Browsing by Author "Almeida, Filipe"
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- Adaptive evolution of PB1 from influenza A(H1N1)pdm09 virus towards an enhanced fitnessPublication . Santos, Luís A.; Almeida, Filipe; Gíria, Marta; Trigueiro-Louro, João; Rebelo-de-Andrade, HelenaPB1 influenza virus retain traces of interspecies transmission and adaptation. Previous phylogenetic analyses highlighted mutations L298I, R386K and I517V in PB1 to have putatively ameliorated the A(H1N1)pdm09 adaptation to the human host. This study aimed to evaluate the reversal of these mutations and infer the role of these residues in the virus overall fitness and adaptation. We generate PB1-mutated viruses introducing I298L, K386R and V517I mutations in PB1 and evaluate their phenotypic impact on viral growth and on antigen yield. We observed a decrease in viral growth accompanied by a reduction in hemagglutination titer and neuraminidase activity, in comparison with wt. Our data indicate that the adaptive evolution occurred in the PB1 leads to an improved overall viral fitness; and such biologic advantaged has the potential to be applied to the optimization of influenza vaccine seed prototypes.
- 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.
- NS1 protein as a novel anti-influenza target: Map-and-mutate antiviral rationale reveals new putative druggable hot spots with an important role on viral replicationPublication . Trigueiro-Louro, João; Santos, Luís A.; Almeida, Filipe; Correia, Vanessa; Brito, Rui M.M.; Rebelo-de-Andrade, HelenaInfluenza NS1 is a promising anti-influenza target, considering its conserved and druggable structure, and key function in influenza replication and pathogenesis. Notwithstanding, target identification and validation, strengthened by experimental data, are lacking. Here, we further explored our previously designed structure-based antiviral rationale directed to highly conserved druggable NS1 regions across a broad spectrum of influenza A viruses. We aimed to identify NS1-mutated viruses exhibiting a reduced growth phenotype and/or an altered cell apoptosis profile. We found that NS1 mutations Y171A, K175A (consensus druggable pocket 1), W102A (consensus druggable pocket 3), Q121A and G184P (multiple consensus druggable pockets) - located at hot spots amenable for pharmacological modulation - significantly impaired A(H1N1)pdm09 virus replication, in vitro. This is the first time that NS1-K175A, -W102A, and -Q121A mutations are characterized. Our map-and-mutate strategy provides the basis to establish the NS1 as a promising target using a rationale with a higher resilience to resistance development.
- Optimization of A(H1N1)pdm09 vaccine seed viruses: The source of PB1 and HA vRNA as a major determinant for antigen yieldPublication . Almeida, Filipe; Santos, Luís A.; Trigueiro-Louro, João M.; Rebelo-de-Andrade, HelenaVaccination prevents and reduces the severity of influenza virus infections. Continuous evolution of influenza hemagglutinin (HA) and neuraminidase (NA) supports the virus to evade pre-existing immunity, which demands vaccines to be reformulated every year. Incorporation of polymerase basic protein 1 (PB1) viral RNA (vRNA) of the same origin of HA and NA vRNA has been observed in previous pandemic viruses and occasionally reported for influenza A vaccine prototype strains of prior seasons. At this point, it remains to be explored whether this phenomenon translates into an improved growth phenotype. In this work, we showed that the HA vRNA of A(H1N1)pdm09 is generally incorporated with the PB1 vRNA of the same origin, establishing the beneficial effect of the presence of PB1 and the pattern of the PB1-HA co-incorporation in the A(H1N1)pdm09 model. We further investigated the putative interplay between PB1 and antigenic proteins regarding the vRNA composition of the progeny and observed that vRNA segregation does not appear to be mainly determined by protein-protein interactions; while vRNA-vRNA interactions can be suggested as the main driving force. Our data also indicate an increase in the hemagglutination capacity and neuraminidase activity due to incorporation of PB1, HA and NA from A(H1N1)pdm09, in comparison with the recombinant virus incorporating only HA and NA from A(H1N1)pdm09 - which have the potential to improve current limitations regarding antigenicity and immunogenicity of influenza vaccines. Further knowledge of the complex vRNA-vRNA interaction network between PB1 and HA will additionally contribute to improve current vaccine formulation, and to gradually optimize the production of A(H1N1)pdm09 reverse genetics vaccine seed virus towards a higher cost-effectiveness.
- Polymorphisms in Inc Proteins and Differential Expression of inc Genes among Chlamydia trachomatis Strains Correlate with Invasiveness and Tropism of Lymphogranuloma Venereum IsolatesPublication . Almeida, Filipe; Borges, Vitor; Ferreira, Rita; Borrego, Maria José; Gomes, João Paulo; Mota, Luis JaimeChlamydia trachomatis is a human bacterial pathogen that multiplies only within an intracellular membrane-bound vacuole, the inclusion. C. trachomatis includes ocular and urogenital strains, usually causing infections restricted to epithelial cells of the conjunctiva and genital mucosa, respectively, and lymphogranuloma venereum (LGV) strains, which can infect macrophages and spread into lymph nodes. However, C. trachomatis genomes display >98% identity at the DNA level. In this work, we studied whether C. trachomatis Inc proteins, which have a bilobed hydrophobic domain that may mediate their insertion in the inclusion membrane, could be a factor determining these different types of infection and tropisms. Analyses of polymorphisms and phylogeny of 48 Inc proteins from 51 strains encompassing the three disease groups showed significant amino acid differences that were mainly due to variations between Inc proteins from LGV and ocular or urogenital isolates. Studies of the evolutionary dynamics of inc genes suggested that 10 of them are likely under positive selection and indicated that most nonsilent mutations are LGV specific. Additionally, real-time quantitative PCR analyses in prototype and clinical strains covering the three disease groups identified three inc genes with LGV-specific expression. We determined the transcriptional start sites of these genes and found LGV-specific nucleotides within their promoters. Thus, subtle variations in the amino acids of a subset of Inc proteins and in the expression of inc genes may contribute to the unique tropism and invasiveness of C. trachomatis LGV strains.