Biology subjects

Rodrigues, M. L.

Publications and source records attributed to Rodrigues, M. L..

9 recordsLinked to original sources

bioRxiv2024

Extracellular vesicles produced during fungal infection in humans are immunologically active

Of the known 1.5 million fungal species, Candida spp., Cryptococcus spp., and Paracoccidioides spp. are the main pathogenic species causing serious diseases with almost two million annual deaths. The diagnosis and treatment of fungal infections are challenging since of the limited access to diagnostic tests and the emergence of antifungal resistance. Extracellular vesicles (EVs) promote the interactions of fungal cells with other organisms and play an important role in the pathogen-host relationship. Owing to the complexity of fungal EVs and the lack of clinical studies on their roles in human infections, we studied the EVs from the serum and urine samples of patients with fungal infections caused by Candida albicans, Cryptococcus neoformans, and Paracoccidioides brasiliensis and determined their roles. Steroids, sphingolipids, and fatty acids were identified as the main secondary metabolites via mass spectrometry analysis. We asked whether these metabolites in EVs could play roles in modulating the host immune response. Our findings revealed the polarization of the proinflammatory profile in murine and human macrophages, with the increased production of cytokines, such as the tumor necrosis factor-, interferon-{gamma}, and interleukin-6, and an increased expression of the inducible nitric oxide synthase gene, a M1 response marker. Therefore, circulating EVs from patients with fungal infections are likely involved in the disease pathophysiology. Our findings provide insights into the roles of EVs in fungal infections in clinical samples and in vitro, suggesting possible targets for systemic mycoses therapy. Significance StatementFungal infections cause approximately 1.6 million deaths annually. Due to therapeutic and diagnostic limitations, it is mandatory to understand and develop new immunological interventions. Despite several in vitro studies on the production of extracellular vesicles (EVs) from fungal pathogens, this study is a pioneer in the identification and characterization of EVs in the course of fungal infection in humans. Our group demonstrated the presence of EVs in clinical samples from patients diagnosed with candidiasis, cryptococcosis, and paracoccidioidomycosis, as well as the EVs interaction produced by host and fungal pathogen with the immune system, resulting in relationships that may be beneficial for the progression or elimination of fungal disease.

microbiology↗

bioRxiv2021

Monoclonal antibodies against cell wall chitooligomers as accessory tools for the control of cryptococcosis

Therapeutic strategies against systemic mycoses can involve antifungal resistance and significant toxicity. Thus, novel therapeutic approaches to fight fungal infections are urgent. Monoclonal antibodies (mAbs) are promising tools to fight systemic mycoses. In this study, mAbs of the IgM isotype were developed against chitin oligomers. Chitooligomers derive from chitin, an essential component of the fungal cell wall and a promising therapeutic target, as it is not synthesized by humans or animals. Surface plasmon resonance (SPR) assays and cell-binding tests showed that the mAbs recognizing chitooligomers have high affinity and specificity for the chitin derivatives. In vitro tests showed that the chitooligomer mAbs increased the fungicidal capacity of amphotericin B against Cryptococcus neoformans. The chitooligomer-binding mAbs interfered with two essential properties related to cryptococcal pathogenesis: biofilm formation and melanin production. In a murine model of C. neoformans infection, the combined administration of the chitooligomer-binding mAb and subinhibitory doses of amphotericin B promoted disease control. The data obtained in this study support the hypothesis that chitooligomer antibodies are of great potential as accessory tools in the control of cryptococcosis.

microbiology↗

bioRxiv2021

POPULATION GENOMIC ANALYSIS OF CRYPTOCOCCUS BRAZILIAN STRAINS REVEALS AN AFRICAN TYPE SUBCLADES DISTRIBUTION

The genomes of a large number of Cryptococcus neoformans isolates have been sequenced and analyzed in recent years. These genomes have been used to understand the global population structure of this opportunistic pathogen. However, only a small number of South American isolates have been considered in these studies, and the population structure of C. neoformans in this part of the world remains elusive. Here, we analyzed the genomic sequences of 53 Brazilian Cryptococcus isolates and deciphered the C. neoformans population structure in this country. Our data reveal an African-like structure that suggested repeated intercontinental transports from Africa to South America. We also identified a mutator phenotype in one VNBII Brazilian isolate, exemplifying how fast-evolving isolates can shape the Cryptococcus population structure. Finally, phenotypic analyses revealed wide diversity but not lineage specificity in the expression of classical virulence traits within the set of isolates.

genomics↗

bioRxiv2021

Extracellular vesicles regulate yeast growth, biofilm formation, and yeast-to-hypha differentiation in Candida albicans

The ability to undergo morphological changes during adaptation to distinct environments is exploited by Candida albicans and has a direct impact on virulence. In this study, we investigated the influence of fungal extracellular vesicles (EVs) during yeast growth, biofilm formation, and morphogenesis in C. albicans. Addition of C. albicans EVs (Ca EVs) to the culture medium positively affected yeast growth. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated that Ca EVs inhibited biofilm formation by C. albicans in vitro. By time-lapse microscopy and SEM, we showed that Ca EV-treatment stops filamentation promoting pseudohyphae formation with multiple sites for yeast budding. The ability of Ca EVs to regulate dimorphism was further compared to EVs isolated from different C. albicans strains, Saccharomyces cerevisiae, and Histoplasma capsulatum. Ca EVs from distinct strains robustly inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 hours. A minor inhibitory effect was promoted by EVs from S. cerevisiae and H. capsulatum only after 24 hours of incubation. The inhibitory effect of Ca EVs was promoted by a combination of lipid compounds identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably, Ca EVs were also able to reverse filamentation, transforming hyphal growth to yeast forms. Transcriptomic analysis demonstrated that treatment with Ca EVs modified the expression of more than 300 genes. The most effectively upregulated pathways were related to DNA metabolism. The downregulated genes were mostly associated with extracellular and adhesion proteins. Finally, yeast cells treated with Ca EVs for 24 hours lost their agar invasive ability and were avirulent when inoculated in Galleria mellonella larvae. In summary, our results indicate that fungal EVs can profoundly modify C. albicans growth and regulate yeast-to-hypha differentiation inhibiting biofilm formation and virulence.

microbiology↗

bioRxiv2020

Phytotoxic tryptoquialanines produced in vivo by Penicillium digitatum are exported in extracellular vesicles

Penicillium digitatum is the most aggressive pathogen of citrus fruits. Tryptoquialanines are major indole alkaloids produced by P. digitatum. It is unknown if tryptoquialanines are involved in the damage of citrus fruits caused by P. digitatum. To investigate the pathogenic roles of tryptoquialanines, we initially asked if tryptoquialanines could affect the germination of Citrus sinensis seeds. Exposure of the citrus seeds to tryptoquialanine A resulted in a complete inhibition of germination and an altered metabolic response. Since this phytotoxic effect requires the extracellular export of tryptoquialanine A, we investigated the mechanisms of extracellular delivery of this alkaloid in P. digitatum. We detected extracellular vesicles (EVs) released by P. digitatum both in culture and during infection of citrus fruits. Compositional analysis of EVs produced during infection revealed the presence of a complex cargo, which included tryptoquialanines and the mycotoxin fungisporin. The EVs also presented phytotoxicity activity in vitro, and caused damage to the tissues of citrus seeds. Through molecular networking, it was observed that the metabolites present in the P. digitatum EVs are produced in all of its possible hosts. Our results reveal a novel phytopathogenic role of P. digitatum EVs and tryptoquialanine A, implying that this alkaloid is exported in EVs during plant infection. IMPORTANCEDuring the post-harvest period, citrus fruits can be affected by phytopathogens such as Penicillium digitatum, which causes the green mold disease and is responsible for up to 90 % of the total citrus losses. Chemical fungicides are widely used to prevent the green mold disease, leading to concerns about environmental and health risks. To develop safer alternatives to control phytopathogens, it is necessary to understand the molecular basis of infection during the host-pathogen interaction. In the P. digitatum model, the virulence strategies are poorly known. Here, we describe the production of phytotoxic extracellular vesicles (EVs) by P. digitatum during the infection of citrus fruits. We also characterized the secondary metabolites in the cargo of EVs and found in this set of molecules an inhibitor of seed germination. Since EVs and secondary metabolites have been related to virulence mechanisms in other host-pathogen interactions, our data are important for the comprehension of how P. digitatum causes damage to its primary hosts.

microbiology↗

bioRxiv2020

New insights into Cryptococcus extracellular vesicles suggest a new structural model and an antifungal vaccine strategy

Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data regarding the structural and compositional aspects of EVs isolated from the fungal pathogenic species Cryptococcus neoformans, C. deneoformans and C. deuterogattii. Using cutting-edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by mannoprotein-based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analyzing EV protein cargo from the three species, we characterized the typical Cryptococcus EV proteome. It contains several membrane-bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of Cryptococcus EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular C. neoformans mutant strain rendered a strong antibody response in mice and significantly prolonged their survival upon C. neoformans infection.

microbiology↗

bioRxiv2020

Characterization of extracellular vesicles produced by Aspergillus fumigatus protoplasts

Extracellular vesicles (EVs) are outer membranous compartments produced by yeast and mycelial forms of several fungal species. One of the difficulties to perceive the role of EVs during the fungal life is the fact that an active secretion of these EVs has not been clearly demonstrated in situ due to the presence of a thick cell wall. One alternative to have a better access to these vesicles is to use protoplasts. This approach has been investigated here with Aspergillus fumigatus, one of the most common opportunistic fungal pathogens worldwide. Analysis of regenerating protoplasts by scanning electron microscopy and fluorescence microscopy indicated the occurrence of outer membrane projections in association with surface components and the release of particles with properties resembling those of fungal EVs. EVs in culture supernatants were characterized by transmission electron microscopy and nanoparticle tracking analysis. Proteomic and glycome analysis of EVs revealed the presence of a complex array of enzymes related to lipid / sugar metabolism, pathogenic processes, and cell wall biosynthesis. Our data indicate that i) EV production is a common feature of different morphological stages of this major fungal pathogen, and ii) protoplastic EVs are a promising tool to undertake studies of vesicle functions in fungal cells. IMPORTANCEFungal cells use extracellular vesicles (EVs) to export biologically active molecules to the outer space. Since fungal cells are encaged in a thick cell wall, it is reasonable to expect that this structure might impact the vesicle-mediated molecular export. In this study, we used protoplasts of Aspergillus fumigatus, a major fungal pathogen, as a model to evaluate EV production in the absence of a cell wall. Our results demonstrated that wall-less A. fumigatus exports plasma membrane-derived EVs containing a complex combination of proteins and glycans. Our study is the first to characterize fungal EVs in the absence of a cell wall. Our results suggest that protoplasts are a promising model for functional studies of fungal vesicles.

microbiology↗

bioRxiv2020

A screening of the MMV Pathogen Box(R) reveals new potential antifungal drugs against the etiologic agents of chromoblastomycosis

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by traumatic implantation of many species of black fungi. Due to the refractoriness of some cases and common recurrence of CBM, a more effective and less time-consuming treatment is mandatory. The aim of this study was to identify compounds with in vitro antifungal activity in the Pathogen Box(R) compound collection against different CBM agents. Synergism of these compounds with drugs currently used to treat CBM was also assessed. An initial screening of the drugs present in this collection at 1M was performed with a Fonsecaea pedrosoi clinical strain according to the EUCAST protocol. The compounds with activity against this fungus were also tested against other seven etiologic agents of CBM (Cladophialophora carrionii, Phialophora verrucosa, Exophiala jeanselmei, Exophiala dermatitidis, Fonsecaea monophora, Fonsecaea nubica, and Rhinocladiella similis) at concentrations ranging from 0.039 to 10 M. The analysis of potential synergism of these compounds with itraconazole and terbinafine was performed by the checkerboard method. Eight compounds inhibited more than 60% of the F. pedrosoi growth: difenoconazole, bitertanol, iodoquinol, azoxytrobin, MMV688179, MMV021013, trifloxystrobin, and auranofin. Iodoquinol produced the lowest MIC values (1.25 - 2.5M) and MMV688179 showed MICs that were higher than all compounds tested (5 - >10M). When auranofin and itraconazole were tested in combination, a synergistic interaction (FICI = 0.37) was observed against the C. carrionii isolate. In summary, auranofin, iodoquinol, and MMV021013 were identified as promising compounds to be tested in CBM models of infection.

microbiology↗

bioRxiv2019

Extracellular vesicle-mediated RNA release in Histoplasma capsulatum

Eukaryotic cells, including fungi, release extracellular vesicles (EVs). These lipid bilayered compartments play essential roles in cellular communication and pathogenesis. EV composition is complex and includes proteins, glycans, pigments, and RNA. RNA classes with putative roles in pathogenesis have been described in EVs produced by fungi. Here we describe the RNA content in EVs produced by the G186AR and G217B strains of Histoplasma capsulatum, an important human fungal pathogen. A total of 124 mRNA were identified in both strains. In this set of RNA classes, 93 transcripts were enriched in EVs from the G217B strain, while 31 enriched in EVs produced by the G186AR strain. This result suggests that there are important strain-specific properties in the mRNA composition of fungal EVs. We also identified short fragments (25-40 long) that were strain-specific, with a greater number of them identified in EVs produced by the G217B strain. Remarkably, the most enriched processes were stress responses and translation. Half of these fragments aligned to the reverse strand of the transcript, suggesting the occurrence of miRNA-like molecules in fungal EVs. We also compared the transcriptome profiles of H. capsulatum with the RNA composition of EVs and no correlation was observed. Altogether, our study provided information about the RNA molecules present in H. capsulatum EVs, and the differences in composition between the G186AR and G217B strains. In addition, we showed that the correlation between the most expressed transcripts in the cell and their presence in the EVs, reinforcing the idea that the RNAs were directed to the EVs by a regulated mechanism.\n\nImportanceExtracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. In pathogenic fungi, EV-associated RNA classes have recently been described; however, only a few studies describing the RNA in fungal EVs are available. An improved knowledge on EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates of Histoplasma capsulatum. Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA.

microbiology↗