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.

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.

Biogenesis of Fungal Extracellular Vesicles: What Do We Know?

So far, extracellular vesicles (EVs) have been described in 15 genera of fungi. They carry molecules that contribute to the interaction of fungal cells with the host. Although the number of studies on fungal EVs has increased, the mechanisms involved in their biogenesis are still poorly understood. The current knowledge of EV biogenesis shows us that they can originate both in the cytoplasm and at the plasma membrane. In this chapter, we will focus on these two cellular sites to review what is known about the biogenesis of fungal EVs.

Identification of four compounds from the Pharmakon library with antifungal activity against Candida auris and species of Cryptococcus.

There is an urgent need to develop novel antifungals. In this study, we screened 1600 compounds for antifungal activity against Cryptococcus neoformans and Candida auris. We evaluated 4 promising compounds against 24 additional isolates of Cr. neoformans, Ca. auris, Cr. deuterogattii, and Cr. gattii. The four compounds, dequalinium chloride (DQC), bleomycin sulfate (BMS), pentamidine isethionate salt (PIS), and clioquinol (CLQ), varied in their efficacy against these pathogens but were generally more effective against cryptococci. The compounds exerted their antifungal effect via multiple mechanisms, including interference with the capsule of cryptococci and induction of hyphal-like morphology in Ca. auris. Our results indicate that DQC, BMS, PIS, and CLQ represent potential prototypes for the future development of antifungals. LAY SUMMARY: Fungal infections can be lethal and the options to fight them are scarce. We tested 1600 molecules for their ability to control the growth of two important fungal pathogens, namely Candida auris and species of Cryptococcus. Four of these compounds showed promising antifungal activities.

Fenbendazole Controls In Vitro Growth, Virulence Potential, and Animal Infection in the Cryptococcus Model.

The human diseases caused by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are associated with high indices of mortality and toxic and/or cost-prohibitive therapeutic protocols. The need for affordable antifungals to combat cryptococcal disease is unquestionable. Previous studies suggested benzimidazoles as promising anticryptococcal agents combining low cost and high antifungal efficacy, but their therapeutic potential has not been demonstrated so far. In this study, we investigated the antifungal potential of fenbendazole, the most effective anticryptococcal benzimidazole. Fenbendazole was inhibitory against 17 different isolates of C. neoformans and C. gattii at a low concentration. The mechanism of anticryptococcal activity of fenbendazole involved microtubule disorganization, as previously described for human parasites. In combination with fenbendazole, the concentrations of the standard antifungal amphotericin B required to control cryptococcal growth were lower than those required when this antifungal was used alone. Fenbendazole was not toxic to mammalian cells. During macrophage infection, the anticryptococcal effects of fenbendazole included inhibition of intracellular proliferation rates and reduced phagocytic escape through vomocytosis. Fenbendazole deeply affected the cryptococcal capsule. In a mouse model of cryptococcosis, the efficacy of fenbendazole to control animal mortality was similar to that observed for amphotericin B. These results indicate that fenbendazole is a promising candidate for the future development of an efficient and affordable therapeutic tool to combat cryptococcosis.

Analysis of Cryptococcus Extracellular Vesicles.

Extracellular vesicles (EVs) are produced by all domains of life. In fungal pathogens, they participate in virulence mechanisms and/or induce protective immunity, depending on the pathogenic species. EVs produced by pathogenic members of the Cryptococcus genus mediate virulence, antifungal resistance, as well as humoral and cell-mediated immunity. The isolation of cryptococcal EVs has been laborious and time-consuming for years. In this chapter, we detail a fast protocol for the isolation and analysis of EVs produced by members of the Cryptococcus genus.

Morphological and pathogenic investigation of the emerging fungal threat Emergomyces africanus.

Emergomyces africanus is a highly fatal fungal pathogen affecting individuals with advanced HIV disease. Molecular patterns and ultrastructural aspects of E. africanus are unknown, and pathogenic models have not been investigated in detail. Since the cell wall of fungi is a determinant for interaction with the host and antifungal development, we characterized the ultrastructural aspects of E. africanus and the general properties of cell wall components under different conditions of growth in vitro and in vivo. We also tested the pathogenic potential of E. africanus in a Galleria mellonella model of infection. Transmission electron microscopy revealed the common intracellular, ultrastructural features of fungi in association with a thick cell wall. Scanning electron microscopy revealed a smooth cell surface, with no apparent decorative structures. Yeast cultures of E. africanus showed the distribution of chitin, chitooligomers, and mannoproteins commonly observed in fungi. However, in mixed microenvironments containing yeast and filamenting forms of E. africanus, the detection of chitooligomers was increased in comparison with isolated yeast cells, while the detection of these components in filamenting forms was markedly reduced. These observations were suggestive of the ability of E. africanus to change its cell wall composition in response to different microenvironments. Although E. africanus was unable to kill G. mellonella, this infection model allowed us to isolate infected hemocytes for further analysis of mannoproteins, chitin, and chitooligomers. Once again, the detection of E. africanus chitooligomers was markedly increased. These results reveal previously unknown ultrastructural features of E. africanus and suggest a high plasticity in the cell wall of this lethal pathogen. IMPORTANCE: The epidemiology of fungal infections is very dynamic, and novel health emergencies are hard to predict. New fungal pathogens have been continuously emerging for the last few decades, and Emergomyces africanus is one of these threats to human health. This complex scenario points to the need for generating knowledge about emerging pathogens so that new therapeutic strategies can be designed. In this study, we characterized the general cellular and pathogenic properties of the emerging fungal pathogen E. africanus. Our results reveal that E. africanus manifests some of the typical properties of fungal cells but also exhibits some unique characteristics that might be helpful for the future development of therapeutic strategies.

Proteomics reveals that the antifungal activity of fenbendazole against Cryptococcus neoformans requires protein kinases.

Cryptococcus neoformans is responsible for over 100 000 deaths annually, and the treatment of this fungal disease is expensive and not consistently effective. Unveiling new therapeutic avenues is crucial. Previous studies have suggested that the anthelmintic drug fenbendazole is an affordable and nontoxic candidate to combat cryptococcosis. However, its mechanism of anticryptococcal activity has been only superficially investigated. In this study, we examined the global cellular response of C. neoformans to fenbendazole using a proteomic approach (data are available via ProteomeXchange with identifier PXD047041). Fenbendazole treatment mostly impacted the abundance of proteins related to metabolic pathways, RNA processing, and intracellular traffic. Protein kinases, in particular, were significantly affected by fenbendazole treatment. Experimental validation of the proteomics data using a collection of C. neoformans mutants led to the identification of critical roles of five protein kinases in fenbendazole's antifungal activity. In fact, mutants lacking the expression of genes encoding Chk1, Tco2, Tco3, Bub1, and Sch9 kinases demonstrated greater resistance to fenbendazole compared to wild-type cells. In combination with the standard antifungal drug amphotericin B, fenbendazole reduced the cryptococcal burden in mice. These findings not only contribute to the elucidation of fenbendazole's mode of action but also support its use in combination therapy with amphotericin B. In conclusion, our data suggest that fenbendazole holds promise for further development as an anticryptococcal agent.

Screening of the Pandemic Response Box Reveals an Association between Antifungal Effects of MMV1593537 and the Cell Wall of Cryptococcus neoformans, Cryptococcus deuterogattii, and Candida auris.

There is an urgent unmet need for novel antifungals. In this study, we searched for novel antifungal activities in the Pandemic Response Box, a collection of 400 structurally diverse compounds in various phases of drug discovery. We identified five molecules which could control the growth of Cryptococcus neoformans, Cryptococcus deuterogattii, and the emerging global threat Candida auris. After eliminating compounds which demonstrated paradoxical antifungal effects or toxicity to mammalian macrophages, we selected compound MMV1593537 as a nontoxic, fungicidal molecule for further characterization of antifungal activity. Scanning electron microscopy revealed that MMV1593537 affected cellular division in all three pathogens. In Cryptococcus, MMV1593537 caused a reduction in capsular dimensions. Treatment with MMV1593537 resulted in increased detection of cell wall chitooligomers in these three species. Since chitooligomers are products of the enzymatic hydrolysis of chitin, we investigated whether surface chitinase activity was altered in response to MMV1593537 exposure. We observed peaks of enzyme activity in C. neoformans and C. deuterogattii in response to MMV1593537. We did not detect any surface chitinase activity in C. auris. Our results suggest that MMV1593537 is a promising, nontoxic fungicide whose mechanism of action, at least in Cryptococcus spp, requires chitinase-mediated hydrolysis of chitin. IMPORTANCE The development of novel antifungals is a matter of urgency. In this study, we evaluated antifungal activities in a collection of 400 molecules, using highly lethal fungal pathogens as targets. One of these molecules, namely, MMV1593537, was not toxic to host cells and controlled the growth of isolates of Cryptococcus neoformans, C. deuterogattii, C. gattii, Candida auris, C. albicans, C. parapsilosis, and C. krusei. We tested the mechanisms of antifungal action of MMV1593537 in the Cryptococcus and C. auris models and concluded that the compound affects the cell wall, a structure which is essential for fungal life. At least in Cryptococcus, this effect involved chitinase, an enzyme which is required for remodeling the cell wall. Our results suggest that MMV1593537 is a candidate for future antifungal development.

Analysis of Cryptococcal Extracellular Vesicles: Experimental Approaches for Studying Their Diversity Among Multiple Isolates, Kinetics of Production, Methods of Separation, and Detection in Cultures of Titan Cells.

Extracellular vesicles (EVs) produced by members of the Cryptococcus genus are associated with fundamental processes of fungal physiology and virulence. However, several questions about the properties of cryptococcal EVs remain unanswered, mostly because of technical limitations. We recently described a fast and efficient protocol of high-yield EV isolation from solid medium. In this study, we aimed at using the solid medium protocol to address some of the open questions about EVs, including the kinetics of EV production, the diversity of EVs produced by multiple isolates under different culture conditions, the separation of vesicles in a density gradient followed by the recovery of functional EVs, the direct detection of EVs in culture supernatants, and the production of vesicles in solid cultures of Titan cells. Our results indicate that the production of EVs is directly impacted by the culture medium and time of growth, resulting in variable detection of EVs per cell and a peak of EV detection at 24 h of growth. Nanoparticle tracking analysis (NTA) of EV samples revealed that multiple isolates produce vesicles with variable properties, including particles of diverging dimensions. EVs were produced in the solid medium in amounts that were separated on a centrifugation density gradient, resulting in the recovery of functional EVs containing the major cryptococcal capsular antigen. We also optimized the solid medium protocol for induction of the formation of Titan cells, and analyzed the production of EVs by NTA and transmission electron microscopy. This analysis confirmed that EVs were isolated from solid cultures of cryptococcal enlarged cells. With these approaches, we expect to implement simple methods that will facilitate the analysis of EVs produced by fungal cells. IMPORTANCE Fungal extracellular vesicles (EVs) are considered to be important players in the biology of fungal pathogens. However, the limitations in the methodological approaches to studying fungal EVs impair the expansion of knowledge in this field. In the present study, we used the Cryptococcus genus as a model for the study of EVs. We explored the simplification of protocols for EV analysis, which helped us to address some important, but still unanswered, questions about fungal EVs.

Pathogenic Delivery: The Biological Roles of Cryptococcal Extracellular Vesicles.

Extracellular vesicles (EVs) are produced by all domains of life. In fungi, these structures were first described in Cryptococcus neoformans and, since then, they were characterized in several pathogenic and non-pathogenic fungal species. Cryptococcal EVs participate in the export of virulence factors that directly impact the Cryptococcus-host interaction. Our knowledge of the biogenesis and pathogenic roles of Cryptococcus EVs is still limited, but recent methodological and scientific advances have improved our understanding of how cryptococcal EVs participate in both physiological and pathogenic events. In this review, we will discuss the importance of cryptococcal EVs, including early historical studies suggesting their existence in Cryptococcus, their putative mechanisms of biogenesis, methods of isolation, and possible roles in the interaction with host cells.