Percorrer por autor "Pinheiro, Miguel"
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- Chlamydia trachomatis In Vivo to In Vitro Transition Reveals Mechanisms of Phase Variation and Down-Regulation of Virulence FactorsPublication . Borges, Vítor; Pinheiro, Miguel; Antelo, Minia; Sampaio, Daniel A.; Vieira, Luís; Ferreira, Rita; Nunes, Alexandra; Almeida, Filipe; Mota, Luís J.; Borrego, Maria José; Gomes, João PauloResearch on the obligate intracellular bacterium Chlamydia trachomatis demands culture in cell-lines, but the adaptive process behind the in vivo to in vitro transition is not understood. We assessed the genomic and transcriptomic dynamics underlying C. trachomatis in vitro adaptation of strains representing the three disease groups (ocular, epithelial-genital and lymphogranuloma venereum) propagated in epithelial cells over multiple passages. We found genetic features potentially underlying phase variation mechanisms mediating the regulation of a lipid A biosynthesis enzyme (CT533/LpxC), and the functionality of the cytotoxin (CT166) through an ON/OFF mechanism. We detected inactivating mutations in CT713/porB, a scenario suggesting metabolic adaptation to the available carbon source. CT135 was inactivated in a tropism-specific manner, with CT135-negative clones emerging for all epithelial-genital populations (but not for LGV and ocular populations) and rapidly increasing in frequency (~23% mutants per 10 passages). RNA-sequencing analyses revealed that a deletion event involving CT135 impacted the expression of multiple virulence factors, namely effectors known to play a role in the C. trachomatis host-cell invasion or subversion (e.g., CT456/Tarp, CT694, CT875/TepP and CT868/ChlaDub1). This reflects a scenario of attenuation of C. trachomatis virulence in vitro, which may take place independently or in a cumulative fashion with the also observed down-regulation of plasmid-related virulence factors. This issue may be relevant on behalf of the recent advances in Chlamydia mutagenesis and transformation where culture propagation for selecting mutants/transformants is mandatory. Finally, there was an increase in the growth rate for all strains, reflecting gradual fitness enhancement over time. In general, these data shed light on the adaptive process underlying the C. trachomatis in vivo to in vitro transition, and indicates that it would be prudent to restrict culture propagation to minimal passages and check the status of the CT135 genotype in order to avoid the selection of CT135-negative mutants, likely originating less virulent strains.
- Genome-scale analysis of the non-cultivable Treponema pallidum reveals extensive within-patient genetic variationPublication . Pinto, Miguel; Borges, Vítor; Antelo, Minia; Pinheiro, Miguel; Nunes, Alexandra; Azevedo, Jacinta; Borrego, Maria José; Mendonça, Joana; Carpinteiro, Dina; Vieira, Luís; Gomes, João Paulo.Insights into the genomic adaptive traits of Treponema pallidum, the causative bacterium of syphilis, have long been hampered due to the absence of in vitro culture models and the constraints associated with its propagation in rabbits. Here, we have bypassed the culture bottleneck by means of a targeted strategy never applied to uncultivable bacterial human pathogens to directly capture whole-genome T. pallidum data in the context of human infection. This strategy has unveiled a scenario of discreet T. pallidum interstrain single-nucleotide-polymorphism-based microevolution, contrasting with a rampant within-patient genetic heterogeneity mainly targeting multiple phase-variable loci and a major antigen-coding gene (tprK). TprK demonstrated remarkable variability and redundancy, intra- and interpatient, suggesting ongoing parallel adaptive diversification during human infection. Some bacterial functions (for example, flagella- and chemotaxis-associated) were systematically targeted by both inter- and intrastrain single nucleotide polymorphisms, as well as by ongoing within-patient phase variation events. Finally, patient-derived genomes possess mutations targeting a penicillin-binding protein coding gene (mrcA) that had never been reported, unveiling it as a candidate target to investigate the impact on the susceptibility to penicillin. Our findings decode the major genetic mechanisms by which T. pallidum promotes immune evasion and survival, and demonstrate the exceptional power of characterizing evolving pathogen subpopulations during human infection.
- Helicobacter pullorum Isolated from Fresh Chicken Meat: Antibiotic Resistance and Genomic Traits of an Emerging Foodborne PathogenPublication . Borges, Vítor; Santos, Andrea; Correia, Cristina Belo; Saraiva, Margarida; Ménard, Armelle; Vieira, Luís; Sampaio, Daniel A.; Pinheiro, Miguel; Gomes, João Paulo; Oleastro, MónicaMeat and meat products are important sources of human intestinal infections. We report the isolation of Helicobacter pullorum strains from chicken meat. Bacteria were isolated from 4 of the 17 analyzed fresh chicken meat samples, using a membrane filter method. MIC determination revealed that the four strains showed acquired resistance to ciprofloxacin; one was also resistant to erythromycin, and another one was resistant to tetracycline. Whole-genome sequencing of the four strains and comparative genomics revealed important genetic traits within the H. pullorum species, such as 18 highly polymorphic genes (including a putative new cytotoxin gene), plasmids, prophages, and a complete type VI secretion system (T6SS). The T6SS was found in three out of the four isolates, suggesting that it may play a role in H. pullorum pathogenicity and diversity. This study suggests that the emerging pathogen H. pullorum can be transmitted to humans by chicken meat consumption/contact and constitutes an important contribution toward a better knowledge of the genetic diversity within the H. pullorum species. In addition, some genetic traits found in the four strains provide relevant clues to how this species may promote adaptation and virulence.
- INSaFLU — uma plataforma bioinformática online para análise e comparação do genoma completo do vírus influenza: um contributo pioneiro para o reforço da vigilância da gripe à escala globalPublication . Borges, Vítor; Pinheiro, Miguel; Pechirra, Pedro; Guiomar, Raquel; Gomes, João PauloA vigilância laboratorial da gripe entrou numa nova era marcada pela análise e caracterização do vírus da gripe à escala do seu genoma completo. Este ar tigo resume o contexto inerente ao desenvolvimento e à aplicação da plataforma bioinformática online INSaFLU (“INSide the FLU”) (https://insaflu.insa.pt/), a qual consiste na primeira ferramenta bioinformática online gratuita especificamente criada para permitir que qualquer laboratório do mundo possa integrar facilmente a análise genoma do vírus na vigilância e investigação da gripe. Este avanço científico constitui um importante alicerce para a operacionalização de uma vigilância à escala global harmonizada pelo uso da sequência do genoma total do vírus da gripe.
- INSaFLU-TELEVIR: an open web-based bioinformatics suite for viral metagenomic detection and routine genomic surveillancePublication . Santos, João Dourado; Sobral, Daniel; Pinheiro, Miguel; Isidro, Joana; Bogaardt, Carlijn; Pinto, Miguel; Eusébio, Rodrigo; Santos, André; Mamede, Rafael; Horton, Daniel L; Gomes, João Paulo; TELEVIR Consortium; Borges, VítorBackground: Implementation of clinical metagenomics and pathogen genomic surveillance can be particularly challenging due to the lack of bioinformatics tools and/or expertise. In order to face this challenge, we have previously developed INSaFLU, a free web-based bioinformatics platform for virus next-generation sequencing data analysis. Here, we considerably expanded its genomic surveillance component and developed a new module (TELEVIR) for metagenomic virus identification. Results: The routine genomic surveillance component was strengthened with new workflows and functionalities, including (i) a reference-based genome assembly pipeline for Oxford Nanopore technologies (ONT) data; (ii) automated SARS-CoV-2 lineage classification; (iii) Nextclade analysis; (iv) Nextstrain phylogeographic and temporal analysis (SARS-CoV-2, human and avian influenza, monkeypox, respiratory syncytial virus (RSV A/B), as well as a "generic" build for other viruses); and (v) algn2pheno for screening mutations of interest. Both INSaFLU pipelines for reference-based consensus generation (Illumina and ONT) were benchmarked against commonly used command line bioinformatics workflows for SARS-CoV-2, and an INSaFLU snakemake version was released. In parallel, a new module (TELEVIR) for virus detection was developed, after extensive benchmarking of state-of-the-art metagenomics software and following up-to-date recommendations and practices in the field. TELEVIR allows running complex workflows, covering several combinations of steps (e.g., with/without viral enrichment or host depletion), classification software (e.g., Kaiju, Kraken2, Centrifuge, FastViromeExplorer), and databases (RefSeq viral genome, Virosaurus, etc.), while culminating in user- and diagnosis-oriented reports. Finally, to potentiate real-time virus detection during ONT runs, we developed findONTime, a tool aimed at reducing costs and the time between sample reception and diagnosis. Conclusions: The accessibility, versatility, and functionality of INSaFLU-TELEVIR are expected to supply public and animal health laboratories and researchers with a user-oriented and pan-viral bioinformatics framework that promotes a strengthened and timely viral metagenomic detection and routine genomics surveillance. INSaFLU-TELEVIR is compatible with Illumina, Ion Torrent, and ONT data and is freely available at https://insaflu.insa.pt/ (online tool) and https://github.com/INSaFLU (code).
- INSaFLU: an automated open web-based bioinformatics suite "from-reads" for influenza whole-genome-sequencing-based surveillancePublication . Borges, Vítor; Pinheiro, Miguel; Pechirra, Pedro; Guiomar, Raquel; Gomes, João PauloBackground: A new era of flu surveillance has already started based on the genetic characterization and exploration of influenza virus evolution at whole-genome scale. Although this has been prioritized by national and international health authorities, the demanded technological transition to whole-genome sequencing (WGS)-based flu surveillance has been particularly delayed by the lack of bioinformatics infrastructures and/or expertise to deal with primary next-generation sequencing (NGS) data. Results: We developed and implemented INSaFLU (“INSide the FLU”), which is the first influenza-oriented bioinformatics free web-based suite that deals with primary NGS data (reads) towards the automatic generation of the output data that are actually the core first-line “genetic requests” for effective and timely influenza laboratory surveillance (e.g., type and sub-type, gene and whole-genome consensus sequences, variants’ annotation, alignments and phylogenetic trees). By handling NGS data collected from any amplicon-based schema, the implemented pipeline enables any laboratory to perform multi-step software intensive analyses in a user-friendly manner without previous advanced training in bioinformatics. INSaFLU gives access to user-restricted sample databases and projects management, being a transparent and flexible tool specifically designed to automatically update project outputs as more samples are uploaded. Data integration is thus cumulative and scalable, fitting the need for a continuous epidemiological surveillance during the flu epidemics. Multiple outputs are provided in nomenclature-stable and standardized formats that can be explored in situ or through multiple compatible downstream applications for fine-tuned data analysis. This platform additionally flags samples as “putative mixed infections” if the population admixture enrolls influenza viruses with clearly distinct genetic backgrounds, and enriches the traditional “consensus-based” influenza genetic characterization with relevant data on influenza sub-population diversification through a depth analysis of intra-patient minor variants. This dual approach is expected to strengthen our ability not only to detect the emergence of antigenic and drug resistance variants but also to decode alternative pathways of influenza evolution and to unveil intricate routes of transmission. Conclusions: In summary, INSaFLU supplies public health laboratories and influenza researchers with an open “one size fits all” framework, potentiating the operationalization of a harmonized multi-country WGS-based surveillance for influenza virus.
- Nova abordagem genómica para a investigação da sífilisPublication . Pinto, Miguel; Borges, Vítor; Antelo, Minia; Pinheiro, Miguel; Nunes, Alexandra; Azevedo, Jacinta; Borrego, Maria José; Mendonça, Joana; Carpinteiro, Dina; Vieira, Luís; Gomes, João PauloA sífilis é uma doença sexualmente transmissível causada pela bactéria Treponema pallidum e constitui um problema de saúde pública mundial, em parte devido à ausência de uma vacina para prevenção da sua transmissão. A investigação desta doença tem sido atrasada pela incapacidade histórica de cultivar T. pallidum in vitro, dificultando por exemplo o desenvolvimento de estudos genómicos. De facto, há uma grande lacuna no conhecimento da epidemiologia molecular deste agente patogénico, assim como da base molecular que medeia a patogénese da sífilis. No estudo aqui apresentado, foi possível implementar uma abordagem inovadora para capturar o genoma de T. pallidum no contexto de infeção humana, evitando-se, assim, a necessidade da cultura da bactéria em modelo animal. Esta estratégia permitiu estudar, pela primeira vez, como é que este agente patogénico vai alterando o seu genoma para se adaptar e sobreviver como agente infecioso humano. Nomeadamente permitiu descodificar os principais mecanismos genéticos pelos quais a bactéria T. pallidum evade o sistema imunitário e se adapta ao Homem nesta complexa e multifásica doença. A aplicação desta estratégia inovadora de monitorização da interação Homem-bactéria poderá ser importante para o desenvolvimento de novas medidas profiláticas e/ou terapêuticas. Acresce que esta abordagem constitui também um ponto de viragem para o aperfeiçoamento de metodologias de diagnóstico e de epidemiologia molecular, o que permitirá aumentar o conhecimento da distribuição geográfica, das vias de transmissão e das propriedades de virulência deste agente patogénico para bem da saúde pública.
- ReporType: A Flexible Bioinformatics Tool for Targeted Loci Screening and Typing of Infectious AgentsPublication . Cruz, Helena; Pinheiro, Miguel; Borges, VítorIn response to the pressing need for continuous monitoring of emergence and circulation of pathogens through genomics, it is imperative to keep developing bioinformatics tools that can help in their rapid characterization and classification. Here, we introduce ReporType, a versatile bioinformatics pipeline designed for targeted loci screening and typing of infectious agents. Developed using the snakemake workflow manager, ReporType integrates multiple software for read quality control and de novo assembly, and then applies ABRicate for locus screening, culminating in the production of easily interpretable reports for the identification of pathogen genotypes and/or screening of specific genomic loci. The pipeline accommodates a range of input formats, from Illumina or Oxford Nanopore Technology (ONT) reads (FASTQ) to Sanger sequencing files (AB1), or FASTA files, making it flexible for application in multiple pathogens and with different purposes. ReporType is released with pre-prepared databases for some viruses and bacteria, yet it remains easily configurable to handle custom databases. ReporType performance and functionality were validated through proof-of-concept exercises, encompassing diverse pathogenic species, including viruses such as measles, Newcastle disease virus (NDV), Dengue virus (DENV), influenza, hepatitis C virus (HCV) and Human T-Cell Lymphotropic virus type 1 (HTLV-1), as well as bacteria like and . In summary, ReporType emerges as a simple, dynamic and pan-pathogen tool, poised to evolve in tandem with the ever-changing needs of the fields of pathogen genomics, infectious disease epidemiology, and one health bioinformatics. ReporType is freely available at GitHub.