Biology subjects

Reis, F.

Publications and source records attributed to Reis, F..

11 recordsLinked to original sources

bioRxiv2025

Impact of storage on the stability and the protective effect of extracellular vesicles released by Candida albicans

Extracellular vesicles (EVs) released by Candida albicans are multi-antigenic compartments considered as promising prototypes for vaccine development. However, their stability, appropriate storage and handling conditions are largely unexplored, which raises questions related to their biotechnological applicability. Here, we evaluated the physical and functional stability of C. albicans EVs under long-term storage. Furthermore, we conducted a comparative analysis of these properties in C. albicans EVs obtained through three commonly utilized isolation protocols documented in the literature. After identifying the most efficient isolation method for optimal yield, we devised a potential quality control for EVs isolation based on protein and sterol ratio. Subsequently, we investigated the impact of drying EVs using vacuum centrifugation at room temperature or -4 {degrees}C and the effect of freeze-thaw cycles in EVs stability. Transmission electron microscopy (TEM) revealed that EVs maintained morphological stability after long-term (up to 4 years) storage at -80 {degrees}C as well as storage at room temperature, 4 {degrees}C and -20 {degrees}C for 7 days with or without vacuum centrifugation, with a tendency of higher recovery when lower temperature is used. Remarkably, all of the C. albicans EVs suspensions maintained their biological properties as demonstrated by their ability to protect Galleria mellonella against C. albicans infection. However, the number of freeze-thaw cycles significantly impacted on the protective effect of the EVs. Overall, our findings demonstrate that C. albicans EVs maintain notable morphological and biological stability of under several conditions, enabling their efficient and reproducible utilization in research and potentially as therapeutic agents. ImportanceExtracellular vesicles (EVs) released by Candida albicans are promising vaccine prototypes due to their multi-antigenic nature. However, their storage and handling conditions are not well understood, raising concerns about their biotechnological use. This study evaluated the long-term physical and functional stability of C. albicans EVs. We compared three isolation methods to identify the most effective one and suggested a quality control measure based on protein and sterol ratios. We also examined the effects of vacuum drying and freeze-thaw cycles on EV stability. Our findings show that C. albicans EVs maintain their biological function after long-term storage at -80 {degrees}C and under various conditions. Notably, their protective effect in an insect model was reduced though repeated freeze-thaw cycles. This research provides valuable insights for the efficient use of these vesicles in future studies.

microbiology↗

bioRxiv2025

Plant age and genotype explain variation in the microbiome of natural Lotus corniculatus populations

In natural populations, plants are associated with a huge diversity of bacteria, fungi and other microbes. There is usually substantial microbiome variation between different plant individuals and populations, and the drivers of this variation are still poorly understood, particularly in wild plants. Here, we were interested in the potential of plant genotype and plant age to explain intraspecific variation in the plant microbiome of Lotus corniculatus. In seven natural populations, we genotyped a total of 168 individuals over four years, determined their ages through growth ring analysis, and then sequenced their root, shoot, flower and seed microbiomes. We found that plant genotypes differed both in the diversity and composition of microbes, and that some microbial taxa were associated with particular plant genotypes. The genotype effects tended to be strongest and most consistent for plant-associated bacteria, with the largest plant genotype differences in the microbiome diversity of flowers and seeds. We found less evidence for an effect of plant age on microbiome diversity: the age of plants explained variation in fungi diversity, and it was associated with the abundance of several microbial taxa. Our study indicates that the genotype of a plant and - to a lesser degree its age - influences the diversity and composition of plant-associated microbiota, even in complex natural environments. ImportanceThe plant microbiome plays a key role in important plant functions such as pathogen resistance, nutrient uptake, and stress tolerance. To fully understand these processes, it is essential to identify the factors that drive microbiome variation. Most research to date has focused on model or crop species under controlled conditions, leaving open questions about the drivers of microbiome diversity and composition in natural populations. In this multi-year field study, we examined how plant genotype and age shape the microbiomes of Lotus corniculatus. We show that the plant genotype strongly influences microbial diversity and composition, while plant age has subtler but still important effects, particularly on fungal communities. To our knowledge, this is the first study to link plant age with microbiome variation in natural populations. These findings demonstrate that plant traits can shape the microbiome even in complex natural environments.

microbiology↗

bioRxiv2025

Organ-specific microbiomes in natural Lotus corniculatus populations: Metacommunity dynamics in the plant endosphere

The structure of plant microbial communities vary due to a broad range of factors such as host and environmental factors, abiotic and biotic perturbations, and various assembly processes occurring at multiple tempo-spatial scales. In natural environments plant microbial communities are constantly exposed to such perturbations and processes. Thus, to attain a systemic understanding of the ecology of plant microbiomes, it is essential to study assembly processes that influence patterns of microbial community structures in natural environments. In this study we examined bacterial, fungal, and eukaryotic communities in plant organs of Lotus corniculatus in natural populations at seven grassland sites for four years. We used the framework of metacommunity theory of ecology to understand assembly processes that shape community structures and variations by defining microbial communities associated with the roots, shoots, flowers, and seeds as distinct communities linked by dispersal. In this study we show the organ-specificity of plant endophytic communities. Our findings suggest that selective filtering by plant organs, microbial interactions, as well as abiotic and biotic factors at tempo-spatial scales result in distinct core microbiomes of plant organs. In addition, transmission of microorganisms from within and outside the plant hosts accounts for the distinct yet overlapping organ microbiomes. We could provide a comprehensive knowledge of the stochastic and deterministic assembly processes that shape plant microbial communities in natural conditions. Understanding these ecological processes is essential for harnessing beneficial effects of plant-associated microbial communities on plant productivity, resilience, and pathogen defense.

microbiology↗

bioRxiv2022

Extracellular vesicle formation in Cryptococcus deuterogattii impacts fungal virulence and requires the NOP16 gene

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants lacking NOP16 expression, we observed that this gene was required for EV production. Analysis of the small molecule composition of EVs produced by wild-type cells and two independent nop16{Delta} mutants revealed that the deletion of NOP16 resulted not only in a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the nop16{Delta} mutants were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the nop16{Delta} cells in G. mellonella. These results reveal a role for NOP16 in EV biogenesis and cargo, and also indicate that the composition of EVs is determinant for cryptococcal virulence.

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↗

bioRxiv2021

Small molecule analysis of extracellular vesicles produced by Cryptococcus gattii: identification of a tripeptide controlling cryptococcal infection in an invertebrate host model

The small molecule (molecular mass < 900 Daltons) composition of extracellular vesicles (EVs) produced by the pathogenic fungus Cryptococcus gattii is unknown, which limits the understanding of the functions of cryptococcal EVs. In this study, we analyzed the composition of small molecules in samples obtained from solid cultures of C. gattii by a combination of chromatographic and spectrometric approaches, and untargeted metabolomics. This analysis revealed previously unknown components of EVs, including small peptides with known biological functions in other models. The peptides found in C. gattii EVs had their chemical structure validated by chemical approaches and comparison with authentic standards, and their functions tested in a Galleria mellonella model of cryptococcal infection. One of the vesicular peptides (isoleucine-proline-isoleucine, Ile-Pro-Ile) improved the survival of G. mellonella lethally infected with C. gattii or C. neoformans. These results indicate that small molecules exported in EVs are biologically active in Cryptococcus. Our study is the first to characterize a fungal EV molecule inducing protection, pointing to an immunological potential of extracellular peptides produced by C. gattii.

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

Shifts in antimalarial drug policy since 2006 have rapidly selected P. falciparum resistance alleles in Angola

BACKGROUNDPlasmodium falciparum resistance to chloroquine (CQ), the most widely used antimalarial drug, has historically posed a major threat to malaria control in Angola and throughout the world. Although Angola replaced CQ with artemisinin combination therapy (ACT) as a frontline treatment in 2006, malaria cases and deaths have recently been rising. CQ-resistance mutations may still be a contributing factor, given that (1) some also modulate resistance to ACT partner drugs and (2) ACT is not yet consistently implemented across Angola. It is important to continue monitoring all known resistance alleles in P. falciparum, but no studies have done so in Angola since 2012. METHODSWe sampled P. falciparum DNA from the blood of 50 hospital patients in Cabinda, Angola in 2018. Each infection was genotyped for 13 alleles in the genes crt, mdr1, dhps, dhfr, and kelch13, which collectively confer resistance to six common drugs. To analyze frequency trajectories over time, we also collated P. falciparum genotype data published from across Angola in the last two decades. RESULTSThe two most important alleles for CQ resistance, crt 72-76CVIET and mdr1 86Y, have both declined in frequency from respective highs of 98% in 1999 and 73% in 2003. However, the former remains at 71% frequency in this sample while the latter has dropped to just 7%. Of seven possible alleles for sulfadoxine-pyrimethamine (SP) resistance in dhps and dhfr, the average total number per isolate increased from 2.9 in 2004 to 4.4 in 2018. Finally, we detected no non-synonymous polymorphisms in kelch13, which is involved in artemisinin resistance in Southeast Asia. CONCLUSIONSChanges in drug policy in Angola since 2006 appear to have exerted strong selection on P. falciparum drug resistance alleles. Resistance to CQ is declining, but due to functional tradeoffs and novel selection at mdr1 loci, resistance to ACT partner drugs appears to be rising. More haplotype-based studies at mdr1 will be needed to understand the changing efficacy of multiple drugs. Finally, SP resistance has jumped rapidly since 2014, consistent with widespread use of intermittent SP treatment during pregnancy. These data can be used to support effective drug policy decisions in Angola.

evolutionary biology↗

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

Fenbendazole controls in vitro growth, virulence potential and animal infection in the Cryptococcusmodel

The human diseases caused by the fungal pathogens Cryptococcus neoformans and C. 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 anti-cryptococcal 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 anti-cryptococcal benzimidazole. Fenbendazole was inhibitory against 30 different isolates of C. neoformans and C. gattii at a low concentration. The mechanism of anti-cryptococcal 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 anti-cryptococcal effects of fenbendazole included inhibition of intracellular proliferation rates and reduced phagocytic escape through vomocytosis. Fenbendazole deeply affected the cryptococcal capsule. In a mice 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.

microbiology↗

bioRxiv2019

A novel protocol for the isolation of fungal extracellular vesicles reveals the participation of a putative scramblase in polysaccharide export and capsule construction in Cryptococcus gattii.

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 C. 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. These results were 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 aim25{Delta} 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 aim25{Delta} 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. IMPORTANCEExtracellular 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.

microbiology↗