MICROTUBULE CYTOSKELETON REMODELING DURING TOXOPLASMA GONDII HOST CELL INVASION

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Toxoplasma gondii Tubulin Cofactor B plays a key role in host cell invasion and replication

Publication . Francisco, Samuel Nuno Furtado da Conceição; Narciso, Sofia Bizarro Nolasco da Silva; Leitão, José Alexandre da Costa Perdigão e Cameira

Tubulin 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 hypothesise that these proteins have a role in the regulation of MT cytoskeleton dynamics during Toxoplasma gondii host cell invasion. In this context, we characterized the Tubulin cofactor B (TBCB) in T. gondii. TBCB is a CAPGly domain-containing protein that together with TBCE, interact with and dissociate the tubulin dimer. The TBCB sub-cellular localization in T. gondii was studied using an in-house anti-TBCB serum. T. gondii lines overexpressing TBCB were obtained by random integration as well as TBCB conditional knockout lines by CRISPR/Cas9 system. TBCB transgenic clones were characterized by growing assays (plaque, invasion, replication and egress assays), western blot analysis and fluorescence microscopy (standard, confocal and super-resolution). TBCB showed a polarized localization, at the anterior region of the parasite, under the conoid and in close association with polar ring and subpellicular MTs. It did not present a clear co-localization with the apical complex secretory vesicles, although the interaction with rhoptries and micronemes cannot be excluded. TBCB overexpression lines showed a significant decrease in the capacity to form plaques, attributable to a proportional reduction in the capacity to invade. No differences were observed in replication and egress assays. The TgTBCB knockout line, showed a complete depletion of the protein and a viability no longer than a week. These lines showed a strong reduction in their capacity to invade the host cell and in their replication rate. In the absence of TBCB, cells have an altered axis of division resulting in abnormal division. Some parasites show the loss of the correct division axis and some parasites have four daughter cells forming inside instead of two. TBCB is a polarity marker in T. gondii and is involved in the invasion and replication processes. Its apical localization, together with TBCB mammalian partners already described (MT associated proteins) and the invasion phenotypes, suggest that TBCB can be involved in the intracellular traffic of secretory vesicles depending on MTs. Importantly, TBCB is an essential protein, constituting a good target for new control strategies.

2020-01-20Tese de doutoramento

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Fundação para a Ciência e a Tecnologia

Número da atribuição

SFRH/BD/79423/2011