Caseinolytic peptidase B (ClpB) plays a pivotal role in suppressing and reversing protein aggregation. Toxoplasma gondii is an intracellular parasitic protozoan that infects a wide variety of mammals and birds and therefore is exposed to a broad range of living condition. We screened ToxoDB (http://ToxoDB.org) and identified 10 putative T. gondii genes encoding members of the Clp superfamily of caseinolytic proteases and chaperones. Of these, we focused on characterizing the Class I ATP-dependent molecular chaperones TgClpB1, TgClpB2, and TgClpB3. We found that TgClpB1, the most divergent of the five T. gondii Class I Clp ATPases, is cytoplasmic, TgClpB2 is found in the mitochondria of the parasites, and TgClpB3 is a ClpB with novel apicoplast localization. Knockout strains of TgClpB1 and TgClpB2 were established by CRISPR/Cas9 mutagenesis, and their complementing strains were constructed with FLAG-tag. Although knockout of TgClpB1 or TgClpB2 did not affect growth under normal circumstances, TgClpB1 was required for T. gondii thermotolerance. The growth, replication, and invasion capabilities of TgClpB1-deficient mutants were significantly inhibited after extracellular parasites were pretreated at 45°C. Moreover, TgClpB1 were observed at the poles of the ΔTgClpB1 FLAG-tagged strain treated at 42°C.
Keywords: AAA+ ATPases; ClpB; caseinolytic proteases and chaperones; thermotolerance; toxoplasma gondii.