Browsing by Author "Nunes, A."
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- Adaptive evolution of the Chlamydia trachomatis dominant antigen reveals distinct evolutionary scenarios for B- and T-cell epitopes: worldwide surveyPublication . Nunes, A.; Nogueira, P.J.; Borrego, M.J.; Gomes, João PauloBackground: Chlamydia trachomatis is one of the most disseminated human pathogens, for which no vaccine is available yet. Understanding the impact of the host pressure on pathogen antigens is crucial, but so far it was only assessed for highly-restricted geographic areas. We aimed to evaluate the evolutionary picture of the chlamydial key antigen (MOMP), which is one of the leading multi-subunit vaccine candidates, in a worldwide basis. Methodology/Principal Findings: Using genetics, molecular evolution methods and mathematical modelling, we analyzed all MOMP sequences reported worldwide, composed by 5026 strains from 33 geographic regions of five continents. Overall, 35.9% of variants were detected. The evolutionary pattern of MOMP amino acid gains/losses was found to differ from the remaining chromosome, reflecting the demanding constraints of this porin, adhesin and dominant antigen. Amino acid changes were 4.3-fold more frequent in host-interacting domains (P,10212), specifically within B-cell epitopes (P,1025), where 25% of them are at fixation (P,1025). According to the typical pathogen-host arms race, this rampant B-cell antigenic variation likely represents neutralization escape mutants, as some mutations were previously shown to abrogate neutralization of chlamydial infectivity in vitro. In contrast, T-cell clusters of diverse HLA specificities are under purifying selection, suggesting a strategy that may lead to immune subversion. Moreover, several silent mutations are at fixation, generating preferential codons that may influence expression, and may also reflect recombination-derived ‘hitchhikingeffect’ from favourable nonsilent changes. Interestingly, the most prevalent C. trachomatis genotypes, E and F, showed a mutation rate 22.3-fold lower than that of the remainder (P,10220), suggesting more fitted antigenic profiles. Conclusions/Significance: Globally, the adaptive evolution of the C. trachomatis dominant antigen is likely driven by its complex pathogenesis-related function and reflects distinct evolutionary antigenic scenarios that may benefit the pathogen, and thus should be taking into account in the development of a MOMP-based vaccine.
- 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.
- Evolution, phylogeny, and molecular epidemiology of ChlamydiaPublication . Nunes, A.; Gomes, João PauloThe Chlamydiaceae are a family of obligate intracellular bacteria characterized by a unique biphasic developmental cycle. It encompasses the single genus Chlamydia, which involves nine species that affect a wide range of vertebral hosts, causing infections with serious impact on human health (mainly due to Chlamydia trachomatis infections) and on farming and veterinary industries. It is believed that Chlamydiales originated 700 mya, whereas C. trachomatis likely split from the other Chlamydiaceae during the last 6 mya. This corresponds to the emergence of modern human lineages, with the first descriptions of chlamydial infections as ancient as four millennia. Chlamydiaceae have undergone a massive genome reduction, on behalf of the deletional bias ‘‘use it or lose it’’, stabilizing at 1–1.2 Mb and keeping a striking genome synteny. Their phylogeny reveals species segregation according to biological properties, with huge differences in terms of host range, tissue tropism, and disease outcomes. Genome differences rely on the occurrence of mutations in the >700 orthologous genes, as well as on events of recombination, gene loss, inversion, and paralogous expansion, affecting both a hypervariable region named the plasticity zone, and genes essentially encoding polymorphic and transmembrane head membrane proteins, type III secretion effectors and some metabolic pathways. Procedures for molecular typing are still not consensual but have allowed the knowledge of molecular epidemiology patterns for some species as well as the identification of outbreaks and emergence of successful clones for C. trachomatis. This manuscript intends to provide a comprehensive review on the evolution, phylogeny, and molecular epidemiology of Chlamydia.
- Genomes of Helicobacter pylori prophagesPublication . Vale, F.F.; Nunes, A.; Oleastro, M.; Gomes, J.P.; Sampaio, D.; Rocha, R.; Vítor, J.; Engstrand, L.; Pascoe, B.; Berthenet, E.; Sheppard, S.; Hitchings, M.D.; Mégraud, F.; Vadivedu, J.; Lehours, P.Nearly 20% of the Helicobacter pylori genomes carry prophages genes. Recently we were able to clearly differentiate four populations of prophages according to geographical origin of host strain. Interestingly we were able to discriminate between Northern Europe and Southern Europe using a phage sequence typing based on 2 prophage genes of H. pylori (integrase and holin) but present in only a minority of strains.
- 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.
- 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
- Normalization strategies for real-time expression data in Chlamydia trachomatisPublication . Borges, V.; Ferreira, R.; Nunes, A.; Nogueira, P.; Borrego, M.J.; Gomes, João PauloChlamydia trachomatis is a widespread obligate intracellular pathogen genetically non-tractable for which transcriptomics is a fundamental tool to better understand 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 real-time quantitative PCR assays at both normal and stress (D-cycloserine treatment) growth conditions throughout the developmental cycle of three C. trachomatis strains with different tissue tropism. Normalization was performed by real-time absolute quantification of the 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 chlamydial normal developmental cycle, which indicates its potential use as endogenous control in relative expression assays. However, it was highly unstable under D-cycloserine treatment (where oppA_2 was top-ranked), suggesting prudence when using ribosomal genes in expression experiments involving stress conditions. 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 that are especially relevant for intracellular organisms, likely constitutes a good option. Moreover, the number of genomes also seems to be less subject to variation than expression of endogenous controls when working under stress conditions. The present study constitutes the first evaluation of putative endogenous controls for real-time expression assays in C. trachomatis.