Biology subjects

Pacheco Fill, T.

Publications and source records attributed to Pacheco Fill, T..

5 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↗

bioRxiv2022

Metabolomic comparison using Streptomyces spp. as a factory of secondary metabolites

Understanding extremophiles and their usefulness in biotechnology involves studying their habitat, physiology and biochemical adaptations, as well as their ability to produce biocatalysts, in environments that are still poorly explored. In northwestern Peru, which has saline lagoons of marine origin Pacific Ocean, the other site is from the coast of Brazil of the Atlantic Ocean. Both environments are considered extreme. The objective of the present work was to compare two different strains isolated from these extreme environments at the metabolic level using molecular network methodology through the Global Natural Products Molecular Social Network (GNPS). In our study, the MS/MS spectra from the network were compared with GNPS spectral libraries, where the metabolites were annotated. Differences were observed in the molecular network presented in the two strains of Streptomyces spp. coming from these two different environments. Within the an-notated compounds from marine bacteria, the metabolites characterized for Streptomyces sp. B-81 from Peruvian marshes were lobophorins A (1) and H (2), as well as divergolides A (3), B (4) and C (5). Streptomyces sp. 796.1 produced different compounds, such as glucopiericidin A (6) and dehy-dro-piericidin A1a (7). The search for new metabolites in underexplored environments may therefore reveal new metabolites with potential application in different areas of biotechnology.

molecular biology↗

bioRxiv2022

Secondary metabolites produced during Aspergillus fumigatus and Pseudomonas aeruginosa biofilm formation

In Cystic Fibrosis (CF), mucus plaques are formed in the patients lung, creating a hypoxic condition and a propitious environment for colonization and persistence of many microorganisms. There is clinical evidence showing that Aspergillus fumigatus can co-colonize CF patients with Pseudomonas aeruginosa, which has been associated with lung function decline. P. aeruginosa produces several compounds with inhibitory and anti-biofilm effects against A. fumigatus in vitro; however, little is known about the fungal compounds produced in counterattack. Here, we annotated fungal and bacterial secondary metabolites (SM) produced in mixed biofilms in normoxia and hypoxia conditions. We detected nine SMs produced by P. aeruginosa. Phenazines and different analogs of pyoverdin were the main compounds produced by P. aeruginosa, and their secretion were increased by the fungal presence. The roles of the two operons responsible for phenazines production (phzA1 and phzA2) were also investigated showing both mutants are able to produce partial sets of phenazines. We detected a total of 20 SMs secreted by A. fumigatus either in monoculture or in co-culture with P. aeruginosa. All these compounds are secreted during biofilm formation either in normoxia or hypoxia. However, only eight compounds (demethoxyfumitremorgin C, fumitremorgin, ferrichrome, ferricrocin, tricetylfusigen, gliotoxin, gliotoxin E, and pyripyropene A) were detected during the biofilm formation by the co-culture of A. fumigatus and P. aeruginosa upon both normoxia and hypoxia conditions. Overall, we showed how diverse is SM secretion during A. fumigatus and P. aeruginosa mixed culture and how this can affect biofilm formation both in normoxia and hypoxia. Author SummaryThe interaction between Pseudomonas aeruginosa and Aspergillus fumigatus has been well-characterized in vitro. In this scenario, the bacterium exerts a strong inhibitory effect against the fungus. However, little is known about the metabolites produced by the fungus to counterattack the bacteria. Our work aimed to annotate secondary metabolites (SM) secreted during co-culture between P. aeruginosa and A. fumigatus during biofilm formation in both normoxia and hypoxia. The bacterium produces several different types of phenazines and pyoverdins, in response to the fungus presence. In contrast, we were able to annotate 29 metabolites produced during A. fumigatus biofilm formation but only eight compounds were detected during biofilm formation by the co-culture of A. fumigatus and P. aeruginosa upon both normoxia and hypoxia. In conclusion, we have detected many SMs secreted during A. fumigatus and P. aeruginosa biofilm formation. This analysis can provide several opportunities to understand the interaction between these two species.

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

EVALUATION OF ANTIMICROBIAL AND ANTIPROLIFERATIVE ACTIVITIES OF ACTINOBACTERIA ISOLATED FROM THE SALINE LAGOONS OF NORTHWEST PERU

The unexplored saline lagoons of the north of Peru harbor a rich microbiome, due to reported studies of different extreme environments around the world. In these regions, there are several ecosystems and microhabitats not yet explored, and little is known about the diversity of actinobacteria and other microorganisms. We suggest that the endemic bacteria present in this extreme environment could be source of active molecules with anticancer, antimicrobial, antiparasitic properties. Using phenotypic and genotypic characterization techniques including the 16S rRNA were identified into the genera Streptomyces 39 (78%), Pseudonocardia 3 (6%), Staphylococcus 4 (8%), Bacillus 2 (4%), and Pseudomonas 2 (4%). All isolated bacteria for the genotypic data were preliminarily identified. Actinobacteria strains were found dominantly in both sites (Lagoon1-3 = 16 isolates and lagoon 4 = 12 isolates). Phylogenetic analysis revealed that 28 isolates were exclusively affiliated to eleven different clusters of Actinobacteria of the major genus Streptomyces. Three Streptomyces sp. strains M-92, B-146, and B-81, were tested for antibacterial and antiproliferative activities. The results showed antiproliferative activities against three tumor cell lines, U251 glioma; MCF7 breast; NCI-H460 lung non-small type of cells, and the antibacterial activity to Staphylococcus aureus ATCC 6538, E. coli ATCC 10536, and Acinetobacter baumanni AC-972 which is resistant to multiple drugs. The promising results belong to Streptomyces sp. B-81 strain in the R2A medium using a doxorubicin with control positive, the best result was from the latter (TGI = 0,57 {micro}g/mL) for glioma; NCI-H460 lung of type non-small cells (TGI = 0,61 {micro}g/mL), and breast cancer (TGI =0,80 {micro}g/mL), this strain was selected to be fractionated because it had better antiproliferative and antibacterial activity, and its fractions were evaluated concerning antiproliferative activity against nine types of tumor cells and one non-tumor. The methanolic fraction showed a better result in the antiproliferative activity and was able to inhibit U251 (glioma) (TGI = 38.3 {micro}g/mL), OVCAR-03 (ovary) (TGI = 62.1 {micro}g/mL), and K562 (leukemia) (TGI = 81.5 {micro}g/mL). The methanol 50% - acetate 50% fraction (Fraction 4) inhibited U251 (glioma) (TGI = 73.5 {micro}g/mL) and UACC-62 (melanoma) (TGI = 89.4 {micro}g/mL). Moreover, the UHPLC-MS/MS data and molecular networking of Streptomyces sp. B-81 isolate extract revealed the production cholic acid, Lobophorin A, Lobophorin B, Lobophorin E, Lobophorin K and compound 6. Extremophilic environments such as the Morrope and Bayovar Salt Flats are promising sources of new bacteria with promising pharmaceutical potential; These compounds could be useful to treat various infectious diseases or even some type of cancer.

molecular biology↗