Molecular characterization of the conoid complex in Toxoplasma reveals its conservation in all apicomplexans, including Plasmodium species

Abstract

The apical complex is the instrument of invasion used by apicomplexan parasites, and the conoid is a conspicuous feature of this apparatus found throughout this phylum. The conoid, however, is believed to be heavily reduced or missing from Plasmodium species and other members of the class Aconoidasida. Relatively few conoid proteins have previously been identified, making it difficult to address how conserved this feature is throughout the phylum, and whether it is genuinely missing from some major groups. Moreover, parasites such as Plasmodium species cycle through 3 invasive forms, and there is the possibility of differential presence of the conoid between these stages. We have applied spatial proteomics and high-resolution microscopy to develop a more complete molecular inventory and understanding of the organisation of conoid-associated proteins in the model apicomplexan Toxoplasma gondii. These data revealed molecular conservation of all conoid substructures throughout Apicomplexa, including Plasmodium, and even in allied Myzozoa such as Chromera and dinoflagellates. We reporter-tagged and observed the expression and location of several conoid complex proteins in the malaria model P. berghei and revealed equivalent structures in all of its zoite forms, as well as evidence of molecular differentiation between blood-stage merozoites and the ookinetes and sporozoites of the mosquito vector. Collectively, we show that the conoid is a conserved apicomplexan element at the heart of the invasion mechanisms of these highly successful and often devastating parasites.

Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid

“…This additional ring is composed of MyoB, which was proposed to fulfil, at least in part, a similar role as the conoidassociated myosin H in T. gondii [38]. Interestingly, while MyoB is also expressed in schizonts [37], the ATR/SAS6L ring is not observed at this stage [36], further confirming that the Plasmodium apical complex shows stage-specific variations in its composition, as previously proposed [29,36]. In line with this, our results also reveal additional species specificities at given stages with the observation of different numbers of subpellicular microtubules between P. berghei and P. falciparum merozoites.…”

“…Altogether, multiple lines of evidence indicate that the ATR is related to the conoid (Fig 6B). First, the ATR colocalises closely with the SAS6L ring [36] that is associated with the conoid in T. gondii [29,35]. Second, the ATR position relative to the APR depends on the activation of ookinete motility and secretion, a characteristic reminiscent of the enigmatic protrusion of the conoid that happens upon activation of secretion and motility in T. gondii tachyzoites [9].…”

“…The third proximal ring, the APR, is much thicker than the 2 other apical rings and serves as a MTOC for the subpellicular microtubules. Despite previous observations of the apical complex by ultrastructural studies, there is relatively limited knowledge of its molecular composition and variation across the different zoite stages in Plasmodium [29]. We thus analysed expanded P. berghei ookinetes similarly stained for α/β tubulin and polyglutamylated tubulin (Fig 4A).…”

“…First, we noticed that the ATR is not polyglutamylated consistent with previous observations for the conoid of T. gondii [16] (Fig 4A). Of relevance, some proteins associated with the conoid in T. gondii [29,35] are also conserved in Plasmodium [29]. This includes the SAS-6-like (SAS6L) protein that has been demonstrated to form an apical ring in Plasmodium ookinetes [36].…”

“…It is possible that additional preconoidal ring proteins create this extension but that the associated structures are not as electron dense as known preconoidal rings in T. gondii tachyzoites or P. falciparum merozoites to be assigned as such by EM or NHS-ester U-ExM. In addition to SAS6L and MyoB, several other proteins associated with the conoid in T. gondii have been recently shown to be expressed in Plasmodium schizonts, ookinetes, and sporozoites [9,29] and may localise at different locations in the apical complex. For example, the recently described PBANKA_1334800 that shows radial spines at the apical end of ookinetes [29] is likely one of the proteins in between the IMC and the apical ends of the subpellicular microtubules.…”