A doublecortin-domain protein of Toxoplasma and its orthologues bind to and modify the structure and organization of tubulin polymers

Abstract

Background: TgDCX is a doublecortin-domain protein associated with the conoid fibers, a set of strongly curved non-tubular tubulin-polymers in Toxoplasma. TgDCX deletion impairs conoid structure and parasite invasion. TgDCX contains two tubulin-binding domains: a partial P25α and the DCX/doublecortin domain. Orthologues are found in apicomplexans and their free-living relatives Chromera and Vitrella. Results: We report that isolated TgDCX-containing conoid fibers retain their pronounced curvature, but loss of TgDCX destabilizes the fibers. We crystallized and determined the 3D-structure of the DCX-domain, which is similar to those of human doublecortin and well-conserved among TgDCX orthologues. However, the orthologues vary widely in targeting to the conoid in Toxoplasma and in modulating microtubule organization in Xenopus cells. Several orthologues bind to microtubules in Xenopus cells, but only TgDCX generates short, strongly curved microtubule arcs. EM analysis shows microtubules decorated with TgDCX bundled into rafts, often bordered on one edge by a "C"-shaped incomplete tube. A Chromera orthologue closely mimics TgDCX targeting in Toxoplasma and binds to microtubules in Xenopus cells, but does not generate arcs or "C"-shaped tubes, and fails to rescue the defects of the TgDCX-knockout parasite. Conclusions: These observations suggest that species-specific features of TgDCX enable it to generate strongly curved tubulin-polymers to support efficient host-cell invasion.

Conservation of theToxoplasmaconoid complex proteome reveals a cryptic conoid inPlasmodiumthat differentiates between blood- and vector-stage zoites

“…Indeed, even in Toxoplasma the function of the conoid is relatively poorly understood. Most studies of individual protein elements have identified molecules required for its assembly and stability [29,[31][32][33][34][35]. But other studies have implicated roles in control processes, including activating motility and exocytosis, both of which are requirements for invasion as well as host egress events [24,28,30].…”

“…The other is that we still know relatively little of the molecular composition of the conoid that would allow the objective testing for the presence of a homologous structure [5]. The conspicuous ultrastructure of conoids such as those of coccidians draw attention to tubulin being a major element, however it is known that there are other conoid proteins responsible for its assembly, stability, and function during invasion [24,[28][29][30][31][32][33][34][35]. To test if a homologous conoid cell feature is present in Aconoidasida, but cryptic by traditional microscopy techniques, fuller knowledge of the molecules that characterise this feature in a 'classic' conoid model is needed.…”

Truncated TPPP – An Endopterygota-specific protein

“…The amino acid sequences needed for tubulin/microtubule binding located in the C-terminus of long-type TPPPs can be found in another TPPP-like protein, apicortin, occurring mostly in apicomplexan parasites [ 21 ]. Indeed, the necessity of this protein for the formation of the structure of the conoid, the nontubular polymeric form of tubulin, was proven [ 22 ]. Since the “truncated” TPPP lacks these amino acids thus it is a logical conclusion that it very probably is not able to bind microtubules.…”

Apicortin, a Constituent of Apicomplexan Conoid/Apical Complex and Its Tentative Role in Pathogen—Host Interaction

“…This domain is also known to play a role in the stabilization of microtubules [ 25 , 26 ]. The two domains, the partial p25alpha and the DCX, have some homology: 22% identity and 30% similarity [ 28 ].…”

“…Otherwise, the sequences are very similar, independently of whether the protein can be found in an apicomplexan species or not. It was shown by Leung et al [ 28 ] that this region of the protein has an outstanding role in tubulin binding.…”