Biology subjects

Pires, M.

Publications and source records attributed to Pires, M..

31 records · Page 2Linked to original sources

bioRxiv2023

Post-Translational Modification Impact on MHC Peptide Binding and TCR Engagement

The human major histocompatibility complex (MHC) plays a crucial role in the presentation of peptidic fragments from proteins; these peptides can be derived from self-proteins or from non-human antigens, such as those produced by viruses or bacteria. To prevent cytotoxicity against healthy cells, thymocytes expressing T cell receptors (TCRs) that recognize self-peptides are removed from circulation in a process called negative selection. However, post-translational modifications (PTMs) are largely excluded from negative selection; this feature opens the door to the possibility that PTMs directly contribute to the development of autoreactive T cells and subsequent autoimmune diseases. Despite it being well-established that PTMs are prevalent in peptides presented on MHCs, the exact mechanisms by which PTMs influence the antigen presentation machinery remains poorly understood. In our work, we introduce chemical modifications mirroring PTMs onto peptides to systematically investigate their impact on MHC binding and TCR recognition. Our findings reveal the numerous ways PTMs alter antigen presentation, which could have implications for tumor neoantigen presentation.

immunology↗

bioRxiv2023

Measurement of Accumulation of Antibiotics to Staphylococcus aureus in Phagosomes

Staphylococcus aureus (S. aureus) has evolved the ability to persist after uptake into host immune cells. This intracellular niche enables S. aureus to potentially escape host immune responses and survive the lethal actions of antibiotics. While the elevated tolerance of S. aureus to small-molecule antibiotics is likely to be multifactorial, we pose that there may be contributions related to permeation of antibiotics into phagocytic vacuoles, which would require translocation across two mammalian bilayers. To empirically test this, we adapted our recently developed permeability assay to determine the accumulation of FDA-approved antibiotics into phagocytic vacuoles of live macrophages. Bioorthogonal reactive handles were metabolically anchored within the surface of S. aureus, and complementary tags were chemically added to antibiotics. Following phagocytosis of tagged S. aureus cells, we were able to specifically analyze the arrival of antibiotics within the phagosomes of infected macrophages. Our findings enabled the determination of permeability differences between extra- and intracellular S. aureus, thus providing a roadmap to dissect the contribution of antibiotic permeability to intracellular pathogens.

microbiology↗

bioRxiv2022

Permeation Across the Mycomembrane in Live Mycobacteria

The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most commonly ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. Nonetheless, there is limited information on the types of molecular scaffolds that can readily permeate past the mycomembrane of mycobacteria. To address this, we describe a novel assay that combines metabolic tagging of the peptidoglycan scaffold, which sits directly beneath the mycomembrane, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in Mycobacterium smegmatis and Mtb. A small panel of molecules was tested and we found a large range in the permeability profile of molecules. Interestingly, the general trend is similar across the two types of mycobacteria, with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria. The methods described, which do not require genetic manipulation, can be generally adopted to other species for which envelope permeability is treatment-limiting.

microbiology↗

bioRxiv2022

Immuno-targeting of Gram-positive Pathogens via a Cell Wall Binding Tick Antifreeze Protein

The human immune system employs several mechanisms to defend against pathogenic bacteria. However, pathogenic bacterial cells have evolved means to counter these responses, rendering our immune system less effective. Immunological agents that supplement or modulate the host immune response have proven to have powerful therapeutic potential, although this modality is less explored against bacterial pathogens. We describe the application of a bacterial binding protein to re-engage the immune system towards pathogenic bacteria. More specifically, a hapten was conjugated to a protein expressed by Ixodes scapularis ticks, called Ixodes scapularis antifreeze glycoprotein (IAFGP), that has high affinity for the D-alanine residue on the peptidoglycan of the bacterial cell wall. We showed that a fragment of this protein retained high surface binding affinity. Moreover, when conjugated to a hapten this conjugate led to the display of haptens on the cell surface of vancomycin-resistant Enterococcus faecalis. Hapten display then induced the recruitment of antibodies and promoted immune-cell mediated uptake of bacterial pathogens. These results demonstrate the feasibility in using cell wall binding agents as the basis of a class of bacterial immunotherapies against bacterial pathogens.

microbiology↗

bioRxiv2022

Estimating sequence diversity of prion protein gene (PRNP) in Portuguese populations of two cervid species: red deer and fallow deer.

Among the transmissible spongiform encephalopathies (TSEs), chronic wasting disease (CWD) in cervids is now a rising concern in wildlife within Europe, after the detection of the first case in Norway in 2016, in a wild reindeer and until June 2022 a total of 34 cases were described in Norway, Sweden and Finland. The definite diagnosis is postmortem, performed in target areas of the brain and lymph nodes. Samples are first screened using a rapid test and, if positive, confirmed by immunohistochemistry and Western immunoblotting. The study of the genetics of the prion protein gene, PRNP, has been proved to be a valuable tool for determining the relative susceptibility to TSEs. In the present study, the exon 3 of PRNP gene of 143 samples from red deer (Cervus elaphus) and fallow deer (Dama dama) of Portugal was analyzed. Three single nucleotide polymorphisms (SNPs) were found in red deer - codon A136A, codon T98A, codon Q226E - and no sequence variation was detected in fallow deer. The low genetic diversity found in our samples is compatible with previous studies in Europe. The comparison with results from North America, suggests that the free-ranging deer from our study may present susceptibility to CWD, although lack of experimental data and the necessity of extensive survey are necessary to evaluate these populations.

genetics↗

bioRxiv2022

Structure Activity Relationship of the Stem Peptide in Sortase A mediated Ligation from Staphylococcus aureus

The surfaces of most Gram-positive bacterial cells, including that of Staphylococcus aureus (S. aureus), are heavily decorated with proteins that coordinate cellular interactions with the extracellular space. In S. aureus, sortase A is the principal enzyme responsible for covalently anchoring proteins, which display the sorting signal LPXTG, onto the peptidoglycan (PG) matrix. Considerable efforts have been made to understand the role of this signal peptide in the sortase-mediated reaction. In contrast, much less is known about how the primary structure of the other substrate involved in the reaction (PG stem peptide) could impact sortase activity. To assess the sortase activity, a library of synthetic analogs of the stem peptide that mimic naturally existing variations found in the S. aureus PG primary sequence were evaluated. Using a combination of two unique assays, we showed that there is broad tolerability of substrate variations that are effectively processed by sortase A. While some of these stem peptide derivatives are naturally found in mature PG, they are not known to be present in the PG precursor, lipid II. These results suggest that sortase A could process both lipid II and mature PG as acyl-acceptor strands, which has not been previously described.

microbiology↗

bioRxiv2021

SaccuFlow: High-throughput Analysis Platform to Investigate Bacterial Cell Wall Interactions

Bacterial cell walls are formidable barriers that protect bacterial cells against external insults and oppose internal turgor pressure. While cell wall composition is variable across species, peptidoglycan is the principal component of all cell walls. Peptidoglycan is a mesh-like scaffold composed of crosslinked strands that can be heavily decorated with anchored proteins. The biosynthesis and remodeling of peptidoglycan must be tightly regulated by cells because disruption to this biomacromolecule is lethal. This essentiality is exploited by the human innate immune system in resisting colonization and by a number of clinically relevant antibiotics that target peptidoglycan biosynthesis. Evaluation of molecules or proteins that interact with peptidoglycan can be a complicated and, typically, qualitative effort. We have developed a novel assay platform (SaccuFlow) that preserves the native structure of bacterial peptidoglycan and is compatible with high-throughput flow cytometry analysis. We show that the assay is facile and versatile as demonstrated by its compatibility with sacculi from Gram-positive bacteria, Gram-negative bacteria, and mycobacteria. Finally, we highlight the utility of this assay to assess the activity of sortase A from Staphylococcus aureus against potential anti-virulence agents.

microbiology↗

bioRxiv2020

Packed Like Sardines - How Surface Crowdedness Impacts Accessibility to Peptidoglycan of Staphylococcus aureus

Bacterial cell walls are essential barriers that protect bacteria against the onslaught of potentially lethal molecules from the outside. Small molecule therapeutics, proteins from bacterial foes, and host immune proteins must navigate past a dense layer of bacterial biomacromolecules (e.g., capsular proteins, teichoic acids, and anchored proteins) to reach the peptidoglycan (PG) layer of Gram-positive bacteria. A subclass of molecules (e.g., antibiotics with intracellular targets) must also permeate through the PG (in a molecular sieving manner) to reach the cytoplasmic membrane. In the case of Staphylococcus aureus (S. aureus), teichoic acids are the major biopolymers that decorate bacterial cell surfaces. Despite the biological and therapeutic importance of surface accessibility, systematic analyses in live bacterial cells have been lacking. We describe a novel live cell fluorescence assay that reports on the permeability of molecules to and within the PG scaffold. The assay has robust reproducibility, is readily adoptable to any Gram-positive organism, and is compatible with high-throughput sample processing. Analysis of the factors controlling permeability to S. aureus and the methicillin resistant MRSA revealed that molecular flexibility plays a central role in molecular permeability. Moreover, teichoic acids impeded permeability of molecules of a wide range of sizes and chemical composition.

microbiology↗

bioRxiv2020

Production of moth sex pheromones for pest control by yeast fermentation

The use of insect sex pheromones is an alternative technology for pest control in agriculture and forestry, which, in contrast to insecticides, does not have adverse effects on human health or environment and is efficient also against insecticide-resistant insect populations.1,2 Due to the high cost of chemically synthesized pheromones, mating disruption applications are currently primarily targeting higher value crops, such as fruits.3 Here we demonstrate a biotechnological method for the production of pheromones of economically important moth pests using engineered yeast cell factories. Biosynthetic pathways towards several pheromones or their precursors were reconstructed in the oleaginous yeast Yarrowia lipolytica, which was further metabolically engineered for improved pheromone biosynthesis by decreasing fatty alcohol degradation and downregulating storage lipid accumulation. The sex pheromone of the cotton bollworm Helicoverpa armigera was produced by oxidation of fermented fatty alcohols into corresponding aldehydes. The resulting pheromone was just as efficient and specific for trapping of H. armigera male moths in cotton fields in Greece as a synthetic pheromone mixture. We further demonstrated the production of the main pheromone component of the fall armyworm Spodoptera frugiperda. Our work describes a biotech platform for the production of commercially relevant titres of moth pheromones for pest control by yeast fermentation. Significance statementAgriculture largely relies on insecticides and genetically modified crops for pest control, however alternative solutions are required due to emerging resistance, toxicity and regulatory issues, and consumer preferences. Mating disruption with sex pheromones that act by preventing insect reproduction is considered the most promising and scalable alternative to insecticides. This method is highly efficient and safe for human health and environment. The likelihood of insect resistance development is very low and can be handled by adjusting the pheromone composition. The high cost of chemically synthesized pheromones is the major barrier for the wider adoption of pheromones. A novel method based on yeast fermentation enables the production of insect sex pheromones as a lower cost from renewable feedstocks.

bioengineering↗

bioRxiv2020

Facile Synthesis and Metabolic Incorporation of m-DAP Bioisosteres Into Cell Walls of Live Bacteria

Bacterial cell walls contain peptidoglycan (PG), a scaffold that provides proper rigidity to resist lysis from internal osmotic pressure and a barrier to protect cells against external stressors. It consists of repeating sugar units with a linkage to a stem peptide that becomes highly crosslinked by cell wall transpeptidases (TP). Because it is an essential component of the bacterial cell, the PG biosynthetic machinery is often the target of antibiotics. For this reason, cellular probes that advance our understanding of PG biosynthesis and its maintenance can be powerful tools to reveal novel drug targets. While synthetic PG fragments containing L-Lysine in the 3rd position on the stem peptide are easier to access, those with meso-diaminopimelic acid (m-DAP) pose a severe synthetic challenge. Herein, we describe a solid phase synthetic scheme based on the widely available Fmoc-protected L-Cysteine building block to assemble meso-cystine (m-CYT), which mimics key structural features of m-DAP. To demonstrate proper mimicry of m-DAP, cell wall probes were synthesized with m-CYT in place of m-DAP and evaluated for their metabolic processing in live bacterial cells. We found that m-CYT-based cell wall probes were properly processed by TPs in various bacterial species that endogenously contain m-DAP in their PG. We anticipate that this strategy, which is based on the use of inexpensive and commercially available building blocks, can be widely adopted to provide greater accessibility of PG mimics for m-DAP containing organisms.

microbiology↗

bioRxiv2019

The impact of leishmaniasis on mental health and psychosocial well-being: A systematic review

BackgroundLeishmaniasis is a neglected tropical parasitic disease endemic in South Asia, East Africa, South America and the Middle East. It is associated with low socioeconomic status (SES) and responsible for considerable mortality and morbidity. Reports suggest that patients with leishmaniasis may have a higher risk of mental illness (MI), psychosocial morbidity (PM) and reduced quality of life (QoL), but this is not well characterised. The aim of this study was to conduct a systematic review to assess the reported impact of leishmaniasis on mental health and psychosocial wellbeing.\n\nMethodsA systematic review of the literature was carried out. Pre-specified criteria were applied to identify publications including observational quantitative studies or systematic reviews. Two reviewers screened all of the titles, abstracts and full-studies and a third reviewer was consulted for disagreements. Data was extracted from papers meeting the criteria and quality appraisal of the methods was performed using the Newcastle-Ottowa Scale or the Risk of Bias in Systematic Review tool.\n\nResultsA total of 14 studies were identified from 12,517 records. Nine cross-sectional, three case-control, one cohort study and one systematic review were included. Eleven assessed MI outcomes and were measured with tools specifically designed for this; nine measured PM and 12 measured QoL using validated measurement tools. Quality appraisal of the studies showed that six were of good quality. Cutaneous leishmaniasis and post kala-azar dermal leishmaniasis showed evidence of associated MI and PM including depression, anxiety and stigma, while all forms of disease showed decreased QoL. The findings were used to inform a proposed model and conceptual framework to show the possible links between leishmaniasis and mental health outcomes.\n\nConclusionThere is evidence that leishmaniasis has an impact on MI, PM or QoL of patients and their families and this occurs in all the main subtypes of the disease. There are however large gaps in the evidence. Further research is required to understand the full extent of this problem and its mechanistic basis.\n\nAUTHOR SUMMARYLeishmaniasis is a parasitic disease prevalent in many low-and middle-income countries worldwide. In this study the authors looked for evidence as to whether leishmaniasis affected the mental health and quality of life of patients. To conduct the review, a wide search of the literature was conducted, where a total of 14 full articles were included and analysed. It was found that different forms of leishmaniasis (visceral leishmaniasis, cutaneous leishmaniasis and post kala-azar dermal leishmaniasis) do cause a significant impact on patients mental health and quality of life through societal factors such as stigma, lack of knowledge, culture and low self-esteem among others. However, no evidence of biological mechanisms was found linking leishmaniasis to mental illness or decreased quality of life. Despite being a very incapacitating disease physically, leishmaniasis also leads to mental illness and decreased quality of life, and should therefore be a priority on the global health agenda for both researchers and policy makers.

epidemiology↗

bioRxiv2019

Niche partitioning and coexistence of two spiders of the genus Peucetia (Araneae, Oxyopidae) inhabiting Trichogoniopsis adenantha plants (Asterales, Asteraceae): a population approach

Niche theory suggests that the coexistence of ecologically similar species at the same site requires some form of resource partitioning that reduces or avoids interspecific competition. Here, we investigated the temporal and spatial niche differentiation of two sympatric congeneric spiders, Peucetia rubrolineata and P. flava, inhabiting Trichogoniopsis adenantha (Asteraceae) plants along an altitudinal gradient in various shaded and open areas in an Atlantic forest in Serra do Japi, SP, Brazil. In theory, the coexistence of two Peucetia species could be explained by: (1) temporal segregation; (2) differential use of the branches of the plant; (3) differential use of specific parts of the branches of the plant; (4) differential distribution in shaded and open areas; and (5) differential altitudinal distribution of the two Peucetia species. With respect to temporal niche, we observed that the two spider species had a similar age structure and similar fluctuation in abundance throughout the year. With respect to micro-habitat use, in both species, different instars used different plant parts, while the same instars of both species used the same type of substrate. However, the two Peucetia species segregated by meso-habitat type, with P. rubrolineata preferring T. adenantha plants in shaded areas and P. flava preferring those in open areas. Our results support the hypothesis of niche partitioning begetting diversity, and highlight the importance of analysing habitat use at multiple scales to understand mechanisms related to coexistence.

ecology↗

bioRxiv2017

Analyzing ecological networks of species interactions

Networks provide one of the best representations for ecological communities, composed of many species with sometimes complex connections between them. Yet the methodological literature allowing one to analyze and extract meaning from ecological networks is dense, fragmented, and unwelcoming. We provide a general overview to the field of using networks in community ecology, outlining both the intent of the different measures, their assumptions, and the contexts in which they can be used. When methodologically justified, we suggest good practices to use in the analysis of ecological networks. We anchor this synopsis with examples from empirical studies, and conclude by highlighting what identified as needed future developments in the field.

ecology↗