Clp family protein: ClpB1 plays a crucial role in thermotolerance.

Caseinolytic peptidase B (ClpB) plays a pivotal role in suppressing and reversing protein aggregation. 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 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 ClpB1, ClpB2, and ClpB3. We found that ClpB1, the most divergent of the five Class I Clp ATPases, is cytoplasmic, ClpB2 is found in the mitochondria of the parasites, and ClpB3 is a ClpB with novel apicoplast localization. Knockout strains of ClpB1 and ClpB2 were established by CRISPR/Cas9 mutagenesis, and their complementing strains were constructed with FLAG-tag. Although knockout of ClpB1 or ClpB2 did not affect growth under normal circumstances, ClpB1 was required for thermotolerance. The growth, replication, and invasion capabilities of ClpB1-deficient mutants were significantly inhibited after extracellular parasites were pretreated at 45°C. Moreover, ClpB1 were observed at the poles of the ΔClpB1 FLAG-tagged strain treated at 42°C.