Browsing by Author "Basto, Afonso P."
Now showing 1 - 6 of 6
Results Per Page
Sort Options
- Balancing act: tubulin glutamylation and microtubule dynamics in Toxoplasma gondiiPublication . Delgado, Inês; Gonçalves, João; Fernandes, Rita; Zúquete, Sara; Basto, Afonso P.; Leitão, Alexandre; Soares, Helena; Nolasco, SofiaThe success of the intracellular parasite Toxoplasma gondii in invading host cells relies on the apical complex, a specialized microtubule cytoskeleton structure associated with secretory organelles. The T. gondii genome encodes three isoforms of both α- and β-tubulin, which undergo specific post-translational modifications (PTMs), altering the biochemical and biophysical proprieties of microtubules and modulating their interaction with associated proteins. Tubulin PTMs represent a powerful and evolutionarily conserved mechanism for generating tubulin diversity, forming a biochemical 'tubulin code' interpretable by microtubule-interacting factors. T. gondii exhibits various tubulin PTMs, including α-tubulin acetylation, α-tubulin detyrosination, Δ5α-tubulin, Δ2α-tubulin, α- and β-tubulin polyglutamylation, and α- and β-tubulin methylation. Tubulin glutamylation emerges as a key player in microtubule remodeling in Toxoplasma, regulating stability, dynamics, interaction with motor proteins, and severing enzymes. The balance of tubulin glutamylation is maintained through the coordinated action of polyglutamylases and deglutamylating enzymes. This work reviews and discusses current knowledge on T. gondii tubulin glutamylation. Through in silico identification of protein orthologs, we update the recognition of putative proteins related to glutamylation, contributing to a deeper understanding of its role in T. gondii biology.
- Balancing act: tubulin glutamylation and microtubule dynamics in Toxoplasma gondiiPublication . Delgado, Inês L.S.; Gonçalves, João; Fernandes, Rita; Zúquete, Sara; Basto, Afonso P.; Leitão, Alexandre; Soares, Helena; Nolasco, SofiaThe success of Toxoplasma gondii (intracellular parasite) host cell invasion relies on the apical complex, a specialized microtubule cytoskeleton structure associated with secretory organelles. The genome encodes three isoforms of both α- and β-tubulin which are altered by specific post-translational modifications (PTMs), changing the biochemical/biophysical proprieties of microtubules, and modulating their interaction with associated proteins. Tubulin PTMs are a powerful and evolutionarily conserved mechanism to generate tubulin diversity, forming a biochemical ‘tubulin code’ that can be ‘read’ by microtubule-interacting factors. The T. gondii tubulin PTMs are: α-tubulin acetylation, α-tubulin detyrosination, Δ5α-tubulin, Δ2α-tubulin, α- and β-tubulin polyglutamylation, and α- and α-tubulin methylation. Tubulin glutamylation is a key candidate to assist microtubule remodeling in Toxoplasma, being involved in the regulation of microtubule stability, dynamics, interaction with motor proteins, and severing enzymes. The correct balance of tubulin glutamylation is achieved by the coordinated action of polyglutamylases and deglutamylating enzymes. In this work, we will review and discuss the current knowledge on T. gondii tubulin glutamylation. By in silico identification of mammalian protein orthologs, we explored and updated the identification of putative proteins related to glutamylation, contributing to a better understanding of the role of tubulin glutamylation in T. gondii.
- Characterization of a MOB1 homolog in the Apicomplexan parasite toxoplasma gondiiPublication . Delgado, Inês L. S.; Tavares, Alexandra; Francisco, Samuel; Santos, Dulce; Coelho, João; Basto, Afonso P.; Zúquete, Sara; Müller, Joachim; Hemphill, Andrew; Meissner, Markus; Soares, Helena; Leitão, Alexandre; Nolasco, S.Monopolar spindle One Binder1 (MOB1) proteins are conserved components of the tumor-suppressing Hippo pathway, regulating cellular processes such as cytokinesis. Apicomplexan parasites present a life cycle that relies on the parasites’ ability to differentiate between stages and regulate their proliferation; thus, Hippo signaling pathways could play an important role in the regulation of the apicomplexan life cycle. Here, we report the identification of one MOB1 protein in the apicomplexan Toxoplasma gondii. To characterize the function of MOB1, we generated gain-of-function transgenic lines with a ligand-controlled destabilization domain, and loss-of-function clonal lines obtained through CRISPR/Cas9 technology. Contrary to what has been characterized in other eukaryotes, MOB1 is not essential for cytokinesis in T. gondii. However, this picture is complex since we found MOB1 localized between the newly individualized daughter nuclei at the end of mitosis. Moreover, we detected a significant delay in the replication of overexpressing tachyzoites, contrasting with increased replication rates in knockout tachyzoites. Finally, using the proximity-biotinylation method, BioID, we identified novel members of the MOB1 interactome, a probable consequence of the observed lack of conservation of some key amino acid residues. Altogether, the results point to a complex evolutionary history of MOB1 roles in apicomplexans, sharing properties with other eukaryotes but also with divergent features, possibly associated with their complex life cycle.
- Combined TLR2/TLR4 activation equip non-mucosal dendritic cells to prime Th1 cells with gut tropismPublication . Zúquete, Sara; Ferreira, Mariana; Delgado, Inês L. S.; Gazalle, Paula; Andaluz, Stephanie; Rosa, Maria Teresa; Mendes, Ana Catarina; Santos, Dulce; Nolasco, Sofia; Graça, Luís; Leitão, Alexandre; Basto, Afonso P.Activated CD4+ T cells located at mucosal surfaces orchestrate local effector immune mechanisms. When properly polarized, these cells contribute to block infections at early stages and may be essential to restrain the local growth of mucosal tumors, playing a critical role in host protection. How CD4+ T cells simultaneously integrate gut-homing instructions and Th polarization signals transmitted by TLR-activated dendritic cells (DCs) is unknown. Here, we show that the combined activation through TLR2, which alone does not induce a clear Th polarization, and TLR4, which alone does not imprint mucosal tropism, equip non-mucosal DCs to prime gut-homing CD4+ T cells with reinforced Th1 polarization. These results show that targeting DCs with combined innate stimuli with distinct properties is a rational strategy to program the outcome of T cell polarization and simultaneously control their tissue tropism. Exploring this strategy could enhance the efficacy of vaccines and immune cell therapies.
- A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulationsPublication . Basto, Afonso P.; Piedade, João; Ramalho, Ruben; Alves, Susana; Soares, Helena; Cornelis, Pierre; Martins, Carlos; Leitão, AlexandreThe conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.
- The apicomplexan parasite Toxoplasma gondiiPublication . Delgado, Inês L. S.; Zúquete, Sara; Santos, Dulce; Basto, Afonso P.; Leitão, Alexandre; Nolasco, SofiaToxoplasma gondii is a ubiquitous zoonotic parasite with an obligatory intracellular lifestyle. It relies on a specialized set of cytoskeletal and secretory organelles for host cell invasion. When infecting its felid definitive host, T. gondii undergoes sexual reproduction in the intestinal epithelium, producing oocysts that are excreted with the feces and sporulate in the environment. In other hosts and/or tissues, T. gondii multiplies by asexual reproduction. Rapidly dividing tachyzoites expand through multiple tissues, particularly nervous and muscular tissues, and eventually convert to slowly dividing bradyzoites which produce tissue cysts, structures that evade the immune system and remain infective within the host. Infection normally occurs through ingestion of sporulated oocysts or tissue cysts. While T. gondii is able to infect virtually all warm-blooded animals, most infections in humans are asymptomatic, with clinical disease occurring most often in immunocompromised hosts or fetuses carried by seronegative mothers that are infected during pregnancy.