Abstract
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.
Highlights
Caseinolytic proteases and chaperones (Clp) play an important role in protein homeostasis and cellular survival during both stress and optimal growth conditions [1]
We focused on characterizing the Class I ATPdependent 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 Caseinolytic peptidase B (ClpB) with novel apicoplast localization
Summary
Caseinolytic proteases and chaperones (Clp) play an important role in protein homeostasis and cellular survival during both stress and optimal growth conditions [1]. T. gondii is a unicellular intracellular protozoan belonging to the parasitic phylum Apicomplexa and subclass Coccidium [7] It is one of the most widespread parasites in the worldwide, potentially capable of infecting all birds and mammals including humans [8]. High temperature stress has a wide range of effects on cells, including changes in membrane fluidity and structure, cell cycle arrest, protein denaturation, and cell death. Such issues can occur when essential proteins are damaged by high temperatures, or when aggregates of damaged proteins become toxic to the cell [9, 10]. Clp superfamily proteins are crucial for protein quality control under these conditions, and yet little is known about this family of proteins in T. gondii
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