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Cryptococcosis is a life-threatening fungal infection, and its current treatment is toxic and subject to resistance. Drug repurposing represents an interesting approach to find drugs to reduce the toxicity of antifungals. In this study, we evaluated the combination of N-acetylcysteine (NAC) with amphotericin B (AMB) for the treatment of cryptococcosis. We examined the effects of NAC on fungal morphophysiology and on the macrophage fungicidal activity 3 and 24 hours post inoculation. The therapeutic effects of NAC combination with AMB were investigated in a murine model with daily treatments regimens. NAC alone reduced the oxidative burst generated by AMB in yeast cells, but did not inhibit fungal growth. The combination NAC + AMB decreased capsule size, zeta potential, superoxide dismutase activity and lipid peroxidation. In macrophage assays, NAC + AMB did not influence the phagocytosis, but induced fungal killing with different levels of oxidative bursts when compared to AMB alone: there was an increased reactive oxygen species (ROS) after 3 hours and reduced levels after 24 hours. By contrast, ROS remained elevated when AMB was tested alone, demonstrating that NAC reduced AMB oxidative effects without influencing its antifungal activity. Uninfected mice treated with NAC + AMB had lower concentrations of serum creatinine and glutamate-pyruvate transaminase in comparison to AMB. The combination of NAC + AMB was far better than AMB alone in increasing survival and reducing morbidity in murine-induced cryptococcosis, leading to reduced fungal burden in lungs and brain and also lower concentrations of pro-inflammatory cytokines in the lungs. In conclusion, NAC + AMB may represent an alternative adjuvant for the treatment of cryptococcosis.

Cryptococcus gattii, an environmental yeast isolated from plants, is one of the agents of cryptococcosis. Here, we aimed to develop a plant model to study C. gattii–plant interaction, since it is unclear how it affects the plant and the yeast. We tested three inoculation methods (scarification, infiltration, and abrasion) in three plant species: Arabidopsis thaliana, Nicotiana tabacum, and N. benthamiana. Cryptococcus gattii was able to grow in all three models, with a peak of yeast cell burden after 7 days, without any pathological effects. Furthermore, the fungal burden was reduced later, confirming that C. gattii is not a phytopathogen. Cryptococcus gattii proliferation was higher in N. benthamiana, which presented an increase in the hydrogen peroxide content, antioxidant system activity, and indoleacetic acid (IAA) production. Cryptococcus gattii colonies recovered from N. benthamiana presented lower ergosterol content, reduced capsule, and increased growth rate in vitro and inside macrophages. In vitro, IAA altered C. gattii morphology and susceptibility to antifungal drugs. We hypothesize that C. gattii can temporarily colonize plant living tissues, which can be a potential reservoir of yeast virulence, with further dissemination to the environment, birds, and mammals. In conclusion, N. benthamiana is suitable for studying C. gattii–plant interaction.

[Background] The fungal cell wall is an essential and robust external structure that protects the cell from the environment. It is mainly composed of polysaccharides with different functions, some of which are necessary for cell integrity. Thus, the process of fractionation and analysis of cell wall polysaccharides is useful for studying the function and relevance of each polysaccharide, as well as for developing a variety of practical and commercial applications. This method can be used to study the mechanisms that regulate cell morphogenesis and integrity, giving rise to information that could be applied in the design of new antifungal drugs. Nonetheless, for this method to be reliable, the availability of trustworthy commercial recombinant cell wall degrading enzymes with non‑contaminating activities is vital., [Results] Here we examined the efficiency and reproducibility of 12 recombinant endo‑β(1,3)‑d‑glucanases for specifically degrading the cell wall β(1,3)‑d‑glucan by using a fast and reliable protocol of fractionation and analysis of the fission yeast cell wall. This protocol combines enzymatic and chemical degradation to fractionate the cell wall into the four main polymers: galactomannoproteins, α‑glucan, β(1,3)‑d‑glucan and β(1,6)‑d‑glucan. We found that the GH16 endo‑β(1,3)‑d‑glucanase PfLam16A from Pyrococcus furiosus was able to completely and reproducibly degrade β(1,3)‑d‑glucan without causing the release of other polymers. The cell wall degradation caused by PfLam16A was similar to that of Quantazyme, a recombinant endo‑β(1,3)‑d‑glucanase no longer commercially available. Moreover, other recombinant β(1,3)‑d‑glucanases caused either incomplete or excessive degradation, suggesting deficient access to the substrate or release of other polysaccharides, [Conclusions] The discovery of a reliable and efficient recombinant endo‑β(1,3)‑d‑glucanase, capable of replacing the previously mentioned enzyme, will be useful for carrying out studies requiring the digestion of the fungal cell wall β(1,3)‑d‑glucan. This new commercial endo‑β(1,3)‑d‑glucanase will allow the study of the cell wall composition under different conditions, along the cell cycle, in response to environmental changes or in cell wall mutants. Furthermore, this enzyme will also be greatly valuable for othern practical and commercial applications such as genome research, chromosomes extraction, cell transformation, protoplast formation, cell fusion, cell disruption, industrial processes and studies of new antifungals that specifically target cell wall synthesis, This work was supported by grants PGC2018-098924-B-I00 (Spanish Ministry of Science and Innovation, MICINN , Spain; and the European Regional Developmental Fund, FEDER, EU), and CSI150P20 and “Escalera de Excelencia” CLU-2017-03 (Regional Government of Castile and Leon, JCYL, Spain; and the European Regional Developmental Fund, FEDER, EU).

[Background] In our previous study the synergism of four combinations of Zuccagnia punctata (ZpE) and Larrea nitida (LnE) exudates with the reliable statistical-based MixLow method was assessed, and the markers of the most anti-C. albicans synergistic ZpE-LnE bi-herbal combination were quantified according to European Medicines Agency (EMA)., [Purpose] To study the mechanisms of action as well as the cytotoxic properties of the ZpE-LnE most synergistic combination found in the previous work., [Materials and methods] Minimum Fungicidal Concentration (MFC) and rate of killing of ZpE-LnE were assessed with the microbroth dilution and the time-kill assays respectively. Morphological alterations were observed with both confocal and fluorescence microscopy on the yeast Schizosaccharomyces pombe. The ergosterol exogenous assay, the quantification of ergosterol, the sorbitol as well as glucan synthase (GS) and chitin synthase (ChS) assays were used to detect the effects on the fungal membrane and cell wall respectively. The capacity of ZpE-LnE of inhibiting Candida virulence factors was assessed with previously reported methods. The effect of ZpE-LnE and of ZpE or LnE alone on cell viability was determined on human hepatoma cells line Huh7., [Results] ZpE-Ln E was fungicidal killing C. albicans in a shorter time than amphotericin B and produced malformations in S. pombe cells. ZpE-LnE showed to bind to ergosterol but not to inhibit any step of the ergosterol biosynthesis. ZpE-LnE showed a low or moderate capacity of inhibiting GS and ChS. Regarding inhibition of virulence factors, ZpE-LnE significantly decreased the capacity of adhesion to eukaryotic buccal epithelial cells (BECs), did not inhibit the germ tube formation and inhibited the secretion of phospholipases and proteinases but not of haemolysins. ZpE-LnE demonstrated very low toxicity on Huh7 cells, much lower than that each extract alone., [Conclusion] The fungicidal properties of ZpE-LnE against C. albicans, its dual mechanism of action targeting the fungal membrane's ergosterol as well as the cell wall, its capacity of inhibiting several important virulence factors added to its low toxicity, make ZpE-LnE a good candidate for the development of a new antifungal bi-Herbal Medicinal Product., S.Z. and M.S. acknowledge Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), PICT2014-1170 and PICT2016-1833 and National University of Rosario, Argentina (UNR) for funds. J.C.R. acknowledges MINECO (BIO2015-69958-P) and Junta de Castilla y León (CSI068P17), Spain for funds. A.D.Q. acknowledges ANPCyT (PICT2013-1440) and UNR for funds. E.B. acknowledges Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) for a doctoral fellowship. M.S., L.S. and A.D.Q. are members of the CONICET Researcher career; M.S., L.S., and E.B. belong to the teaching staff of Pharmacognosy area and M.S. belongs to the teaching staff of Mycological Area Faculty of Biochemical and Pharmaceutical Sciences, UNR.

The effects of fire on the vegetation vary across continents. However, in Neotropical fire-prone grasslands, the relationship between fire and seed germination is still poorly understood, while their regeneration, especially after strong anthropogenic disturbance, is challenging for their conservation. In the present study, we assessed diversity of germination strategies in 15 dominant herbaceous species from Neotropical altitudinal grasslands (locally known as campos rupestres). We exposed seeds to several fire-related treatments. We also compared germination between regularly and post-fire fruiting species. Finally, we investigated the diversity of dormancy classes aiming at better understanding the biogeography and phylogeny of seed dormancy. Germination strategies varied among families. Velloziaceae and Xyridaceae produced non-dormant, fast-germinating seeds. Cyperaceae and Poaceae showed an extremely low or null germination due to a high proportion of unviable or embryo-less seeds. The seeds of campo rupestre grasslands are fire resistant, but there is no evidence that fire triggers germination in this fire-prone ecosystem. Although heat and charred wood did not promote germination, smoke enhanced germination in one grass species and decreased the mean germination time and improved synchrony in Xyridaceae and Velloziaceae. Fire had a positive effect on post-fire regeneration by stimulating fruit set in some Cyperaceae and Poaceae species. These species produced faster germinating seeds with higher germination percentage and synchrony compared to regularly fruiting Cyperaceae and Poaceae species. This strategy of dispersion and regeneration seems to be an alternative to the production of seeds with germination triggered by fire. Physiological dormancy is reported for the first time in several clades of Neotropical plants. Our data help advance the knowledge on the role of fire in the regeneration of Neotropical grasslands.

Cryptococcus gattii and Cryptococcus neoformans are environmental fungi that cause cryptococcosis, which is usually treated with amphotericin B and fluconazole. However, therapeutic failure is increasing because of the emergence of resistant strains. Because these species are constantly isolated from vegetal materials and the usage of agrochemicals is growing, we postulate that pesticides could be responsible for the altered susceptibility of these fungi to clinical drugs. Therefore, we evaluated the influence of the pesticide tebuconazole on the susceptibility to clinical drugs, morphophysiology, and virulence of C. gattii and C. neoformans strains. The results showed that tebuconazole exposure caused in vitro cross-resistance (CR) between the agrochemical and clinical azoles (fluconazole, itraconazole, and ravuconazole) but not with amphotericin B. In some strains, CR was observed even after the exposure ceased. Further, tebuconazole exposure changed the morphology, including formation of pseudohyphae in C. neoformans H99, and the surface charge of the cells. Although the virulence of both species previously exposed to tebuconazole was decreased in mice, the tebuconazole-exposed colonies recovered from the lungs were more resistant to azole drugs than the nonexposed cells. This in vivo CR was confirmed when fluconazole was not able to reduce the fungal burden in the lungs of mice. The tolerance to azoles could be due to increased expression of the ERG11 gene in both species and of efflux pump genes ( AFR1 and MDR1 ) in C. neoformans . Our study data support the idea that agrochemical usage can significantly affect human pathogens present in the environment by affecting their resistance to clinical drugs.

In the past three decades invasive mycoses have globally emerged as a persistent source of healthcare-associated infections. The cell wall surrounding the fungal cell opposes the turgor pressure that otherwise could produce cell lysis. Thus, the cell wall is essential for maintaining fungal cell shape and integrity. Given that this structure is absent in host mammalian cells, it stands as an important target when developing selective compounds for the treatment of fungal infections. Consequently, treatment with echinocandins, a family of antifungal agents that specifically inhibits the biosynthesis of cell wall (1-3)β-D-glucan, has been established as an alternative and effective antifungal therapy. However, the existence of many pathogenic fungi resistant to single or multiple antifungal families, together with the limited arsenal of available antifungal compounds, critically affects the effectiveness of treatments against these life-threatening infections. Thus, new antifungal therapies are required. Here we review the fungal cell wall and its relevance in biotechnology as a target for the development of new antifungal compounds, disclosing the most promising cell wall inhibitors that are currently in experimental or clinical development for the treatment of some invasive mycoses., We thank Emma Keck for language revision. J.C.G.C acknowledges support from a Postdoctoral Contract granted by the University of Salamanca, Spain. This publication was supported by the grants BIO2015-69958-P from the Ministry of Science, Innovation and Universities (MICIU) (http://www.ciencia.gob.es/) and grants CSI068P17 and Escalera de Excelencia CLU-2017-03 from the Regional Government of Castile and Leon (JCyL/FEDER) and the European Regional Development Fund (ERDF) (https://www.jcyl.es/).

Os campos rupestres da Cadeia do Espinhaco destacam-se por sua grande biodiversidade e alta proporcao de especies endemicas. Estudos sobre a restauracao ecologica desse ecossistema sao urgentemente necessarios, tendo em vista sua resiliencia extremamente baixa e especial vulnerabilidade frente a degradacao do solo. Investigamos experimentalmente o desempenho de sobrevivencia, crescimento em altura e fenologia reprodutiva de mudas de oito especies nativas de campo rupestre plantadas em areas degradadas quartziticas. O plantio foi feito em 2011, e as mudas foram monitoradas por 12 a 18 meses. Todas as especies apresentaram elevada sobrevivencia (90-100%) durante o periodo considerado e crescimento significativo em altura, o que indicou o estabelecimento bem-sucedido e adaptacao as condicoes rigorosas encontradas nessas areas degradadas. Alem disso, quatro especies arbustivas completaram o ciclo reprodutivo, atingindo a dispersao de sementes com aproximadamente dois anos de idade. Os resultados indicam que o plantio de mudas de especies nativas deve ser considerado como uma importante ferramenta na restauracao de areas degradadas quartziticas em campos rupestres. Palavras-chave: Cerrado, crescimento de mudas, facilitacao, restauracao ecologica, Serra do Cipo, solos quartziticos.