Acanthamoeba castellanii (Ac) are ubiquitously distributed in nature, and by contaminating medical devices such as heart valves and contact lenses, they cause a broad range of clinical presentations to humans. Although several molecules have been described to play a role in Ac pathogenesis, including parasite host-tissue invasion and escaping of host-defense, little information is available on their mechanisms of secretion. Herein, we describe the molecular components secreted by Ac, under different protein availability conditions to simulate host niches. Ac extracellular vesicles (EVs) were morphologically and biochemically characterized. Dynamic light scattering analysis of Ac EVs identified polydisperse populations, which correlated to electron microscopy measurements. High-performance thin liquid chromatography of Ac EVs identified phospholipids, steryl-esters, sterol and free-fatty acid, the last two also characterized by GC-MS. Secretome composition (EVs and EVs-free supernatants) was also determined and proteins biological functions classified. In peptone-yeast-glucose (PYG) medium, a total of 179 proteins were identified (21 common proteins, 89 exclusive of EVs and 69 in EVs-free supernatant). In glucose alone, 205 proteins were identified (134 in EVs, 14 common and 57 proteins in EVs-free supernatant). From those, stress response, oxidative and protein and amino acid metabolism proteins prevailed. Qualitative differences were observed on carbohydrate metabolism enzymes from Krebs cycle and pentose phosphate shunt. Serine proteases and metalloproteinases predominated. Analysis of the cytotoxicity of Ac EVs (upon uptake) and EVs-free supernatant to epithelial and glioblastoma cells revealed a dose-dependent effect. Therefore, the Ac secretome differs depending on nutrient conditions, and is also likely to vary during infection.

Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this

The majority of therapeutic strategies for mycosis require the protracted administration of antifungals, which can result in significant toxicities and have unacceptable failure rates. Hence, there is an urgent need for the development of improved therapeutic approaches, and monoclonal antibody-based drugs are potentially a powerful alternative to standard antifungals. To develop a broad antibody-like reagent against mycosis, wheat germ agglutinin (WGA) was linked to the effector Fc region of murine IgG2a. The resultant WGA-Fc displayed high affinity to purified chitin and bound efficiently to fungal cell walls, co-localizing with chitin, in patterns ranging from circular (Histoplasma capsulatum) to punctate (Cryptococcus neoformans) to labeling at the bud sites (Candida albicans and Saccharomyces cerevisiae). WGA-Fc directly inhibited fungal growth in standard cultures. WGA-Fc opsonization increased fungal phagocytosis, as well augmented the antifungal functions by macrophages. Prophylactic administration of WGA-Fc fully protected mice against H. capsulatum, correlating with a reduction in lung, spleen and liver fungal burdens. Administration of WGA-Fc also dramatically diminished pulmonary inflammation. Hence, the opsonic activity of WGA-Fc effectively modulates fungal cell recognition and promotes the elimination of fungal pathogens. Therefore, we propose WGA-Fc as a potential “pan-fungal” therapeutic that should be further developed for use against invasive mycoses.

Cryptococcus neoformans (Cn) and Histoplasma capsulatum (Hc) co-exist in the environment and occasionally co-infect individuals, which can lead to severe disease/lethal outcomes. We investigated specific interactions between Cn-Hc to determine the impact of synchronous infection in virulence and disease. Co-infected mice had significantly higher mortality than infection with either species or acapsular Cn-Hc. Coating of Hc with cryptococcal glycans (Cn-gly) resulted in higher pulmonary fungal burden in co-infected animals relative to control. Co-cultivation or addition of Cn-gly resulted in enhanced pellicle formation with a hybrid polysaccharide matrix with higher reactivity to GXM mAbs. Transfer and incorporation of Cn polysaccharide onto Hc surface was time and temperature dependent. Cn-gly transfer altered the zeta potential of Hc and was associated with increased resistance to phagocytosis and killing by macrophages. Mice infected with Hc and subsequently injected with purified Cn-gly died significantly more rapidly than Hc alone infected, establishing the precedent that virulence factors from one fungus can enhance the virulence of unrelated species. These findings suggest a new mechanism of microbial interaction involving the transfer of virulence traits that translates into enhanced lethality during mixed fungal infections and highlights the importance of studying heterogeneous microbial populations in the setting of infection.

No abstract

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Fungal infections have increased in the last years, particularly associated to an increment in the number of immunocompromised individuals and the emergence of known or new resistant species, despite the difficulties in the often time-consuming diagnosis. The controversial efficacy of the currently available strategies for their clinical management, apart from their high toxicity and severe side effects, have renewed the interest in the research and development of new broad antifungal alternatives. These encompass vaccines and passive immunization strategies with monoclonal antibodies (mAbs), recognizing ubiquitous fungal targets, such as fungal cell wall β-1,3-glucan polysaccharides, which could be used in early therapeutic intervention without the need for the diagnosis at species-level. As additional alternatives, based on the Dectin-1 great affinity to β-1,3-glucan, our group developed broad antibody-like Dectin1-Fc(IgG)(s) from distinct subclasses (IgG2a and IgG2b) and compared their antifungal in vitro and passive immunizations in vivo performances. Dectin1-Fc(IgG2a) and Dectin1-Fc(IgG2b) demonstrated high affinity to laminarin and the fungal cell wall by ELISA, flow cytometry and microscopy. Both Dectin-1-Fc(IgG)(s) inhibited H. capsulatum and C. neoformans growth in a dose-dependent fashion. For C. albicans, such inhibitory effect was observed with concentrations as low as 0.098 and 0.049 µg/mL, respectively, which correlated with the impairment of the kinetics and lengths of germ tubes in comparison to controls. Previous opsonization with Dectin-1-Fc(IgG)(s) enhanced considerably the macrophage antifungal effector functions, increasing the fungi macrophages-interactions and significantly reducing the intraphagosome fungal survival, as lower CFUs were observed. The administration of both Dectin1-Fc(IgG)(s) reduced the fungal burden and mortality in murine histoplasmosis and candidiasis models, in accordance with previous evaluations in aspergillosis model. These results altogether strongly suggested that therapeutic interventions with Dectin-1-Fc(IgG)(s) fusion proteins could directly impact the innate immunity and disease outcome in favor of the host, by direct neutralization, opsonization, phagocytosis, and fungal elimination, providing interesting information on the potential of these new strategies for the control of invasive fungal infections.

No abstract