Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, hard to reproduce, and produce low yields. In an attempt to improve the protocols used for EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii and Cryptococcus neoformans on solid media. Nanoparticle tracking analysis in combination with transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in solid media. The properties of cryptococcal vesicles varied according to the culture medium used and the EV-producing species. EV detection was reproduced with an acapsular mutant of C. neoformans, as well as with isolates of Candida albicans, Histoplasma capsulatum, and Saccharomyces cerevisiae. Cryptococcal EVs produced in solid media were biologically active and contained regular vesicular components, including the major polysaccharide glucuronoxylomannan (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular methods used for the isolation of fungal EVs, we asked if it would be applicable to address fundamental questions related to cryptococcal secretion. On the basis that polysaccharide export in Cryptococcus requires highly organized membrane traffic culminating with EV release, we analyzed the participation of a putative scramblase (Aim25; CNBG_3981) in EV-mediated GXM export and capsule formation in C. gattii. EVs from a C. gattii aim25Δ strain differed from those obtained from wild-type (WT) cells in physical-chemical properties and cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, EVs obtained from the aim25Δ mutant were more efficiently used as a source of capsular polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and increased capsular dimensions. These results associate the description of a novel protocol for the isolation of fungal EVs with the identification of a previously unknown regulator of polysaccharide release. IMPORTANCE Extracellular vesicles (EVs) are fundamental components of the physiology of cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of transfer of antifungal resistance and virulence, as well as in immune stimulation and prion transmission. However, studies on the functions of fungal EVs are still limited by the lack of efficient methods for isolation of these compartments. In this study, we developed an alternative protocol for isolation of fungal EVs and demonstrated an application of this new methodology in the study of the physiology of the fungal pathogen Cryptococcus gattii. Our results describe a fast and reliable method for the study of fungal EVs and reveal the participation of scramblase, a phospholipid-translocating enzyme, in secretory processes of C. gattii.

No abstract

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects millions of individuals around the world. Although it has been known for more than a century, the study of T. cruzi has been a challenge, particularly due to the scarcity of tools for genome inquiries. Recently, strategies have been described allowing gene disruption in T. cruzi by the CRISPR/Cas9 nuclease system. Although these strategies demonstrated success in deleting some genes, several aspects could be improved to increase the efficiency of the CRISPR/Cas9 system in T. cruzi. Here, we report a strategy, based on adaptations and improvements of the two previously described systems, that results in efficient gene disruption that can be applied to any target, including the study of essential genes.

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.

No abstract

A heteropolysaccharide was isolated by cold aqueous extraction from edible mushroom Pleurotus eryngii ("King Oyster") basidiocarps and its biological properties were evaluated. Structural assignments were carried out using mono- and bidimensional NMR spectroscopy, monosaccharide composition, and methylation analyses. A mannogalactan having a main chain of (1→6)-linked α-d-galactopyranosyl and 3-O-methyl-α-d-galactopyranosyl residues, both partially substituted at OH-2 by β-d-Manp (MG-Pe) single-unit was found. Biological effects of mannogalactan from P. eryngii (MG-Pe) were tested against murine melanoma cells. MG-Pe was non-cytotoxic, but reduced in vitro melanoma cells invasion. Also, 50mg/kg MG-Pe administration to melanoma-bearing C57BL/6 mice up to 10days decreased in 60% the tumor volume compared to control. Additionally, no changes were observed when biochemical profile, complete blood cells count (CBC), organs, and body weight were analyzed. Mg-Pe was shown to be a promising anti-melanoma molecule capable of switching melanoma cells to a non-invasive phenotype with no toxicity to melanoma-bearing mice.

22Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, 23 hard to reproduce, and produce low yields. In an attempt to improve the protocols used for 24 EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii 25 and C. neoformans on solid media. Nanoparticle tracking analysis in combination with 26 transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in 27 solid media. These results were reproduced with an acapsular mutant of C. neoformans, as 28 well as with isolates of Candida albicans, Histoplasma capsulatum, and Saccharomyces 29 cerevisiae. Cryptococcal EVs produced in solid media were biologically active and contained 30 regular vesicular components, including the major polysaccharide glucuronoxylomannan 31 (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular 32 methods used for the isolation of fungal EVs, we asked if it would be applicable to address 33 fundamental questions related to cryptococcal secretion. On the basis that polysaccharide 34 export in Cryptococcus requires highly organized membrane traffic culminating with EV 35 release, we analyzed the participation of a putative scramblase (Aim25, CNBG_3981) in EV-36 mediated GXM export and capsule formation in C. gattii. EVs from a C. gattii aim25 strain 37 differed from those obtained from wild-type (WT) cells in physical-chemical properties and 38 cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, 39EVs obtained from the aim25 mutant were more efficiently used as a source of capsular 40 polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and 41 increased capsular dimensions. These results associate the description of a novel protocol 42 for the isolation of fungal EVs with the identification of a previously unknown regulator of 43 polysaccharide release. 44 45 IMPORTANCE. Extracellular vesicles (EVs) are fundamental components of the physiology of 46 cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of 47 results describe a fast and reliable method for the study of fungal EVs and reveal the 53 participation of scramblase, a phospholipid translocating enzyme, in secretory processes of 54 C. gattii. 55 56 57

Dengue is endemic in Brazil. The dengue surveillance system's reliance on passive reporting may underestimate disease incidence and cannot detect asymptomatic/pauci-symptomatic cases. In this 3-year prospective cohort study (NCT01391819) in 5-to 13-year-old children from nine schools in Fortaleza (N = 2,117), we assessed dengue virus (DENV) infection seroprevalence by IgG indirect ELISA at yearly visits and disease incidence through active and enhanced passive surveillance. Real-time quantitative polymerase chain reaction (RT-qPCR) and DENV IgM/IgG capture ELISA were used for diagnosis. We further characterized confirmed and probable cases with a plaque reduction neutralization test. At enrollment, 54.1% (95% CI: 46.6, 61.4) of children were DENV IgG positive. The annual incidence of laboratory-confirmed symptomatic dengue cases was 11.0 (95% CI: 7.3, 14.7), 18.1 (10.4, 25.7), and 10.2 (0.7, 19.7), and of laboratory-confirmed or probable dengue cases with neutralizing antibody profile evocative of dengue exposure was 13.