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Unraveling the replication process of Toxoplasma gondii through the MOB1 protein
Publication . L. S. Delgado, Inês; Narciso, Sofia Bizarro Nolasco da Silva; Leitão, José Alexandre da Costa Perdigão e Cameira
ABSTRACT - MOB1 is a conserved protein that regulates cellular proliferation versus apoptosis,
centrosome duplication and cellular differentiation in multicellular eukaryotes and also
cytokinesis and division axis orientation in unicellular and multicellular eukaryotes. Toxoplasma
gondii, an obligate intracellular parasite of veterinary and medical importance, presents one
MOB1 protein. T. gondii interconverts between several cellular stages during its life cycle,
namely between fast replicating tachyzoite and slow replicating bradyzoite stages during its
asexual cycle, a key ability for its success as a parasite. Bradyzoites produce tissue cysts,
establishing a chronic infection that enables recrudescence. Conversion is dependent on cell
cycle regulation and involves cell differentiation and regulation of replication. This led us to
select MOB1 as a strong candidate to be involved in the Toxoplasma replication process. We
employed reverse genetics to assess the Mob1 function in T. gondii. In opposition to what was
observed in other unicellular eukaryotes, as Tetrahymena and Trypanosoma, Mob1 knockout in
T. gondii showed no cytokinesis impairment in its asexual cycle. Instead, we observed an
increase in replication, a decrease in parasitophorous vacuole regularity and a significant loss in
tachyzoite to bradyzoite conversion. Additionally, recombinant MOB1 accumulates in a midline
between the daughter nuclei at the end of mitosis, suggesting MOB1 may be involved in this
process. To elucidate how MOB1 acts in T. gondii, we employed a proximity biotinylation
method and identified the MOB1 interactome. This analysis detected proteins related to several
functional categories, indicating a multivalent role for MOB1 regulated by the ubiquitin proteasome system. We also verified that the Mob1 locus is transcribed from both genomic
strands and gives rise to alternatively spliced variants. Our results indicate that MOB1 is tightly
regulated along the cell cycle and along the life cycle of T. gondii, contributing to the control of
replication and tachyzoite-bradyzoite differentiation.
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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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
EXPL/CVT-EPI/1945/2013