Browsing by Author "Francisco, S."
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- Characterization of the Tubulin-binding cofactor B in Toxoplasma gondii, a zoonotic parasitePublication . Delgado, I.; Francisco, S.; Santos, D.; Zúquete, S.; Leitão, A.; Soares, Helena; Nolasco, SofiaTubulin cofactors participate in the folding, dimerization, and dissociation pathways of the tubulin dimer, being implicated in the control of tubulin proteostasis and consequently in the control of microtubule (MT) dynamics in vivo. We identified a gene coding for a Tubulin cofactor B in the T. gondii genome and characterized its protein (tgTBCB) in the tachyzoite stage.
- Estudo do interactoma da proteína MOB1 de Toxoplasma gondii usando ferramentas bioinformáticas e tecnologia BioIDPublication . Delgado, I. L.; Tavares, A.; Francisco, S.; Santos, D.; Zúquete, S.; Leitão, A.; Soares, Helena; Nolasco, SofiaO protozoário Toxoplasma gondii é um parasita intracelular obrigatório pertencente ao filo Apicomplexa, único pela sua capacidade de infetar qualquer vertebrado. As cinases MOB integram o módulo nuclear da Hippo (humano) e da mitotic exit network (levedura), vias sinalizadoras reguladoras de citocinese, proliferação celular e apoptose, em eucariotas. Através de BLAST, identificámos uma proteína MOB1 em T. gondii. Combinando as bases de dados STRING e ToxoDB.org e a tecnologia BLAST, identificámos como prováveis interactores da proteína MOB1 em T. gondii: 6 proteínas homólogas de membros nucleares das vias Hippo/MEN (YAP, TAZ, LATS/Dbf2, MST/Cdc15, Tem1, Nud1) e 7 proteínas relacionadas com sinalização celular (cinases e ativadores de GTPase). Esta rede de proteínas constitui uma base de trabalho para a caracterização dos mecanismos reguladores do ciclo celular de T. gondii. Para identificar experimentalmente interactores da MOB1, utilizámos a técnica BioID. Consiste na expressão da proteína de interesse em fusão com BirA que irá biotinilar proteínas na sua proximidade. Construímos duas estirpes transgénicas de T. gondii a sobrexpressar as proteínas de fusão Myc-BirA (controlo) e Myc-MOB1-BirA (teste). Isolámos as proteínas biotiniladas por imunoprecipitação com streptavidina e a sua identificação foi feita por espectrometria de massa. Detetámos 3 proteínas ainda não caracterizadas em T. gondii: 2 proteínas transmembranares e uma proteína com domínios funcionais relacionados com a manutenção estrutural de cromossomas. Apesar destas proteínas não terem sido identificadas pelos métodos bioinformáticos, já foram descritas, em outros organismos, proteínas MOB no centrossoma/corpo polar do fuso, cinetocóros e fuso acromático. Os resultados bioinformáticos indicam que as vias sinalizadoras Hippo/mitotic exit network poderão ser ativas em T. gondii, apoiando o provável papel de MOB1 na replicação de T. gondii. Porém, os resultados experimentais, embora preliminares, sugerem que neste parasita estas proteínas poderão ter papéis ainda não descritos em outros organismos. Estão em curso experiências adicionais para testar estes candidatos a interactores da MOB1.
- Study of Mob1 in the apicomplexan Toxoplasma gondii: at the crossroads of asexual and sexual developmentPublication . Delgado, I. L.; Francisco, S.; Zúquete, S.; Leitão, A.; Soares, Helena; Nolasco, SofiaToxoplasma gondii, the causative agent of toxoplasmosis, is the uttermost member of apicomplexan parasites due to its zoonotic character. To achieve a sustainable infection, parasites must avoid overgrowth given that uncontrolled parasite growth may lead to the death of both the host and the parasite. Indeed, understanding how parasites regulate their replication rate, namely balancing proliferation versus cell death, is essential to understanding and controlling parasitic diseases.
- The two apicomplexa Besnoitia besnoiti and Toxoplasma gondii fifferentially after intrinsic host cell polarity by manipulating centrosome and Golgi apparatusPublication . Cardoso, R.; Francisco, S.; Delgado, Inês; Nolasco, Sofia; Leitão, Alexandra; Soares, HelenaObligate intracellular parasites have co-evolved with hosts to be able to invade their cells and flourish. To be successful they need to establish specific molecular parasite-host cell interactions and then manipulate the host cell structures, mechanisms and pathways in order to replicate and grow. In previous work, we described that upon interaction with the host cell, the apicomplexan Besnoitia besnoiti undergoes dramatic modifications of shape and surface, as revealed by atomic force microscopy, accompanied by a distinct tubulin labeling on the posterior region. In the host cell, the microtubule cytoskeleton shows a re-arrangement around the parasitophorous vacuole (PV). This phenomenon was also observed in the closely related parasite Toxoplasma gondii. During this event, our data suggest that this parasite modulates the levels of tubulin polyglutamylation by controlling the factors that regulate the levels and pattern of tubulin post-translation modifications namely TTLLs, CCPs and severing microtubule enzymes such as spastin, katanin and fidgitin. Also, we have observed that T. gondii recruits the host cell centrosome towards the PV, whereas B. besnoiti does not. Notably, both parasites recruit the host Golgi apparatus to the PV but its organization is differentially affected. Moreover, T. gondii replication rate decreases in cells over-expressing TBCCD1 but not in TBCCD1 depleted cells, while for B. besnoiti no differences were found. However, B. besnoiti promotes a reorganization of the Golgi ribbon previously fragmented by TBCCD1 depletion. In fact, as described by us, TBCCD1 is involved in centrosome positioning and Golgi apparatus integrity Furthermore, the T. gondii tubulin cofactor B (TBCB) gene, a member of tubulin folding pathway that also controls microtubule dynamics through the recycling/degradation of the native tubulin heterodimers is involved in the invasion process of T. gondii. Taken together our results strongly support the importance that a successful establishment of the PVs in the host cell requires a cross-talk between the parasite and the host cytoskeleton through the regulation of the factors that control cytoskeleton specific functions and dynamics. Lastly, the differences found in how T. gondii and B. besnoiti interact with their host cells may indicate different evolutionary paths.