Functional characterization of apically localized calmodulins that regulate motility and cell invasion in Toxoplasma gondii
Authors: Long Shaojun 
Published: November 2023

Calcium has long been recognized as an important second messenger in eukaryotes. Toxoplasma gondii, a species of the phylum of Apicomplexa, infects warm‐blooded animals including an estimated 25% of the human population. Apicomplexans are characterized by an apical complex consisting of a unique microtubule rich apical cap, called the conoid, which serves or organize apical secretory organelles and singlet microtubules that underlie the membrane. The T. gondii genome contains many genes encoding proteins with EF hands, a conserved motif of acidic residues that binds to calcium, as first described in calmodulin (CaM). These CaM‐like proteins include those that function as light chain kinases for myosins, as centrins involved in cell division, or that regulate other activities. Few studies have been done on the function of these CaM‐like proteins in T. gondii. Using CRIPSR/Cas9‐mediated gene editing, we tagged CaM1 and CaM2, two proteins that were previously described from a conoid‐enriched fraction, as well as a third newly discovered CaM3 protein that is also localized to the conoid. Although CaM1 and CaM2 were easily disrupted individually by CRISPR/Cas9, the combined knockout was not viable. CaM3 was also refractory to disruption, suggesting it plays an important role. Although CRISRP/Cas9 mediated gene disruption provides a powerful means of testing the importance of genes, it does not lend itself to functional analysis of specific phenotypes in cases where the gene is refractory to disruption. To study the synthetic lethal phenotype of TgCaM1 combined withTgCaM2, and the apparently essential function of TgCaM3, we adapted the plant auxin‐inducible degron (AID) system as described for T. gondii in an accompanying abstract (Brown K.M., et al.,). In combination with CRISPR/Cas9 tagging, we generated AID‐fusion proteins and efficiently targeted the proteins for degradation by addition of auxin in a genetic background expressing the response protein Tir1. In a Δcam2 deletion strain, addition of auxin led to rapid degradation of TgCaM1‐AID resulting in a moderate decrease in egress, a strong defect in parasite motility, decreased invasion of host cells, and a failure to form plaques on monolayers. In contrast, auxin‐induced degradation of TgCaM3‐AID had a moderate effect on egress and invasion, and stunted plaque formation. These phenotypes mimic those for the MyoH, a molecular motor localized at the conoid, which led us to pursue the interactions of these CaMs with MyoH. The interactions were arguably confirmed by biotinylation of MyoH in the CaMs‐BirA strains and by the fact that these CaMs were either mis‐targeted to the cytosol (CaM1, CaM2) or degraded (CaM3) in the MyoH‐AID upon auxin induction. Taken together, the auxin‐inducible degron system combined with CRISPR technology allows for rapid dissection of protein interactions that regulate important steps in parasite motility and cell invasion.Support or Funding InformationThis work was supported by the National Institutes of Health, National Institute of Allergy and Infectious Diseases grant