The roles of Centrin 2 and Dynein Light Chain 8a in apical secretory organelles discharge of Toxoplasma gondii

Abstract

To efficiently enter host cells, apicomplexan parasites such as Toxoplasma gondii rely on an apical complex composed of tubulin‐based structures as well as two sets of secretory organelles named micronemes and rhoptries. The trafficking and docking of these organelles to the apical pole of the parasite is crucial for the discharge of their contents. Here, we describe two proteins typically associated with microtubules, Centrin 2 (CEN2) and Dynein Light Chain 8a (DLC8a), that are required for efficient host cell invasion. CEN2 localizes to four different compartments, and remarkably, conditional depletion of the protein occurs in stepwise manner, sequentially depleting the protein pools from each location. This phenomenon allowed us to discern the essential function of the apical pool of CEN2 for microneme secretion, motility, invasion and egress. DLC8a localizes to the conoid, and its depletion also perturbs microneme exocytosis in addition to the apical docking of the rhoptry organelles, causing a severe defect in host cell invasion. Phenotypic characterization of CEN2 and DLC8a indicates that while both proteins participate in microneme secretion, they likely act at different steps along the cascade of events leading to organelle exocytosis.

Distinct Calcium Binding and Structural Properties of Two Centrin Isoforms from Toxoplasma gondii

“…This differential distribution of TgCEN2 points to versatile functions of TgCEN2 in the four distinct structures. Experimental evidence based on gradual depletion and knockdown of TgCEN2 revealed that this protein is involved in several Ca 2+ -mediated processes, namely microneme secretion, motility, host cell attachment, invasion, and egress [21]. In addition, these analyses also indicate that the depletion of TgCEN2 from the four locations follows different kinetics, first from the pre-conoidal rings and peripheral annuli, leading to major invasion defects, and later from basal complex and centrioles, resulting in replication deficiency [21,22].…”

“…Experimental evidence based on gradual depletion and knockdown of TgCEN2 revealed that this protein is involved in several Ca 2+ -mediated processes, namely microneme secretion, motility, host cell attachment, invasion, and egress [21]. In addition, these analyses also indicate that the depletion of TgCEN2 from the four locations follows different kinetics, first from the pre-conoidal rings and peripheral annuli, leading to major invasion defects, and later from basal complex and centrioles, resulting in replication deficiency [21,22]. In this scenario, identifying the physical interactors of TgCEN2 and characterizing the magnitude of the reciprocal interactions between Ca 2+ , TgCEN2 and TgCEN2-binding targets will be crucial to unravel the exact Ca 2+ -sensing properties, mode of action and biological function of this centrin isoform.…”

“…As with centrins in other organisms, TgCEN1 and TgCEN3 are mainly restricted to the centrioles. Though TgCEN2 possesses high sequence similarity with TgCEN1 (~50% sequence identity) and TgCEN3 (~46% sequence identity), it is also found in various cytoskeletal components at the apical and basal portions of the parasite, where it has been demonstrated to function in various aspects of parasite physiology, including secretion, invasion, and replication [19,21,22]. However, little is known about the structural properties of centrins from T. gondii and how they are affected by Ca 2+ .…”

Loss of a conserved MAPK causes catastrophic failure in assembly of a specialized cilium-like structure inToxoplasma gondii

“…In apicomplexan parasites, the cilium was likely adapted to form the organizing core of the "apical complex" of cytoskeletal structures and secretory organelles for which the phylum is named (Figure 1; de Leon et al, 2013). The protein components of the apical complex include orthologs of cilium-associated proteins including centrins (Hu et al, 2006;Lentini et al, 2019), a SAS6 cartwheel protein (de Leon et al, 2013;Lévêque et al, 2016), an associated rootlet fiber (Francia et al, 2012), and a distributed microtubule-organizing center called the apical polar ring (Leung et al, 2017). During the asexual stage, Apicomplexa such as Toxoplasma have a specialized cilium-like structure called the conoid, and typical motile flagella grow at this site during the sexual stage (Francia et al, 2015).…”

The apical annuli ofToxoplasma gondiiare composed of coiled-coil and signaling proteins embedded in the IMC sutures

“…We identify four new annuli proteins, AAP2, AAP3, AAP5, and the AAMT. AAP4 has been previously detected in BioID data sets of the alveolar suture component ISC4 (Chen et al, 2016), of the conoid (Long et al, 2017a) and a protein above the conoid, SAS6L (Lentini et al, 2019). Lentini et al further co-localized AAP4 with Centrin2 and describe a relationship between Centrin2 knockdown and AAP4 abundance in the parasite (Lentini et al, 2019).…”