Biology subjects

Silva, L. P.

Publications and source records attributed to Silva, L. P..

9 recordsLinked to original sources

bioRxiv2025

Establishment of chicken muscle and adipogenic cell cultures for cultivated meat production

Cultured meat aims to replicate the sensory and functional properties of conventional meat by developing structured muscle tissue using cell culture. This study focuses on the culture of chicken embryonic and mesenchymal stem cells (MSCs) to derive muscle, and fat, optimizing conditions for differentiation and integration. We utilized monolayer and three-dimensional microcarrier-based cultures to produce muscle fibers and adipocytes while maintaining the extracellular matrix (ECM) integrity essential for tissue cohesion. Key pluripotency and myogenic markers (e.g., cOCT4, cMYOD, cMYH1E) were analyzed during differentiation, revealing dynamic gene expression patterns that underscore myogenesis. Myoblast differentiation into mature myotubes demonstrated decreased cPAX7 and increased cMYMK, confirming lineage commitment and muscle fiber formation. Adipogenesis was induced in MSCs using food-grade lecithin, which activated PPAR{gamma}, C/EBP, and FABP4, resulting in robust lipid droplet accumulation. To scale production, microcarriers facilitated cell proliferation, while transglutaminase-based stabilization enabled the formation of three-dimensional tissue structures comparable to native meat. Our findings highlight advances in culture protocols, genotypic and phenotypic expression analyses of multinucleated chicken muscle and adipocyte cells for cultured meat production.

bioengineering↗

bioRxiv2024

Altered host pyruvate metabolism fuels and regulates fungal asexual reproduction

Powdery mildew (PM) fungi are widespread pathogens impacting agricultural productivity. As obligate biotrophs, PMs get all nutrients from their host plant. Concurrent with asexual reproduction, the PM Golovinomyces orontii induces host lipid body accumulation in plant mesophyll cells underlying the epidermal cell that houses the fungal feeding structure. Herein, we show a PM-induced shift in plant primary metabolism, to bypass the pyruvate dehydrogenase complex (PDHc), acts in a dose-dependent manner to support host storage lipid production for PM acquisition. Not only are plant PDHc bypass-derived lipids incorporated into spore storage lipids, but they also act in a regulatory capacity to control the number of fungal reproductive structures per colony, ensuring the fitness of spores that are formed. Evolutionarily distant plant and animal obligate biotrophs manipulate host lipid metabolism for their own acquisition and we propose altered host pyruvate metabolism, bypassing the host PDHc bottleneck, as a common strategy to increase host source flux to acetate/acetyl-CoA for obligate microbial lipid acquisition.

plant biology↗

bioRxiv2021

Aspergillus fumigatus acetate utilisation impacts virulence traits and pathogenicity

Aspergillus fumigatus is a major opportunistic fungal pathogen of immunocompromised and immunocompetent hosts. To successfully establish an infection, A. fumigatus needs to use host carbon sources, such as acetate, present in the body fluids and peripheral tissues. However, utilisation of acetate as a carbon source by fungi in the context of infection has not been investigated. This work shows that acetate is metabolised via different pathways in A. fumigatus and that acetate utilisation is under the regulatory control of a transcription factor (TF), FacB. A. fumigatus acetate utilisation is subject to carbon catabolite repression (CCR), although this is only partially dependent on the TF and main regulator of CCR CreA. The available extracellular carbon source, in this case glucose and acetate, significantly affected A. fumigatus virulence traits such as secondary metabolite secretion and cell wall composition, with the latter having consequences for resistance to oxidative stress, to anti-fungal drugs and to human neutrophil-mediated killing. Furthermore, deletion of facB significantly impaired the in vivo virulence of A. fumigatus in both insect and mammalian models of invasive aspergillosis. This is the first report on acetate utilisation in A. fumigatus and this work further highlights the importance of available host-specific carbon sources in shaping fungal virulence traits and subsequent disease outcome, and a potential target for the development of anti-fungal strategies. ImportanceAspergillus fumigatus is an opportunistic fungal pathogen in humans. During infection, A. fumigatus is predicted to use host carbon sources, such as acetate, present in body fluids and peripheral tissues, to sustain growth and promote colonisation and invasion. This work shows that A. fumigatus metabolises acetate via different pathways, a process that is dependent on the transcription factor FacB. Furthermore, the type and concentration of the extracellular available carbon source were determined to shape A. fumigatus virulence determinants such as secondary metabolite secretion and cell wall composition. Subsequently, interactions with immune cells are altered in a carbon source-specific manner. FacB is required for A. fumigatus in vivo virulence in both insect and mammalian models of invasive aspergillosis. This is the first report that characterises acetate utilisation in A. fumigatus and highlights the importance of available host-specific carbon sources in shaping virulence traits and potentially subsequent disease outcome.

microbiology↗

bioRxiv2021

Screening of chemical libraries for new antifungal drugs against Aspergillus fumigatus reveals the potential mechanism of action of miltefosine

Aspergillus fumigatus is an important fungal pathogen and the main etiological agent of aspergillosis, a disease characterized by a noninvasive process that can evolve to a more severe clinical manifestation called invasive pulmonary aspergillosis (IPA) in immunocompromised patients. The antifungal arsenal to threat aspergillosis is very restricted. Azoles are the main therapeutic approach to control IPA, but the emergence of azole-resistant A. fumigatus isolates has significantly increased over the last decades. Therefore, new strategies are necessary to combat aspergillosis and drug repurposing has emerged as an efficient and alternative approach for identifying new antifungal drugs. Here, we used a screening approach to analyze A. fumigatus in vitro susceptibility to 1,127 compounds. A. fumigatus was more susceptible to 10 compounds, including miltefosine, a drug that displayed fungicidal activity against A. fumigatus. By screening an A. fumigatus transcription factor null library, we identified a single mutant, which has the rmiA (resistant to miltefosine) gene deleted, conferring a phenotype of susceptibility to miltefosine. The transcriptional profiling (RNA-seq) of the wild-type and the {Delta}rmiA strains and the Chromatin Immunoprecipitation coupled to next generation sequencing (ChIP-Seq) of a RmiA-tagged strain exposed to miltefosine revealed genes of the sphingolipids pathway that are directly or indirectly regulated by RmiA. Sphingolipids analysis demonstrated that the mutant has overall decreased levels of sphingolipids when growing in the presence of miltefosine. The identification of RmiA represents the first genetic element described and characterized which plays a direct role in miltefosine response in fungi. Author summaryThe filamentous fungus Aspergillus fumigatus causes a group of diseases named aspergillosis and their development occurs after the inhalation of conidia dispersed in the environment. Very few classes of antifungal drugs are available for aspergillosis treatment, e.g., azoles, but the emergence of global resistance to azoles in A. fumigatus clinical isolates has increased over the last decades. Repositioning or repurposing drugs already available on the market is an interesting and faster opportunity for the identification of novel antifungals agents. By using a repurposing strategy, we identified 10 different compounds that impact A. fumigatus survival. One of these compounds, miltefosine, demonstrated fungicidal activity against A. fumigatus. The mechanism of action of miltefosine is unknown and aiming to get more insights about it, we identified a transcription factor RmiA (Resistant to miltefosine) important for miltefosine resistance. Our results suggest that miltefosine plays antifungal activity against A. fumigatus interfering in the sphingolipids biosynthesis.

microbiology↗

bioRxiv2021

An evolutionary genomic approach reveals both conserved and species-specific genetic elements related to human disease in closely related Aspergillus fungi

Aspergillosis is an important opportunistic human disease caused by filamentous fungi in the genus Aspergillus. Roughly 70% of infections are caused by Aspergillus fumigatus, with the rest stemming from approximately a dozen other Aspergillus species. Several of these pathogens are closely related to A. fumigatus and belong in the same taxonomic section, section Fumigati. Pathogenic species are frequently most closely related to non-pathogenic ones, suggesting Aspergillus pathogenicity evolved multiple times independently. To understand the repeated evolution of Aspergillus pathogenicity, we performed comparative genomic analyses on 18 strains from 13 species, including 8 species in section Fumigati, which aimed to identify genes, both ones previously connected to virulence as well as ones never before implicated, whose evolution differs between pathogens and non-pathogens. We found that most genes were present in all species, including approximately half of those previously connected to virulence, but a few genes were section- or species-specific. Evolutionary rate analyses identified hundreds of genes in pathogens that were faster-evolving than their orthologs in non-pathogens. For example, over 25% of all single-copy genes examined in A. fumigatus were faster-evolving. Functional testing of deletion mutants of 17 transcription factor-encoding genes whose evolution differed between pathogens and non-pathogens identified eight genes that affect either fungal survival in a model of phagocytic killing, host survival in an animal model of fungal disease, or both. These results suggest that the evolution of pathogenicity in Aspergillus involved both conserved and species-specific genetic elements, illustrating how an evolutionary genomic approach informs the study of fungal disease.

evolutionary biology↗

bioRxiv2020

Aspergillus fumigatus G-protein coupled receptors GprM and GprJ are important for the regulation of the cell wall integrity pathway, secondary metabolite production, and virulence

G-protein coupled receptors (GPCRs) are extracellular signalling receptors that sense environmental cues to coordinate a biological response. Fungi sense their environment primarily through GPCR-mediated signalling pathways, which in turn regulate fungal development, metabolism, virulence and mycotoxin biosynthesis. A. fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate when compared to the wild-type (WT) strain, and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the {Delta}gprM and {Delta}gprJ strains and decreased in the overexpression mutants when compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. RNA-sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the {Delta}gprM and {Delta}gprJ strains, and that this regulation occurs, at least partially, through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signalling. Finally, we show that the {Delta}gprM and {Delta}gprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis. This work further contributes to unravelling functions of A. fumigatus GPCRs and shows that GprM and GprJ are essential for CWI, secondary metabolite production and virulence. Author summaryA. fumigatus is the main ethiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immuno-compromised humans. Withstanding the host environment is essential for A. fumigatus virulence and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which in turn regulate fungal development, metabolism, virulence and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella. This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.

microbiology↗

bioRxiv2020

Genomic and phenotypic heterogeneity of clinical isolates of the human pathogens Aspergillus fumigatus, Aspergillus lentulus and Aspergillus fumigatiaffinis

Fungal pathogens are a global threat to human health. For example, fungi from the genus Aspergillus cause a spectrum of diseases collectively known as aspergillosis. Most of the >200,000 life-threatening aspergillosis infections per year worldwide are caused by Aspergillus fumigatus. Recently, molecular typing techniques have revealed that aspergillosis can also be caused by organisms that are phenotypically similar to A. fumigatus but genetically distinct, such as Aspergillus lentulus and Aspergillus fumigatiaffinis. Importantly, some of these so-called cryptic species exhibit different virulence and drug susceptibility profiles than A. fumigatus, however, our understanding of their biology and pathogenic potential has been stymied by the lack of genome sequences and phenotypic profiling. To fill this gap, we phenotypically characterized the virulence and drug susceptibility of 15 clinical strains of A. fumigatus, A. lentulus, and A. fumigatiaffinis from Spain and sequenced their genomes. We found heterogeneity in virulence and drug susceptibility across species and strains. Genes known to influence drug susceptibility (cyp51A and fks1) vary in paralog number and sequence among these species and strains and correlate with differences in drug susceptibility. Similarly, genes known to be important for virulence in A. fumigatus showed variability in number of paralogs across strains and across species. Characterization of the genomic similarities and differences of clinical strains of A. lentulus, A. fumigatiaffinis, and A. fumigatus that vary in disease-relevant traits will advance our understanding of the variance in pathogenicity between Aspergillus species and strains that are collectively responsible for the vast majority of aspergillosis infections in humans.

microbiology↗

bioRxiv2020

Functional characterization of clinical isolates of the opportunistic fungal pathogen Aspergillus nidulans

Aspergillus nidulans is an opportunistic fungal pathogen in patients with immunodeficiency and virulence of A. nidulans isolates has mainly been studied in the context of the chronic granulomatous disease (CGD), with characterization of clinical isolates obtained from non-CGD patients remaining elusive. This study therefore carried out a detailed biological characterization of two A. nidulans clinical isolates (CIs), obtained from a patient with breast carcinoma and pneumonia and from a patient with cystic fibrosis that underwent lung transplantation, and compared them to the reference, non-clinical A4 strain. Both CIs presented increased growth in comparison to the reference strain in the presence of physiologically-relevant carbon sources. Metabolomic analyses showed that the three strains are metabolically very different from each other in these carbon sources. Furthermore, the CIs were highly susceptible to cell wall perturbing agents but not to other physiologically-relevant stresses. Genome analyses identified several frame-shift variants in genes encoding cell wall integrity (CWI) signalling components. Significant differences in CWI signalling were confirmed by western blotting among the three strains. In vivo virulence studies using several different models revealed that strain MO80069 had significantly higher virulence in hosts with impaired neutrophil function when compared to the other strains. In summary, this study presents detailed biological characterization of two A. nidulans sensu stricto clinical isolates. Just like in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits. Further studies are required to fully characterize A. nidulans strain-specific virulence traits and pathogenicity. ImportanceImmunocompromised patients are susceptible to infections with opportunistic filamentous fungi from the genus Aspergillus. Although A. fumigatus is the main etiological agent of Aspergillus spp.-related infections, other species, such as A. nidulans are prevalent in a condition-specific manner. A. nidulans is a predominant infective agent in patients suffering from chronic granulomatous disease (CGD). A. nidulans isolates have mainly been studied in the context of CGD, although infection with A. nidulans also occurs in non-CGD patients. This study carried out a detailed biological characterization of two non-CGD A. nidulans clinical isolates and compared it to a reference strain. Phenotypic, metabolomic and genomic analyses highlight fundamental differences in carbon source utilization, stress responses and maintenance of cell wall integrity among the strains. One clinical strain had increased virulence in models with impaired neutrophil function. Just as in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits.

microbiology↗

bioRxiv2018

Characterizing the pathogenic, genomic, and chemical traits of Aspergillus fischeri, the closest sequenced relative of the major human fungal pathogen Aspergillus fumigatus

Aspergillus fischeri is closely related to Aspergillus fumigatus, the major cause of invasive mold infections. Even though A. fischeri is commonly found in diverse environments, including hospitals, it rarely causes invasive disease; why that is so is unclear. Comparison of A. fischeri and A. fumigatus for diverse pathogenic, genomic, and secondary metabolic traits revealed multiple differences for pathogenesis-related phenotypes, including that A. fischeri is less virulent than A. fumigatus in multiple animal models of disease, grows slower in low oxygen environments, and is more sensitive to oxidative stress. In contrast, the two species exhibit high genomic similarity; ~90% of the A. fumigatus proteome is conserved in A. fischeri, including 48/49 genes known to be involved in A. fumigatus virulence. However, only 10/33 A. fumigatus biosynthetic gene clusters (BGCs) likely involved in secondary metabolite production are conserved in A. fischeri and only 13/48 A. fischeri BGCs are conserved in A. fumigatus. Detailed chemical characterization of A. fischeri cultures grown on multiple substrates identified multiple secondary metabolites, including two new compounds and one never before isolated as a natural product. Interestingly, an A. fischeri deletion mutant of laeA, a master regulator of secondary metabolism, produced fewer secondary metabolites and in lower quantities, suggesting that regulation of secondary metabolism is at least partially conserved. These results suggest that the non-pathogenic A. fischeri possesses many of the genes important for A. fumigatus pathogenicity but is divergent with respect to its ability to thrive under host-relevant conditions and its secondary metabolism.\n\nImportanceAspergillus fumigatus is the primary cause of aspergillosis, a devastating ensemble of diseases associated with severe morbidity and mortality worldwide. A. fischeri is a close relative of A. fumigatus, but is not generally observed to cause human disease. To gain insights into the underlying causes of this remarkable difference in pathogenicity, we compared two representative strains (one from each species) for a range of host-relevant biological and chemical characteristics. We found that disease progression in multiple A. fischeri mouse models was slower and caused less mortality than A. fumigatus. The two species also exhibited different growth profiles when placed in a range of stress-inducing conditions encountered during infection, such as low levels of oxygen and the presence of reactive oxygen species-inducing agents. Interestingly, we also found that the vast majority of A. fumigatus genes known to be involved in virulence are conserved in A. fischeri, whereas the two species differ significantly in their secondary metabolic pathways. These similarities and differences that we identified are the first step toward understanding the evolutionary origin of a major fungal pathogen.

microbiology↗