Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion.
Author summary One of the most common motifs that binds calcium to transduce intracellular signals is called an EF hand- named after the globular domain structure first characterized in ovalbumin. A conserved cluster of four EF hands, each of which that binds one calcium atom, is a conserved feature of calmodulin, centrins, and calmodulin-like proteins, including myosin light chains. Although the presence of EF hands is predictive of calcium binding, it alone does not allow classification of biological function as this set of conserved proteins have very diverse functions. Here we used modified editing procedures based on CRISPR/Cas9 combined with a plant-like degradation system to define the roles of three calmodulin-like proteins in T. gondii. These proteins all localized to a specialized apical structure called the conoid where they overlap with the motor protein called MyoH. Additionally, biochemical and genetic studies suggest they coordinately regulate cell invasion. These new genomic editing tools, combined with an efficient system for protein degradation, expand the functional tool kit for an analysis of essential genes and proteins in T. gondii.