17th International Congress on Toxoplasmosis

Professor

A Kinase / Phosphatase Negative Feedback Loop Controls Toxoplasma Egress

Host-Parasite Interaction & Signalling

cAMP-dependent protein kinase A (PKAc1) plays a critical role as a negative regulator of egress by rapidly suppressing calcium signaling after invasion. The activity of protein kinase A (PKAc1) is governed by adenylate cyclases and phosphodiesterases that serve to produce and hydrolyze cAMP, respectively. Herein, we investigated the phosphodiesterase PDE2, which one of two essential isoforms out of a total 18 PDE genes in T. gondii. Immunoprecipitated PDE2 preferentially hydrolyzed cAMP in vitro and knockdown of PDE2 led to elevated levels of cytosolic cAMP in vivo, consistent with its role as a cAMP-specific PDE. Knockdown of PKAc1 also resulted in higher cytosolic cAMP levels suggesting that PKAc1 is likely required for PDE2 activation and cAMP turnover. PKAc1 phosphorylation of PDE2 was confirmed in vitro and differentially phosphorylated residues were identified using a conditional PKAc1 knockdown line expressing epitope tagged PDE2. Phospho-mimetic and -ablative forms of PDE2 were purified and tested for activity. Select phospho-mimetic mutations in PDE2 retained activity whereas the corresponding phospho-ablative mutations lost activity, confirming that PKAc1 phosphorylation activates PDE2, while dephosphorylation inhibits activity. Finally, to examine the impact of PDE2 on PKAc1 function, PDE2 depleted parasites were stimulated with zaprinast or ionomycin and then assessed for egress. Zaprinast, but not ionomycin, failed to induce Ca2+ mobilization and egress in PDE2 depleted parasites. These defects were partially rescued when PKAc1 was inhibited by H89 confirming that PKAc1 is primarily responsible inhibiting egress. These data support a model in which PKAc1 targets a process downstream of cGMP and upstream of Ca2+ release to prevent egress, and this block is relieved when PKAc1 phosphorylates PDE2 leading to consumption of cAMP and inhibition of PKAc1. The resulting negative feedback loop counteracts the PKG pathway that drives motility, invasion and egress, thus allowing equilibrium conditions during intracellular development.

Oral presentation

Professor Dr.

Exploring the role an Apical Polar Ring Protein in controlling F-actin translocation and gliding motility in Toxoplasma gondii

Cell Biology

In Toxoplasma gondii, the conoid comprises a cone with spiraling tubulin fibers, preconoidal rings (PCRs), and intraconoidal microtubules (ICMTs). This dynamic organelle undergoes extension and retraction through the apical polar ring (APR) during egress, gliding, and invasion processes. The forces involved in conoid extrusion start to be better understood and its role in directing the F-actin flux to the pellicular space, therefore controlling parasite motility, has been proposed1. However, the contribution of the APR and its interactions with the conoid remain unclear. To advance our comprehension of the APR's architecture, Ultrastructure Expansion Microscopy (U-ExM) was applied to pinpoint known and newly identified APR proteins (APR2-APR7). Our results indicate that the APR forms a fixed multilayered structure. Intriguingly, the conditional depletion of APR7 resulted in significant impairments in both motility and invasion. Electron microscopy revealed a disrupted upper layer of the APR in the absence of APR7, and the use of F-actin binding chromobodies uncovered abnormal flux of F-actin. In summary, this study provides valuable insights into APR assembly and help elucidating how its structure regulates parasite motility. 1. Haase, R et al. Nanoscale imaging of the conoid and functional dissection of its dynamics in Apicomplexa. Curr Opin Microbiol. 2022 Dec:70:102226.

Prof.

Revealing cellular and molecular organization in Toxoplasma's invasion machinery after ionophore stimulation

Host cell invasion by Toxoplasma gondii and other important species within the phylum Apicomplexa, is an active process involving the coordinated action of a remarkable machine at their anterior end known as the apical complex (AC), for which the phylum is named. To date, fundamental details of the AC and capturing how the various apical organelles interact during invasion have been relatively elusive due, in part, to the fact that the entire complex is approximately a single diffraction-limited volume in size (in the visible). The conoid, a dynamic structure within the AC of Toxoplasma gondii and related coccidia, comprises a barrel of uniquely arranged tubulin-based filaments that protrudes during invasion. Numerous proteins have been shown to localize to the conoid, but many of their functions and precise locations have yet to be determined. Using cryogenic electron tomography, a powerful tool to study cellular architecture in situ, we visualize the parasite's AC in a near-native state. By averaging subvolumes of interest, we generated a density map that allowed us to accurately assign tubulins in the conoid fibrils and reveal the organization of tubulin-associated proteins. Using single-molecule, super-resolution fluorescence microscopy, we have determined with high resolution the molecular organization of Myosin H (Myo-H), a motor protein that associates with the conoid and is indispensable for parasite invasion. We similarly imaged tubulin in the parasites to provide context at the spatial scale necessary to determine how the Myo-H distribution organizes with respect to the AC as a whole. By imaging conoid-protruded and -retracted parasites, we monitor Myo-H reorganization in preparation for host cell invasion, bringing greater detail to this critical biological process.

Unveiling the role of conoid gliding protein in Toxoplasma gondii invasion and egress: insights from cryogenic correlative light and electron microscopy (cryo-CLEM)

Toxoplasma gondii exhibits numerous cellular adaptations facilitating the invasion of host cells and the subsequent egress from them after multiplication inside of parasitophorous vacuoles. The apical complex comprised of cytoskeletal machinery and secretory organelles is pivotal in these processes. While the overall morphology of the parasites and the apical complex have been extensively studied using various techniques [1-3], the mechanistic understanding and structural composition of these components remain elusive. In a previous study, we identified novel genes implicated in invasion and egress, notably cgp, which encodes the conoid gliding protein. This protein colocalizes with RNG2, an integral component of Toxoplasma"s conoid. Conditional knockout of the cgp gene resulted in significantly reduced egress rates compared to wild-type parasites. To elucidate the impact of cgp depletion on the structure of the conoid and to gain insight into the underlying molecular mechanisms, we developed cryogenic correlative light and electron microscopy (cryo-CLEM) workflows to target and visualize the molecular architecture of the conoid within intact, cryo-preserved parasites. Our in situ cryo-electron tomography analyses revealed a loss of PCR structure in cgp knockout parasites compared with the wild-type phenotype, while other components of the conoid remained unaffected. Our results show the structural role of cgp protein for proper PCR formation and demonstrate the potential of cryo-CLEM targeted in situ tomography for studying the architecture and life cycle of native apicomplexa at unprecedented detail. 1) Tosetti et al., Essential function of the alveolin network in the subpellicular microtubules and conoid assembly in Toxoplasma gondii, eLife, 2020 2) Koreny et al., Molecular characterization of the conoid complex in Toxoplasma reveals its conservation in all apicomplexans, including Plasmodium species, PLoSBiol, 2021 3) Gui et al., Cryo-tomography reveals rigid-body motion and organization of apicomplexan invasion machinery, Nature Communications, 2023

Bumped Kinase Inhibitors (BKI) inhibit Toxoplasma gondii MAPKL-1 and CDPK1

Metabolism, Biochemistry & Drug Development

Bumped Kinase Inhibitors (BKIs) based on a 5‑aminopyrazole-4-carboxamide (AC) core are a series of compounds that can be optimized to inhibit the calcium-dependent protein kinase 1 (CDPK1) of Toxoplasma gondii, Cryptosporidium species, and other apicomplexan pathogens. Extensive preclinical development of BKIs has identified AC-BKI-1748 as a promising lead compound for toxoplasmosis that is highly effective and non-toxic in animal models. Phenotypic and therapeutic effects of BKIs and previous experiments have suggested other potential inhibitory mechanisms of action in addition to TgCDPK1. The mechanism of BKI-1748 was therefore investigated using a forward genetic screen of chemical mutagenesis, selection of BKI-1748 resistant clones, and whole genome sequencing. Three clones were isolated with unique single nucleotide changes in the ATP binding site of T. gondii mitogen-activated protein kinase-like 1 (MAPKL1) gene in BKI-1748 resistant clones. One of these mutations, Leu162Gln, was introduced into a wild-type strain, resulting in 2- to 9-fold resistance to BKI-1748 and other BKIs including 1-NM-PP1. Because these compounds also were potent inhibitors of the isolated TgCDPK1 enzyme, we introduced a Gly128Met substitution into the TgCDPK1 gatekeeper position and found 2- to 8-fold resistance across BKIs. To further investigate the significance of these targets, we created a clone with both the above substitutions, which demonstrated a 14-fold to 153-fold resistance to BKIs. The identification of TgMAPKL-1 and TgCDPK1 as dual targets of multiple BKIs provides a greater understanding of the BKI mechanism of action and suggests a high genetic barrier to meaningful drug resistance for this promising class of compounds.

Myosin F controls actin organization and dynamics in Toxoplasma gondii

In eukaryotes, intracellular cargo transport typically involves molecular motors associating with the surface of membrane-bound cargo and transporting it along microtubule and actin tracks. In Toxoplasma gondii (T. gondii), intracellular cargo transport relies on actin and the unconventional myosin, myosin F (MyoF). Loss of MyoF disrupts vesicle transport, endomembrane organelle positioning, and apicoplast inheritance, yet the mechanism by which this actomyosin system's facilitates this process remains unclear. Using live-cell imaging, we determined that MyoF-EmFP has a dynamic and filamentous-like localization in the parasite cytosol, reminiscent of cytosolic actin filament dynamics. MyoF was not associated with Golgi, apicoplast or dense granule surfaces, suggesting that MyoF does not function using the canonical cargo transport mechanism. Instead, we find that loss of MyoF results in a dramatic rearrangement of the actin cytoskeleton in interphase parasites accompanied by significantly reduced actin dynamics, while actin organization during replication and motility were unaffected. Thus, these findings reveal that MyoF is an actin-organizing protein in T. gondii which facilitates cargo movement using an unconventional transport mechanism.

Session III: Cell Biology I

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