Browsing by Author "Borges, V."
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- A Bioinformática na Identificação dos Processos Moleculares de Interacção entre o Agente Patogénico e o HomemPublication . Borges, V.; Nunes, A.; Ferreira, R.; Borrego, M.J.; Gomes, João PauloQualquer processo infeccioso caracteriza-se por um “braço de ferro” contínuo entre o agente patogénico e o Homem. Se por um lado, o Homem tenta debelar a infecção através da complexa rede celular que caracteriza a resposta imunitária, por outro, o agente patogénico tenta escapar a essa resposta através da acumulação de mutações no seu genoma. De um modo geral, as mutações num gene podem ser sinónimas (quando não originam uma alteração de aminoácido) ou não-sinónimas (quando a proteína correspondente é alterada), sendo estas últimas, quando vantajosas, as principais responsáveis pela adaptação do agente infeccioso ao hospedeiro. A fixação de alterações não-sinónimas vantajosas resulta de um processo evolutivo denominado de “selecção positiva”. A bioinformática surge nos últimos anos como uma ferramenta acessível e indispensável para a análise dos dados genómicos que diariamente são gerados de forma exponencial. Neste âmbito, a bioinformática tem sido essencial, por exemplo, para a identificação dos processos moleculares de interacção entre o agente patogénico e o Homem, que decorrem durante o processo infeccioso. Na presente comunicação, a utilidade desta valência computacional é ilustrada tomando como modelo o processo infeccioso despoletado pela bactéria Chlamydia trachomatis. De facto, esta bactéria intracelular é caracterizada por um perfil bem definido de infecções no Homem, cujos diversos genótipos afectam distintamente o tecido ocular, os órgãos genitais e os nódulos linfáticos inguinais (via macrófagos), orgãos estes que exercem uma pressão selectiva distinta (resposta imunitária, pH, flora comensal, etc) sobre a bactéria. Através da utilização de várias plataformas de bioinformática para a análise específica das mutações que distinguem os vários genótipos de Chlamydia trachomatis, é possível identificar genes, que por força de uma selecção positiva, estão hipoteticamente envolvidos na adaptação/invasão aos vários tipos de células humanas infectadas, nomeadamente, células epiteliais das mucosas ocular e genital, e macrófagos. Noutras vertentes, este tipo de análise de detecção de selecção positiva teve já aplicações no desenvolvimento de uma vacina para o VIH bem como no esclarecimento da evolução da virulência do vírus Influenza, e pode ser aplicado a todo o sistema biológico.
- Complete Genome Sequence of Chlamydia trachomatis Ocular Serovar C Strain TW-3Publication . Borges, V.; Pinheiro, M.; Vieira, Luís; Sampaio, Daniel A.; Nunes, A.; Borrego, M.J.; Gomes, João PauloChlamydia trachomatis is the etiological agent of trachoma, the leading infectious cause of blindness worldwide. We report here the first complete and annotated genome of a C. trachomatis trachoma-causing serovar C strain (strain TW-3). The chromosome and plasmid are 1,043,554 bp and 7,501 bp in length, respectively.
- Congenital SARS-CoV-2 Infection in a Neonate With Severe Acute Respiratory SyndromePublication . Correia, C.R.; Marçal, M.; Vieira, F.; Santos, E.; Novais, C.; Maria, A.T.; Malveiro, D.; Prior, A.R.; Aguiar, M.; Salazar, A.; Pinto, C.G.; Rodrigues, L.C.; Pessanha, M.A.; Borges, V.; Isidro, J.; Gomes, J.P.; Duarte, S.; Vieira, L.; Costa, I.; Alves, M.J.; Calhau, C.; Guiomar, R.; Tuna, M.L.Coronavirus disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is mainly transmitted through droplets, but other ways of transmission have been hypothesized. We report a case of vertical transmission of SARS-CoV-2 in a preterm born to an infected mother, confirmed by the presence of the virus in the neonatal blood, nasopharyngeal and oropharyngeal swabs collected in the first half an hour of life. The neonate presented with acute respiratory distress, similar to the findings in severely affected adults. This case highlights the importance of pregnancy, labor and neonatal period surveillance of affected mothers and their newborns.
- Identification of type III secretion substrates of Chlamydia trachomatis using Yersinia enterocolitica as a heterologous systemPublication . da Cunha, M.; Milho, C.; Almeida, F.; Pais, S.V.; Borges, V.; Borrego, M.J.; Gomes, João Paulo; Mota, L.J.Background: Chlamydia trachomatis is an obligate intracellular human pathogen causing ocular and urogenital infections that are a significant clinical and public health concern. This bacterium uses a type III secretion (T3S) system to manipulate host cells, through the delivery of effector proteins into their cytosol, membranes, and nucleus. In this work, we aimed to find previously unidentified C. trachomatis T3S substrates. Results: We first analyzed the genome of C. trachomatis L2/434 strain for genes encoding mostly uncharacterized proteins that did not appear to possess a signal of the general secretory pathway and which had not been previously experimentally shown to be T3S substrates. We selected several genes with these characteristics and analyzed T3S of the encoding proteins using Yersinia enterocolitica as a heterologous system. We identified 23 C. trachomatis proteins whose first 20 amino acids were sufficient to drive T3S of the mature form of β-lactamase TEM-1 by Y. enterocolitica. We found that 10 of these 23 proteins were also type III secreted in their full-length versions by Y. enterocolitica, providing additional support that they are T3S substrates. Seven of these 10 likely T3S substrates of C. trachomatis were delivered by Y. enterocolitica into host cells, further suggesting that they could be effectors. Finally, real-time quantitative PCR analysis of expression of genes encoding the 10 likely T3S substrates of C. trachomatis showed that 9 of them were clearly expressed during infection of host cells. Conclusions: Using Y. enterocolitica as a heterologous system, we identified 10 likely T3S substrates of C. trachomatis (CT053, CT105, CT142, CT143, CT144, CT161, CT338, CT429, CT656, and CT849) and could detect translocation into host cells of CT053, CT105, CT142, CT143, CT161, CT338, and CT429. Therefore, we revealed several C. trachomatis proteins that could be effectors subverting host cell processes.
- Identification of type III secretion substrates of Chlamydia trachomatis using Yersinia enterocolitica as a heterologous systemPublication . da Cunha, M.; Milho, C.; Almeida, F.; Pais, S.V.; Borges, V.; Maurício, R.; Borrego, M.J.; Gomes, João Paulo; Mota, L.J.Background: Chlamydia trachomatis is an obligate intracellular human pathogen causing ocular and urogenital infections that are a significant clinical and public health concern. This bacterium uses a type III secretion (T3S) system to manipulate host cells, through the delivery of effector proteins into their cytosol, membranes, and nucleus. In this work, we aimed to find previously unidentified C. trachomatis T3S substrates. Results: We first analyzed the genome of C. trachomatis L2/434 strain for genes encoding mostly uncharacterized proteins that did not appear to possess a signal of the general secretory pathway and which had not been previously experimentally shown to be T3S substrates. We selected several genes with these characteristics and analyzed T3S of the encoding proteins using Yersinia enterocolitica as a heterologous system. We identified 23 C. trachomatis proteins whose first 20 amino acids were sufficient to drive T3S of the mature form of β-lactamase TEM-1 by Y. enterocolitica. We found that 10 of these 23 proteins were also type III secreted in their full-length versions by Y. enterocolitica, providing additional support that they are T3S substrates. Seven of these 10 likely T3S substrates of C. trachomatis were delivered by Y. enterocolitica into host cells, further suggesting that they could be effectors. Finally, real-time quantitative PCR analysis of expression of genes encoding the 10 likely T3S substrates of C. trachomatis showed that 9 of them were clearly expressed during infection of host cells. Conclusions: Using Y. enterocolitica as a heterologous system, we identified 10 likely T3S substrates of C. trachomatis (CT053, CT105, CT142, CT143, CT144, CT161, CT338, CT429, CT656, and CT849) and could detect translocation into host cells of CT053, CT105, CT142, CT143, CT161, CT338, and CT429. Therefore, we revealed several C. trachomatis proteins that could be effectors subverting host cell processes.
- In silico scrutiny of genes revealing phylogenetic congruence with clinical prevalence or tropism properties of Chlamydia trachomatis strainsPublication . Ferreira, R.; Antelo, M.; Nunes, A.; Borges, V.; Damião, V.; Borrego, M.J.; Gomes, João PauloMicrobes possess a multiplicity of virulence factors that confer them the ability to specifically infect distinct biological niches. Contrary to what is known for other bacteria, for the obligate intracellular human pathogen Chlamydia trachomatis, the knowledge of the molecular basis underlying serovars’ tissue specificity is scarce. We examined all ~900 genes to evaluate the association between individual phylogenies and cell-appetence or ecological success of C. trachomatis strains. Only ~1% of the genes presented a tree topology showing the segregation of all three disease groups (ocular, urogenital, and lymphatic) into three wellsupported clades. Approximately 28% of the genes, which include the majority of the genes encoding putative type III secretion system effectors and Inc proteins, present a phylogenetic tree where only lymphogranuloma venereum strains form a clade. Similarly, an exclusive phylogenetic segregation of the most prevalent genital serovars was observed for 61 proteins. Curiously, these serovars are phylogenetically cosegregated with the lymphogranuloma venereum serovars for ~20% of the genes. Some clade-specific pseudogenes were identified (novel findings include the conserved hypothetical protein CT037 and the predicted a-hemolysin CT473), suggesting their putative expendability for the infection of particular niches. Approximately 3.5% of the genes revealed a significant overrepresentation of nonsynonymous mutations, and the majority encode proteins that directly interact with the host. Overall, this in silico scrutiny of genes whose phylogeny is congruent with clinical prevalence or tissue specificity of C. trachomatis strains may constitute an important database of putative targets for future functional studies to evaluate their biological role in chlamydial infections.
- In Silico Scrutiny of Genes Revealing Phylogenetic Congruence with Clinical Prevalence or Tropism Properties of Chlamydia trachomatis StrainsPublication . Ferreira, R.; Antelo, M.; Nunes, A.; Borges, V.; Damião, V.; Borrego, M.J.; Gomes, João PauloMicrobes possess a multiplicity of virulence factors that confer them the ability to specifically infect distinct biological niches. Contrary to what is known for other bacteria, for the obligate intracellular human pathogen Chlamydia trachomatis, the knowledge of the molecular basis underlying serovars' tissue specificity is scarce. We examined all ~900 genes to evaluate the association between individual phylogenies and cell-appetence or ecological success of C. trachomatis strains. Only ~1% of the genes presented a tree topology showing the segregation of all three disease groups (ocular, urogenital, and lymphatic) into three well-supported clades. Approximately 28% of the genes, which include the majority of the genes encoding putative type III secretion system effectors and Inc proteins, present a phylogenetic tree where only lymphogranuloma venereum strains form a clade. Similarly, an exclusive phylogenetic segregation of the most prevalent genital serovars was observed for 61 proteins. Curiously, these serovars are phylogenetically cosegregated with the lymphogranuloma venereum serovars for ~20% of the genes. Some clade-specific pseudogenes were identified (novel findings include the conserved hypothetical protein CT037 and the predicted α-hemolysin CT473), suggesting their putative expendability for the infection of particular niches. Approximately 3.5% of the genes revealed a significant overrepresentation of nonsynonymous mutations, and the majority encode proteins that directly interact with the host. Overall, this in silico scrutiny of genes whose phylogeny is congruent with clinical prevalence or tissue specificity of C. trachomatis strains may constitute an important database of putative targets for future functional studies to evaluate their biological role in chlamydial infections.
- Molecular features underlying the higher ecological success of C. trachomatis E and F genotypesPublication . Nunes, A.; Ferreira, R.; Borges, V.; Borrego, M.J.; Gomes, João PauloIn the light of the >98% genomic similarity among Chlamydia trachomatis serovars, the higher worldwide ecological success of E and F is enigmatic. We intend to provide a quick overview of the molecular data that distinguish these from the remaining strains. Examples are: - E and F possess a similar chromosomal genetic make-up distinct from the remaining genotypes. Some loci linked to this independent co-segregation comprehend membrane proteins, hypothetical virulence factors, and regulatory regions (published data). - Some loci reveal nonrandom mutational patterns, where mutations exclusive of E and F are clustered in specific protein domains, likely promoting strains functional and/or structural attributes (published data). - Based on data from a worldwide survey, MOMP of E and F exhibit the lowest mutation rate (22.3-fold lower), implying more fitted antigenic profiles to deal with host immunity (published data). - The likelihood of E and F strains to undergo genetic recombination is about 12-fold lower than that of the other genotypes (P<10-2), suggesting a putative clonal evolution, where superimposed favorable clones may be strongly maintained in vivo (preliminary data from our lab). - Strains E and F do not seem to originate higher infectious load in vivo, when compared with other genital genotypes (published data). Full-genomic data from multiple and diverse clinical isolates will be essential to decipher the secret behind the higher ecological success of E and F strains.
- Mutation rate of SARS-CoV-2 and emergence of mutators during experimental evolutionPublication . Amicone, M.; Borges, V.; Alves, M.J.; Isidro, J.; Zé-Zé, L.; Duarte, S.; Gomes, J. P.; Gordo, I.Background and objectives: To understand how organisms evolve, it is fundamental to study how mutations emerge and establish. Here, we estimated the rate of mutation accumulation of SARS-CoV- 2 in vitro and investigated the repeatability of its evolution when facing a new cell type but no immune or drug pressures. Methodology: We performed experimental evolution with two strains of SARS-CoV-2, one carrying the originally described spike protein (CoV-2-D) and another carrying the D614G mutation that has spread worldwide (CoV-2-G). After 15 passages in Vero cells and whole genome sequencing, we characterized the spectrum and rate of the emerging mutations and looked for evidences of selection across the genomes of both strains. Results: From the frequencies of the mutations accumulated, and excluding the genes with signals of selection, we estimate a spontaneous mutation rate of 1.3 10 6 6 0.2 10 6 per-base per-infection cycle (mean across both lineages of SARS-CoV-262SEM). We further show that mutation accumulation is larger in the CoV-2-D lineage and heterogeneous along the genome, consistent with the action of positive selection on the spike protein, which accumulated five times more mutations than the corresponding genomic average. We also observe the emergence of mutators in the CoV-2-G background, likely linked to mutations in the RNA-dependent RNA polymerase and/or in the error-correcting exonuclease protein. Conclusions and implications: These results provide valuable information on how spontaneous mutations emerge in SARS-CoV-2 and on how selection can shape its genome toward adaptation to new environments. Lay Summary: Each time a virus replicates inside a cell, errors (mutations) occur. Here, via laboratory propagation in cells originally isolated from the kidney epithelium of African green monkeys, we estimated the rate at which the SARS-CoV-2 virus mutates—an important parameter for understanding how it can evolve within and across humans. We also confirm the potential of its Spike protein to adapt to a new environment and report the emergence of mutators—viral populations where mutations occur at a significantly faster rate.
- Normalization strategies for real-time expression data in Chlamydia trachomatisPublication . Borges, V.; Ferreira, R.; Noguerira, P.; Nunes, A.; Borrego, M.J.; Gomes, João PauloSince Chlamydia trachomatis is a genetically non-tractable pathogen, transcriptomics assumes a fundamental role for the better understanding of its biology. However, the suitability of endogenous controls for normalization of transcriptomic data in this bacterium still needs validation. We aimed to assess the stability of 10 genes for their potential use as endogenous controls in qPCR at both normal and stress (antibiotic treatment) growth conditions throughout the developmental cycle of three strains with different cell-appetence. Normalization was performed using the quantified bacterial genomes. We also tested the applicability of two widely used softwares (geNorm and Normfinder) to our data. For all strains, we found that 16SrRNA was the most stably expressed gene throughout the normal developmental cycle, but it was highly unstable under antibiotic exposure, suggesting prudence when using ribosomal genes as endogenous controls in expression experiments involving stress environments. The geNorm and Normfinder algorithms revealed contrasting results and seem inappropriate for the selected pool of genes. Considering the multiplicity of experimental conditions, there should be an in loco validation of endogenous controls, where 16SrRNA appears to be in the front line. Alternatively, normalization of expression data against genomic DNA, which is less influenced by experimental constraints (especially relevant for intracellular organisms) and stress conditions, likely constitutes a good option. The present study constitutes the first evaluation of putative endogenous controls for real-time expression assays in C. trachomatis