TgCentrin2 is required for invasion and replication in the human parasite Toxoplasma gondii

Abstract

Centrins are EF-hand containing proteins ubiquitously found in eukaryotes and are key components of centrioles/basal bodies as well as certain contractile fibers. We previously identified three centrins in the human parasite Toxoplasma gondii, all of which localized to the centrioles. However, one of them, TgCentrin2 (CEN2), is also targeted to ring-shaped structures at the apical and basal ends of the parasite, as well as to multiple annuli at the junction between the apical cap and the rest of the membrane cortex. The role(s) that TgCentrin2 plays in these locations was unknown. Here we report the functional characterization of TgCentrin2 in the parasite lytic cycle. After multiple unsuccessful attempts to knock out or knock down the TgCentrin2 gene with existing tools, we designed a new conditional knockdown method that combines transcriptional and protein stability control to achieve tight regulation of TgCentrin2 levels in the parasite. We discovered that under knockdown conditions, there was an ordered loss of TgCentrin2 from its four compartments, due to differences in incorporation kinetics and structural inheritance over successive generations. This was correlated with the development of major invasion deficiency at early stages of CEN2 knockdown, and replication defects at later stages. These results indicate that TgCentrin2 is incorporated into multiple cytoskeletal structures to serve distinct functions in T. gondii that are required for parasite survival.