Biochemical and Genetic Analysis of the Chlamydia GroEL Chaperonins

Chaperonins are essential for cellular growth under normal and stressful conditions and consequently represent one of the most conserved and ancient protein classes. The paradigm Escherichia coli chaperonin, EcGroEL, and its cochaperonin, EcGroES, assist in the folding of proteins via an ATP-dependent mechanism. In addition to the presence of groEL and groES homologs, groEL paralogs are found in many bacteria, including pathogens, and have evolved poorly understood species-specific functions. Chlamydia spp., which are obligate intracellular bacteria, have reduced genomes that nonetheless contain three groEL genes, Chlamydia groEL ( ChgroEL ), ChgroEL2 , and ChgroEL3 . We hypothesized that ChGroEL is the bona fide chaperonin and that the paralogs perform novel Chlamydia -specific functions. To test our hypothesis, we investigated the biochemical properties of ChGroEL and its cochaperonin, ChGroES, and queried the in vivo essentiality of the three ChgroEL genes through targeted mutagenesis in Chlamydia trachomatis . ChGroEL hydrolyzed ATP at a rate 25% of that of EcGroEL and bound with high affinity to ChGroES, and the ChGroEL-ChGroES complex could refold malate dehydrogenase (MDH). The chlamydial ChGroEL was selective for its cognate cochaperonin, ChGroES, while EcGroEL could function with both EcGroES and ChGroES. A P35T ChGroES mutant (ChGroESP35T) reduced ChGroEL-ChGroES interactions and MDH folding activities but was tolerated by EcGroEL. Both ChGroEL-ChGroES and EcGroEL-ChGroESP35T could complement an EcGroEL-EcGroES mutant. Finally, we successfully inactivated both paralogs but not ChgroEL , leading to minor growth defects in cell culture that were not exacerbated by heat stress. Collectively, our results support novel functions for the paralogs and solidify ChGroEL as a bona fide chaperonin that is biochemically distinct from EcGroEL. IMPORTANCE Chlamydia is an important cause of human diseases, including pneumonia, sexually transmitted infections, and trachoma. The chlamydial chaperonin ChGroEL ...