Erg6 affects membrane composition and virulence of the human fungal pathogen Cryptococcus neoformans.

Ergosterol is the most important membrane sterol in fungal cells and a component not found in the membranes of human cells. We identified the ERG6 gene in the AIDS-associated fungal pathogen, Cryptococcus neoformans, encoding the sterol C-24 methyltransferase of fungal ergosterol biosynthesis. In this work, we have explored its relationship with high-temperature growth and virulence of C. neoformans by the construction of a loss-of-function mutant. In contrast to other genes involved in ergosterol biosynthesis, C. neoformans ERG6 is not essential for growth under permissive conditions in vitro. However, the erg6 mutant displayed impaired thermotolerance and increased susceptibility to osmotic and oxidative stress, as well as to different antifungal drugs. Total lipid analysis demonstrated a decrease in the erg6Δ strain membrane ergosterol content. In addition, this mutant strain was avirulent in an invertebrate model of C. neoformans infection. C. neoformans Erg6 was cyto-localized in the endoplasmic reticulum and Golgi complex. Our results demonstrate that Erg6 is crucial for growth at high temperature and virulence, likely due to its effects on C. neoformans membrane integrity and dynamics. These pathogen-focused investigations into ergosterol biosynthetic pathway components reinforce the multiple roles of ergosterol in the response of diverse fungal species to alterations in the environment, especially that of the infected host. These studies open perspectives to understand the participation of ergosterol in mechanism of resistance to azole and polyene drugs. Observed synergistic growth defects with co-inhibition of Erg6 and other components of the ergosterol biosynthesis pathway suggests novel approaches to treatment in human fungal infections.

Mebendazole Inhibits Histoplasma capsulatum In Vitro Growth and Decreases Mitochondrion and Cytoskeleton Protein Levels.

Histoplasmosis is a frequent mycosis in people living with HIV/AIDS and other immunocompromised hosts. Histoplasmosis has high rates of mortality in these patients if treatment is unsuccessful. Itraconazole and amphotericin B are used to treat histoplasmosis; however, both antifungals have potentially severe pharmacokinetic drug interactions and toxicity. The present study determined the minimal inhibitory and fungicidal concentrations of mebendazole, a drug present in the NIH Clinical Collection, to establish whether it has fungicidal or fungistatic activity against Histoplasma capsulatum. Protein extracts from H. capsulatum yeasts, treated or not with mebendazole, were analyzed by proteomics to understand the metabolic changes driven by this benzimidazole. Mebendazole inhibited the growth of 10 H. capsulatum strains, presenting minimal inhibitory concentrations ranging from 5.0 to 0.08 µM. Proteomics revealed 30 and 18 proteins exclusively detected in untreated and mebendazole-treated H. capsulatum yeast cells, respectively. Proteins related to the tricarboxylic acid cycle, cytoskeleton, and ribosomes were highly abundant in untreated cells. Proteins related to the nitrogen, sulfur, and pyrimidine metabolisms were enriched in mebendazole-treated cells. Furthermore, mebendazole was able to inhibit the oxidative metabolism, disrupt the cytoskeleton, and decrease ribosomal proteins in H. capsulatum. These results suggest mebendazole as a drug to be repurposed for histoplasmosis treatment.

Future perspectives for cryptococcosis treatment.

INTRODUCTION: Cryptococcosis is one of the most devastating human fungal infections. Despite its impact, none of the standard antifungals were developed after 1990. New, improved, less toxic, affordable and widely available treatment is, therefore, imperative. AREAS COVERED: This review offers an insight into technological developments for cryptococcosis disclosed in patent literature. From a broad search of patent documents claiming cryptococcosis treatment and having earliest priority between 1995 and 2015, we selected and summarized compounds/molecules (i) revealed in documents disclosing in vivo activity against Cryptococcus spp. or (ii) found in the pipeline of companies that appeared as assignees in our patent search. This information was complemented with data on compounds under development for this indication from the database Integrity (Clarivate Analytics). EXPERT OPINION: This review demonstrates that drug development against cryptococcosis is discrete. However, it also shows that the existing development is not focused on a single class of molecules, but on different types of molecules with distinct fungal targets, reflecting the complexity of generating novel anti-cryptococcal tools. Given the intrinsic difficulties and high costs of drug development and the evident market failure in this field, we consider drug repurposing the most promising avenue for cryptococcosis treatment.

A Predicted Mannoprotein Participates in Cryptococcus gattii Capsular Structure.

The yeast-like pathogen Cryptococcus gattii is an etiological agent of cryptococcosis. The major cryptococcal virulence factor is the polysaccharide capsule, which is composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MPs). The GXM and GalXM polysaccharides have been extensively characterized; however, there is little information about the role of mannoproteins in capsule assembly and their participation in yeast pathogenicity. The present study characterized the function of a predicted mannoprotein from C. gattii, designated Krp1. Loss-of-function and gain-of-function mutants were generated, and phenotypes associated with the capsular architecture were evaluated. The null mutant cells were more sensitive to a cell wall stressor that disrupts beta-glucan synthesis. Also, these cells displayed increased GXM release to the culture supernatant than the wild-type strain did. The loss of Krp1 influenced cell-associated cryptococcal polysaccharide thickness and phagocytosis by J774.A1 macrophages in the early hours of interaction, but no difference in virulence in a murine model of cryptococcosis was observed. In addition, recombinant Krp1 was antigenic and differentially recognized by serum from an individual with cryptococcosis, but not with serum from an individual with candidiasis. Taken together, these results indicate that C. gattii Krp1 is important for the cell wall structure, thereby influencing capsule assembly, but is not essential for virulence in vivoIMPORTANCECryptococcus gattii has the ability to escape from the host's immune system through poorly understood mechanisms and can lead to the death of healthy individuals. The role of mannoproteins in C. gattii pathogenicity is not completely understood. The present work characterized a protein, Kpr1, that is essential for the maintenance of C. gattii main virulence factor, the polysaccharide capsule. Our data contribute to the understanding of the role of Kpr1 in capsule structuring, mainly by modulating the distribution of glucans in C. gattii cell wall.