Biology subjects

Piraine, R. E. A.

Publications and source records attributed to Piraine, R. E. A..

7 recordsLinked to original sources

bioRxiv2025

RAB5c controls the assembly of non-canonical autophagy machinery to promote phagosome maturation and microbicidal function of macrophages

Non-canonical conjugation of ATG8 proteins, including LC3, to single membranes implicates the autophagy machinery in cell functions unrelated to metabolic stress. One such pathway is LC3-associated phagocytosis (LAP), which aids in phagosome maturation and subsequent signaling upon cargo uptake mediated by certain innate immunity-associated receptors. Here, we show that a specific isoform of RAB5 GTPases, the molecular switches controlling early endosome traffic, is necessary for LAP. We demonstrate that RAB5c regulates phagosome recruitment and function of complexes required for phosphatidylinositol-3-phosphate [PI(3)P] and reactive oxygen species (ROS) generation by macrophages. RAB5c facilitates phagosome translocation of the V-ATPase transmembrane core, which is needed for ATG16L1 binding and consequent LC3 conjugation. RAB5c depletion impaired macrophage elimination of the fungal pathogen Aspergillus fumigatus and disruption of the V-ATPase-ATG16L1 axis increased susceptibility in vivo. Therefore, early endosome-to-phagosome traffic is differentially regulated to promote LAP and ROS contributes to resistance against A. fumigatus by effecting LAP. HIGHLIGHTSO_LIRAB5c is required for LC3-associated phagocytosis C_LIO_LIRAB5c finetunes NAPDH oxidase assembly and ROS generation in the phagosome C_LIO_LIRAB5c regulates V-ATPase assembly on the phagosome C_LIO_LIRAB5c and V-ATPase-ATG16L1 axis are required for the killing of A. fumigatus C_LI

immunology↗

bioRxiv2025

Cross-Species Communication via Fungal Extracellular Vesicles

Extracellular vesicles (EVs) play crucial roles in fungal communication and host immune modulation, representing potential therapeutic targets for fungal infections. This study investigated the role of fungal EVs in both intra- and interspecies communication, focusing on their effects on virulence and immune responses. Co-incubation experiments were performed using EVs derived from Candida albicans and Candida auris to assess interactions with C. albicans planktonic cells and biofilms, as well as Cryptococcus neoformans and Cryptococcus gattii EVs interacting with C. neoformans cultures. EVs were observed associating with recipient cell surfaces, suggesting subsequent internalization. Functional assays revealed that EV exposure led to increased expression of Cap59, Lac1, Ure1, and Erg11 genes, correlating with reduced antifungal susceptibility in both planktonic and biofilm forms. Additionally, EVs facilitated cross-species communication, enhancing biofilm adhesion and dispersion, which underscores their role in phenotypic modulation. Macrophages stimulated with fungal EVs exhibited receptor-specific gene expression changes, notably the upregulation of galectin-3, along with a pro-inflammatory phenotype marked by increased iNOS expression and elevated cytokine levels (IL-1{beta}, IL-6, and IL-8). Collectively, these findings underscore a critical role for fungal EVs in interspecies communication, biofilm regulation, and immune modulation, offering valuable insights into fungal pathogenicity mechanisms. ImportanceCurrently, no vaccines exist to prevent fungal infections, underscoring the need for new therapies. As fungal diseases increase globally, understanding fungal biology is essential to identifying treatment targets. Fungi use EVs to communicate and evade immune responses. EVs mediate cell-cell communication, transporting proteins, polysaccharides, lipids, and nucleic acids - serving as "messages" exchanged within a fungal network. Understanding how these vesicles facilitate communication not only within a single species but also across different fungal species can shed light on their contribution to infection persistence and cross-species adaptability. Moreover, EVs may have a broader role in inter-kingdom communication, influencing how fungi interact with host immune cells. The impact of fungal EVs on human innate immune responses remains a largely underexplored area, with significant gaps in our knowledge. This study aims to examine how fungal EVs affect immune responses and whether their signaling varies across species, potentially revealing new therapeutic targets.

microbiology↗

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↗

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

Immunostimulatory activity of Pichia kluyveri, Hanseniaspora uvarum, Candida intermedia, Saccharomyces boulardii and their derivatives on RAW 264.7 macrophages

Non-Saccharomyces yeasts emerge as possible new probiotics with a beneficial effect equal to or greater than the reference probiotic yeast, Saccharomyces boulardii. In this work, we evaluated the immunomodulation effect caused by Candida intermedia in mice vaccinated with inactivated SARS-CoV-2. We conducted preliminary tests using murine macrophages (RAW 264.7) stimulated with viable and heat-killed yeast cells, culture supernatant, and DNA, using qPCR to detect the mRNA transcription. Next, mice were supplemented with C. intermedia before each dose of the SARS-CoV-2 vaccine, and then antibody production was measured by ELISA. The probiotic strain S. boulardii CNCM I-745 was used as a control. We also explored the differences in fecal microbiomes between the non-supplemented and supplemented groups. Live cells of C. intermedia increased the transcription of IL-4, IL-13, and STAT3 by macrophages RAW 264.7, while heat-killed cells up-regulated TNF and Bcl6, and the culture supernatant positively impacted TLR2 transcription. Concanavalin, zymosan, and lipopolysaccharide were used to stimulate splenocytes from C. intermedia-supplemented animals, which showed increased transcription of TNF, IFN{gamma}, IL-4, Bcl6, and STAT3. Sera from these animals showed enhanced levels of anti-SARS-CoV-2 IgG, as well as IgG1 and IgM isotypes, and sIgA in fecal samples. The microbiome of the C. intermedia-supplemented group showed a higher abundance of Bacteroides spp. and Clostridium spp., impacting the Bacteroidetes/Firmicutes balance. We concluded that C. intermedia and S. boulardii could stimulate and impact the gene expression of cells important for innate immunity, influence the composition of the gastrointestinal microbiome, and primarily boost the humoral response after vaccination. Statements and Declarations FundingThe present work was carried out with the support of Conselho Nacional de Desenvolvimento Cientifico (CNPq, Brazil), grant number 150538/2021-9.

microbiology↗

bioRxiv2021

Mixed culture metagenomics of the microbes making sour beer

Mixed microbial cultures create sour beers, but many brewers do not know which microbes comprise their cultures. The objective of this work was to use deep sequencing to identify microorganisms in sour beers brewed by spontaneous and non-spontaneous methods. Twenty samples were received from brewers, which were processed for microbiome analysis by next generation sequencing. For bacteria, primers were used to amplify the V3-V4 region of the 16S rRNA gene; fungal DNA detection was performed using primers to amplify the entire internal transcribed spacer region. The sequencing results were then used for taxonomy assignment, sample composition, and diversity analyses, as well as nucleotide BLAST searching. We identified 60 genera and 140 species of bacteria, of which the most prevalent were Lactobacillus acetotolerans, Pediococcus damnosus, and Ralstonia picketti/mannitolilytica. In fungal identification, 19 genera and 26 species were found, among which the most common yeasts were Brettanomyces bruxellensis and Saccharomyces cerevisiae. In some cases, genetic material from more than 60 microorganisms was found in a single sample. In conclusion, we were able to determine the microbiomes of various mixed cultures used to produce beer, providing useful information to better understand the sour beer fermentation process and brewing techniques.

microbiology↗

bioRxiv2021

Isolation of wild yeasts from Olympic National Park and Moniliella megachiliensis ONP131 physiological characterization for beer fermentation

Thousands of yeasts have the potential for industrial application, though many were initially considered contaminants in the beer industry. However, these organisms are currently considered important components in beers because they contribute new flavors. Non-Saccharomyces wild yeasts can be important tools in the development of new products, and the objective of this work was to obtain and characterize novel yeast isolates for their ability to produce beer. Wild yeasts were isolated from environmental samples from Olympic National Park and analyzed for their ability to ferment malt extract medium and beer wort. Six different strains were isolated, of which Moniliella megachiliensis ONP131 displayed the highest levels of attenuation during fermentations. We found that M. megachiliensis could be propagated in common yeast media, tolerated incubation temperatures of 37{degrees}C and a pH of 2.5, and was able to grow in media containing maltose as the sole carbon source. Yeast cultivation was considerably impacted (p<0.05) by lactic acid, ethanol, and high concentrations of maltose, but ONP131 was tolerant to high salinity and hop acid concentrations. This is one of the first physiological characterizations of M. megachiliensis, which has potential for the production of beer and other fermented beverages.

microbiology↗