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Projeto de investigação
The katanin role in Mt cytoskeleton remodeling during host cell invasion by Toxoplasma gondii
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Besnoitia besnoiti and Toxoplasma gondii invasion : the role of the parasite's tubulin folding pathway and manipulation of host cell organization
Publication . Cardoso, Rita Isabel de Amorim; Leitão, José Alexandre da Costa Perdigão e Cameira; Soares, Maria Helena Antunes
Besnoitia besnoiti and Toxoplasma gondii, the etiological agents of besnoitiosis and toxoplasmosis, respectively, are two apicomplexan parasites unable to replicate outside the host cell. In order to survive inside the host cell, these parasites have developed strategies to subvert the cytoskeleton and the endomembrane system of the host cell. In this work, the strategies used by B. besnoiti and T. gondii to manipulate the cytoskeleton, remodeling microtubules (MTs), and interfering with the centrosome and Golgi apparatus of the host cell are studied. We observed that the parasitophorous vacuole (PV) of both parasites is surrounded by host MTs, but only T. gondii recruits the host cell centrosome towards the PV. However, the host Golgi apparatus is recruited to the PV by both parasites but its organization is affected in different ways. The differences found between these two parasites are most likely a result of two distinct evolutionary mechanisms and might reflect the different tissue tropism and pathogeny.
Since not only the host cell cytoskeleton, but also the cytoskeleton of the parasite, participate in the establishment of infection, this work also addresses the role of the parasite cytoskeleton during entry and development inside the host cell. For this we propose that components of the tubulin folding pathway are good candidates to regulate cytoskeleton dynamics and reorganization during host invasion. Thus, we started the characterization of the gene structure and expression patterns of the components of tubulin folding pathway (CCTα, TBCB, TBCE and α-tubulin). These studies suggest that these proteins have an important role in parasite replication.
Overall, our results contribute to the present knowledge of the mechanisms underlying host cell invasion by these parasites, which might be important for the definition of future therapeutic strategies.
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.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/CVT/105470/2008