Biology subjects

Alves, L. R.

Publications and source records attributed to Alves, L. R..

8 recordsLinked to original sources

bioRxiv2024

Deciphering Cargo Contents in Extracellular Vesicles of Candida haemulonii var. vulnera

ABSTRACTCandida haemulonii comprises a group of pathogenic fungi known for their resistance to primary antifungal treatments. Infections caused by these pathogens present substantial challenges due to the difficulties in accurate identification. Extracellular vesicles (EVs) released by these fungi play a critical role in the pathogen-host interaction, potentially influencing antifungal resistance and virulence. Previous research by our group indicates that EVs contain immunogenic particles capable of impacting the hosts immune response. Understanding the composition of these EVs is crucial for elucidating the mechanisms underlying resistance and virulence in C. haemulonii var. vulnera. This study aims to investigate the contents of EVs from C. haemulonii var. vulnera using proteomic and microRNA sequencing tools, providing insights into their role in adaptation, survival, and the progression of infections. Our findings reveal key proteins transported by EVs, including BMH1, TEF1, CDC19, and PDC11. These proteins are involved in various cellular processes, such as the alteration of cell wall structure, biofilm formation, and facilitation of morphological changes, among others. Additionally, we observed that miRNA-like molecules transported within EVs are linked to the electron transport chain and regulation of the citric acid cycle, which are metabolic processes associated with virulence factors and rapid adaptation to diverse hosts or environments. In this context, our findings provide a novel perspective on fungal EVs, highlighting their potential as targets for therapies. Therefore, these vesicles may reflect the expression levels of regulatory molecules crucial for the survival, pathogenicity, and virulence of C. haemulonii var. vulnera. IMPORTANCEThe study of Candida haemulonii complex holds substantial clinical significance due to its notable resistance to conventional antifungal therapies and the complex challenges inherent in its specific identification. This research focuses on cargo of EVs released by these fungi, which play an essential role in pathogen-host interactions, influencing fungal pathogenicity. EVs contain immunogenic particles that can modulate the hosts immune response. Proteomic and microRNA analyses of EVs from Candida haemulonii var. vulnera have identified key proteins and miRNAs involved in cellular processes such as metabolic adjustment, biofilm formation, and modulation of cytoplasmic functions. These components are essential for the adaptation, survival, and progression of infections. This study offers novel insights into fungal EVs, underscoring their potential as targets for therapeutic intervention. By elucidating the mechanisms underlying the rapid adaptation of Candida haemulonii, the research enhances our understanding of the pathogenicity of this emerging yeast.

microbiology↗

bioRxiv2023

TcZC3HTTP, a regulatory element that contributes to Trypanosoma cruzi cell proliferation

Post-transcriptional regulation of gene expression is a critical process for adapting and surviving Trypanosoma cruzi, a parasite with a complex life cycle. RNA binding proteins (RBPs) are key players in this regulation, forming ribonucleoprotein complexes (mRNPs) and RNA granules that control transcript stability, localization, degradation, and translation modulation. Understanding the specific roles of individual RBPs is crucial for unraveling the details of this regulatory network. In this study, we generated null mutants of the TcZC3HTTP gene, a specific RBP in the Trypanosoma family, characterized by a C3H zinc finger and a DNAJ domain associated with RNA and protein binding, respectively. Through cell growth assays, we demonstrated that the absence of TcZC3HTTP or the expression of an additional tagged version significantly impacted epimastigote growth, indicating its contribution to cell proliferation. TcZC3HTTP was found to associate with mRNAs involved in cell cycle and division in epimastigotes, while nutritionally stressed parasites exhibited associations with mRNAs coding for other RBPs and rRNA. Furthermore, our analysis of TcZC3HTTP protein partners revealed the presence of several enzymes during normal growth conditions, whereas starvation conditions enriched ribosomal proteins and other RBPs. This study provides insights into the post-transcriptional regulation of gene expression in T. cruzi, highlighting the role of TcZC3HTTP as an RBP involved in cell proliferation and uncovering its versatile functions in different cellular contexts. ImportanceUnderstanding how Trypanosoma cruzi, the causative agent of Chagas disease, regulates gene expression is crucial for developing targeted interventions. In this study, we investigated the role of TcZC3HTTP, an RNA binding protein, in post-transcriptional regulation. Our findings demonstrate that TcZC3HTTP is essential for the growth and proliferation of epimastigotes, a stage of the parasites life cycle. We identified its associations with specific mRNAs involved in cell cycle and division and its interactions with enzymes and other RBPs under normal and starvation conditions. These insights shed light on the regulatory network underlying gene expression in T. cruzi and reveal the multifaceted functions of RBPs in this parasite. Such knowledge enhances our understanding of the parasites biology and opens avenues for developing novel therapeutic strategies targeting post-transcriptional gene regulation in T. cruzi.

microbiology↗

bioRxiv2021

Fungal extracellular vesicles are involved in intraspecies intracellular communication

Fungal infections are associated with high mortality rates in humans. The risk of fungal diseases creates the urgent need to broaden the knowledge base regarding their pathophysiology. In this sense, the role of extracellular vesicles (EVs) has been described to convey biological information and participate in the fungal-host interaction process. EVs play many roles, including cellular physiology, responding to environmental cues, mediating a complex circuit of cellular communication in bidirectional crosstalk with other organisms, and the communication between fungal cells has been speculated. This study demonstrated the intra species uptake of EVs in fungi, including Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis, and the effects triggered by EVs in fungal cells. In C. albicans, we evaluated the involvement of EVs in yeast to hyphae transition, whilst in P. brasiliensis and A. fumigatus the function of EVs as stress transducers was investigated. Both P. brasiliensis and A. fumigatus were exposed to an inhibitor of glycosylation or UV light, respectively. The results demonstrated the role of EVs in regulating the expression of target genes and phenotype features. The EVs treatment induced cellular proliferation and boosted the transition yeast to hyphal transition in C. albicans, while they enhanced stress signals in A. fumigatus and P. brasiliensis, establishing a role for EVs in fungal intra species communication. Thus, fungal EVs regulate the virulence and adaptive traits in fungal interaction systems as potent message effectors, and understanding their effects and mechanism(s) of action could be exploited in antifungal therapies. Author SummaryExtracellular vesicles (EVs) play an important role in export systems. They act as vehicles for the transference of complex cargoes with broad biological functions, such as proteins, carbohydrates, pigments, nucleic acids, and lipids. EVs can contribute to fungal infection outcomes. The EV content exerts immunomodulatory functions during fungus-host interactions. Furthermore, the participation of EVs in communication between fungal cells has been speculated. This study investigated the capacity of EVs to mediate intra-species in three genera of human pathogenic fungi and established a regulatory function of EVs. We also assessed the features of this regulation by analyzing the cellular morphological aspects of fungi after stimulation with EVs. Our data suggest fungal EVs can function as potent signal mediators that mediate virulence and adaptive responses.

microbiology↗

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

Integrated transcriptional analysis of the cellular and extracellular vesicle RNA content of Candida auris in response to caspofungin

Candida auris has emerged as a serious worldwide threat by causing invasive infections in humans that are frequently resistant to one or more conventional antifungal medications, resulting in high mortality rates. Against this backdrop, health warnings around the world have focused efforts on understanding C. auris fungal biology and effective treatment approaches to combat this fungus. To date, there is little information about C. auris gene expression regulation in response to antifungal treatment. Our integrated analyses focused on the comparative transcriptomics of C. auris in the presence and absence of caspofungin as well as a detailed analysis of the yeasts extracellular vesicle (EV)-RNA composition. The results showed that genes coding oxidative stress response, ribosomal proteins, cell wall, and cell cycle were significantly upregulated in the presence of caspofungin, whereas transcriptional regulators and proteins related to the nucleus were downregulated. The mRNAs in the EVs were associated with stress responses induced by caspofungin and the ncRNA content of the EVs shifted during caspofungin treatment. Altogether, the results provide further insights into the fungal response to caspofungin and demonstrate that analyses of C. auris growth under antifungal stress can elucidate resistance and survival mechanisms of this fungus in response to medical therapy.

molecular biology↗

bioRxiv2020

Comparative molecular and immunoregulatory analysis of extracellular vesicles from Candida albicans and Candida auris.

Candida auris is a recently described multidrug-resistant pathogenic fungus that is increasingly responsible for healthcare associated outbreaks across the world. Bloodstream infections of this fungus cause death in up to 70% of the cases. Aggravating this scenario, C. auris disease-promoting mechanisms are poorly understood. Fungi release extracellular vesicles (EVs) carrying a broad range of molecules including proteins, lipids, carbohydrates, pigments, and RNA, many of which are virulence factors. Here, we carried out a comparative molecular characterization of C. auris and C. albicans EVs and evaluated their capacity to modulate effector mechanisms of host immune defense. Using proteomics, lipidomics, and transcriptomics, we found that C. auris released EVs with payloads that were strikingly different from EVs released by C. albicans. EVs released by C. auris potentiated the adhesion of this yeast to an epithelial cell monolayer. C. auris EVs also induced the expression of surface activation markers and cytokines by bone marrow-derived dendritic cells. Altogether, our findings show distinct profiles and properties of EVs released by C. auris and by C. albicans, and highlight the potential contribution of C. auris EVs to the pathogenesis of this emerging pathogen.

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↗

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↗