Proteasome Inhibitors block intracellular growth and replication of Toxoplasma gondii

Lactacystin, a specific inhibitor of proteasomes in eukaryotic cells, did n ot block parasite entry or the establishment of the parasitophorous vacuole , but did inhibit parasite growth and daughter cell budding, as well as DNA synthesis. Two other proteasome inhibitors, MG-132 and proteasome inhibito r 1, also blocked parasite growth and intracellular development. Adding lac tacystin to established, dividing parasites, rapidly blocked parasite growt h and daughter cell budding at all stages in the process. Pre-treating host cells with lactacystin did not block parasite entry or development. Moreov er, under the conditions used, the host cells appeared not to be adversely affected indicating that host cell proteasome activity was not essential fo r parasite entry or development. Concomitant with these effects on parasite growth and division were morphological changes in the parasite including t he appearance of whorls of ER-derived membranes presumably related to the f ailure to breakdown misfolded proteins. These changes were specific to lact acystin and were not seen in parasites treated with other protease inhibito rs. Although the ER-derived structures resembled autophagic bodies, similar structures could not be induced by serum starvation nor did the membranous whorls acidify or undergo morphological changes consistent with autophagos omal maturation. These results highlight the possible role of proteasome ac tivity in Toxoplasma in intracellular development and the regulation of par asite replication. However, how the dividing parasite recycles its organell es and the functional relationship between any lysosomal-autophagic pathway and proteasomes in the parasite remains unresolved.