkably, the most highly enriched processes were stress responses and translation. Half of these fragments aligned to the reverse strand of the transcript, suggesting the occurrence of microRNA (miRNA)-like molecules in fungal EVs. We also compared the transcriptome profiles of H. capsulatum with the RNA composition of EVs, and no correlation was observed. Taking the results together, our study provided information about the RNA molecules present in H. capsulatum EVs and about the differences in composition between the strains. In addition, we found no correlation between the most highly expressed transcripts in the cell and their presence in the EVs, reinforcing the idea that the RNAs were directed to the EVs by a regulated mechanism. IMPORTANCE Extracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. EV-associated RNA classes have recently been described in pathogenic fungi; however, only a few reports of studies describing the RNAs in fungal EVs are available. Improved knowledge of EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates of Histoplasma capsulatum Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA., (Copyright © 2019 Alves et al.)

Recognition and internalisation of intracellular pathogens by host cells is a multifactorial process, involving both stable and transient interactions. The plasticity of the host cell plasma membrane is fundamental in this infectious process. Here, the participation of macrophage lipid microdomains during adhesion and internalisation of the fungal pathogen Histoplasma capsulatum (Hc) was investigated. An increase in membrane lateral organisation, which is a characteristic of lipid microdomains, was observed during the first steps of Hc-macrophage interaction. Cholesterol enrichment in macrophage membranes around Hc contact regions and reduced levels of Hc-macrophage association after cholesterol removal also suggested the participation of lipid microdomains during Hc-macrophage interaction. Using optical tweezers to study cell-to-cell interactions, we showed that cholesterol depletion increased the time required for Hc adhesion. Additionally, fungal internalisation was significantly reduced under these conditions. Moreover, macrophages treated with the ceramide-glucosyltransferase inhibitor (P4r) and macrophages with altered ganglioside synthesis (from B4galnt1 -/- mice) showed a deficient ability to interact with Hc. Coincubation of oligo-GM1 and treatment with Cholera toxin Subunit B, which recognises the ganglioside GM1, also reduced Hc association. Although purified GM1 did not alter Hc binding, treatment with P4 significantly increased the time required for Hc binding to macrophages. The content of CD18 was displaced from lipid microdomains in B4galnt1 -/- macrophages. In addition, macrophages with reduced CD18 expression (CD18 low ) were associated with Hc at levels similar to wild-type cells. Finally, CD11b and CD18 colocalised with GM1 during Hc-macrophage interaction. Our results indicate that lipid rafts and particularly complex gangliosides that reside in lipid rafts stabilise Hc-macrophage adhesion and mediate efficient internalisation during histoplasmosis., (© 2018 John Wiley & Sons Ltd.)

Fire ants are widely studied, invasive and venomous arthropod pests. There is significant biomedical interest in immunotherapy against fire ant stings. However, mainly due to practical reasons, the physiological effects of envenomation has remained poorly characterized. The present study takes advantage of a recently-described venom protein extract to delineate the immunological pathways underlying the allergic reaction to fire ant venom toxins. Mice were injected with controlled doses of venom protein extract. Following sensitization and a second exposure, a marked footpad swelling was observed. Based on eosinophil recruitment and production of Th2 cytokines, we hereby establish that fire ant proteins per se can lead to an allergic response, which casts a new light into the mechanism of action of these toxins.

Diverse pathogenic fungi secrete extracellular vesicles (EV) that contain macromolecules, including virulence factors that can modulate the host immune response. We recently demonstrated that the binding of monoclonal antibodies (mAb) modulates how Histoplasma capsulatum load and releases its extracellular vesicles (EV). In the present paper, we addressed a concentration-dependent impact on the fungus' EV loading and release with different mAb, as well as the pathophysiological role of these EV during the host-pathogen interaction. We found that the mAbs differentially regulate EV content in concentration-dependent and independent manners. Enzymatic assays demonstrated that laccase activity in EV from H. capsulatum opsonized with 6B7 was reduced, but urease activity was not altered. The uptake of H. capsulatum by macrophages pre-treated with EV, presented an antibody concentration-dependent phenotype. The intracellular killing of yeast cells was potently inhibited in macrophages pre-treated with EV from 7B6 (non-protective) mAb-opsonized H. capsulatum and this inhibition was associated with a decrease in the reactive-oxygen species generated by these macrophages. In summary, our findings show that opsonization quantitatively and qualitatively modifies H. capsulatum EV load and secretion leading to distinct effects on the host's immune effector mechanisms, supporting the hypothesis that EV sorting and secretion are dynamic mechanisms for a fine-tuned response by fungal cells.

Flippases are responsible for the asymmetric distribution of phospholipids in biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the putative flippase Apt1 is an important regulator of polysaccharide secretion and pathogenesis in mice by unknown mechanisms. In this study, we analyzed the role of C. neoformans Apt1 in intracellular membrane architecture and synthesis of polysaccharide and lipids. Analysis of wild type (WT), apt1Δ (mutant) and apt1Δ::APT1 (complemented) strains by transmission electron microscopy revealed that deletion of APT1 resulted in the formation of irregular vacuoles. Disorganization of vacuolar membranes in apt1Δ cells was accompanied by a significant increase in the amounts of intra-vacuolar and pigment-containing vesicles. Quantitative immunogold labeling of C. neoformans cells with a monoclonal antibody raised to a major capsular component suggested impaired polysaccharide synthesis. APT1 deletion also affected synthesis of phosphatidylserine, phosphatidylethanolamine, inositolphosphoryl ceramide, glucosylceramide and ergosterylglycoside. These results reveal novel functions of Apt1 and are in agreement with the notion that this putative flippase plays an important role in the physiology of C. neoformans., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Heat shock proteins (Hsps) are highly conserved biomolecules that are constitutively expressed and generally upregulated in response to various stress conditions (biotic and abiotic). Hsps have diverse functions, categorizations, and classifications. Their adaptive expression in fungi indicates their significance in these diverse species, particularly in dimorphic pathogens. Histoplasma capsulatum and Paracoccidioides species are dimorphic fungi that are the causative agents of histoplasmosis and paracoccidioidomycosis, respectively. This minireview focuses on the pathobiology of Hsps, with particular emphasis on their roles in the morphogenesis and virulence of Histoplasma and Paracoccidioides and the potential roles of active and passive immunization against Hsps in protection against infection with these fungi., (Copyright © 2017 American Society for Microbiology.)

Aim: We investigated the involvement of the autophagy protein 7 (Atg7) in physiology and pathogenic potential of Cryptococcus neoformans., Materials & Methods: The C. neoformans gene encoding Atg7 was deleted by biolistic transformation for characterization of autophagy mechanisms, pigment formation, cell dimensions, interaction with phagocytes and pathogenic potential in vivo., Results & Conclusion: ATG7 deletion resulted in defective autophagy mechanisms, enhanced pigmentation and increased cellular size both in vitro and in vivo. The atg7Δ mutant had decreased survival in the lung of infected mice, higher susceptibility to the killing machinery of different host phagocytes and reduced ability to kill an invertebrate host. These results connect Atg7 with mechanisms of pathogenicity in the C. neoformans model.

Group V (GV) phospholipase A2 (PLA2) is a member of the family of secreted PLA2 (sPLA2) enzymes. This enzyme has been identified in several organs, including the kidney. However, the physiologic role of GV sPLA2 in the maintenance of renal function remains unclear. We used mice lacking the gene encoding GV sPLA2 (Pla2g5-/-) and wild-type breeding pairs in the experiments. Mice were individually housed in metabolic cages and 48-h urine was collected for biochemical assays. Kidney samples were evaluated for glomerular morphology, renal fibrosis, and expression/activity of the (Na+ + K+)-ATPase α1 subunit. We observed that plasma creatinine levels were increased in Pla2g5-/- mice following by a decrease in creatinine clearance. The levels of urinary protein were higher in Pla2g5-/- mice than in the control group. Markers of tubular integrity and function such as γ-glutamyl transpeptidase, lactate dehydrogenase, and sodium excretion fraction (FENa+) were also increased in Pla2g5-/- mice. The increased FENa+ observed in Pla2g5-/- mice was correlated to alterations in cortical (Na+ + K+) ATPase activity/ expression. In addition, the kidney from Pla2g5-/- mice showed accumulation of matrix in corticomedullary glomeruli and tubulointerstitial fibrosis. These data suggest GV sPLA2 is involved in the maintenance of tubular cell function and integrity, promoting sodium retention through increased cortical (Na+ + K+)-ATPase expression and activity.

Autoantibodies against the M2 receptors (M2AChR) have been associated with Dilated Cardiomyopathy (DCM). In the heart, P2×7 receptors influence electrical conduction, coronary circulation and response to ischemia. They can also trigger pro-inflammatory responses and the development of neurological, cardiac and renal disorders. Here, P2×7(-/-) mice displayed an increased heart rate and ST segment depression, but similar exercise performance when compared to wild type (WT) animals. After immunization with plasmid containing M2AChR cDNA sequence, WT mice produced anti-M2AChR antibodies, while P2×7(-/-) mice showed an attenuated production. Despite this, WT and P2×7(-/-) showed left ventricle cavity enlargement and decreased exercise tolerance. Transfer of serum from M2AChR WT immunized mice to näive recipients led to an alteration in heart shape. P2×7(-/-) mice displayed a significant increase in the frequency of spleen regulatory T cells population, which is mainly composed by the FoxP3(+)CD25(-) subset. M2AChR WT immunized mice showed an increase in IL-1β, IFNγ and IL-17 levels in the heart, while P2×7(-/-) group produced lower amounts of IL-1β and IL-17 and higher amounts of IFNγ. These results pointed to previously unnoticed roles of P2×7 in cardiovascular and immune systems, and underscored the participation of IL-17 and IFNγ in the progress of autoimmune DCM.

The probiotic yeast Saccharomyces cerevisiae var boulardii is widely used as a low cost and efficient adjuvant against gastrointestinal tract disorders such as inflammatory bowel disease and treatment of several types of diarrhea, both in humans and animals. S. boulardii exerts its protective mechanisms by binding and neutralizing enteric pathogens or their toxins, by reducing inflammation and by inducing the secretion of sIgA. Although several S. cerevisiae strains have proven probiotic potential in both humans and animals, only S. boulardii is currently licensed for use in humans. Recently, some researchers started using S. boulardii as heterologous protein expression systems. Combined with their probiotic activity, the use of these strains as prophylactic and therapeutic proteins carriers might result in a positive combined effort to fight specific diseases. Here, we provide an overview of the current use of S. cerevisiae strains as probiotics and their mechanisms of action. We also discuss their potential to produce molecules with biotherapeutic application and the advantages and hurdles of this approach. Finally, we suggest future directions and alternatives for which the combined effort of specific immunomodulatory effects of probiotic S. cerevisiae strains and ability to express desired foreign genes would find a practical application.

Cyclooxygenase (COX)-2-derived prostaglandin (PG)E(2) controls many aspects of colon cancer development, modulating from apoptosis resistance and cell proliferation to angiogenesis, invasion, and metastasis. Here, we investigated the role of different phospholipases (PL)A(2) in supplying arachidonic acid (AA) for COX-2-dependent PGE(2) generation and signaling pathways involved in activation of colon cancer cells by a physiologically relevant stimulus. To emulate the hypertonic environment found physiologically in colon, the human colon cancer cell line Caco-2 was maintained in hypertonic complete DMEM medium. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked AA release, COX-2 induction and PGE(2) generation. Selective secretory (s)PLA(2) and calcium-independent (i)PLA(2) inhibitors did not modify PGE(2) generation, while either COX-2 or cytosolic (c)PLA(2) inhibitors completely inhibited PGE(2) generation. cPLA(2)-alpha was responsible for AA supply for PGE(2) generation, but had no role in COX-2 induction. Mitogen-activated protein (MAP) kinases, ERK 1/2, p38, and JNK, participated in the signaling events that lead to PGE(2) generation by modulating AA release, but only ERK 1/2 was involved in COX-2 upregulation. Our results indicate that hypertonic stress activates PGE(2) generation by Caco-2 cells through a mechanism dependent on MAP kinase-regulated AA mobilization, increased cPLA(2)-alpha activity, and COX-2 induction., (Copyright 2009 Elsevier Ltd. All rights reserved.)