title: Biochemical and Genetic Analysis of the Chlamydia GroEL Chaperonins.
abstract: Chaperonins are essential for cellular growth under normal and stressful conditions and consequently represent one of the most conserved and ancient protein classes. The paradigmEscherichia colichaperonin, EcGroEL, and its cochaperonin, EcGroES, assist in the folding of proteins via an ATP-dependent mechanism. In addition to the presence ofgroELandgroEShomologs,groELparalogs are found in many bacteria, including pathogens, and have evolved poorly understood species-specific functions.Chlamydiaspp., which are obligate intracellular bacteria, have reduced genomes that nonetheless contain threegroELgenes,Chlamydia groEL(ChgroEL),ChgroEL2, andChgroEL3We hypothesized that ChGroEL is the bona fide chaperonin and that the paralogs perform novelChlamydia-specific functions. To test our hypothesis, we investigated the biochemical properties of ChGroEL and its cochaperonin, ChGroES, and queried thein vivoessentiality of the threeChgroELgenes through targeted mutagenesis inChlamydia trachomatisChGroEL 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 notChgroEL, 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.IMPORTANCEChlamydiais an important cause of human diseases, including pneumonia, sexually transmitted infections, and trachoma. The chlamydial chaperonin ChGroEL and chaperonin paralog ChGroEL2 have been associated with survival under stress conditions, and ChGroEL is linked with immunopathology elicited by chlamydial infections. However, their exact roles in bacterial survival and disease remain unclear. Our results further substantiate the hypotheses that ChGroEL is the primary chlamydial chaperonin and that the paralogs play specialized roles during infection. Furthermore, ChGroEL and the mitochondrial GroEL only functioned with their cochaperonin, in contrast to the promiscuous nature of GroEL fromE. coliandHelicobacter pylori, which might indicate a divergent evolution of GroEL during the transition from a free-living organism to an obligate intracellular lifestyle.
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.
authors.forename: Melissa
authors.initials: M
authors.lastname: Illingworth
authors.affiliation: Department of Microbiology, Southern Illinois University, Carbondale, Illinois, USA.
authors.forename: Anna J
authors.initials: AJ
authors.lastname: Hooppaw
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.
authors.forename: Lu
authors.initials: L
authors.lastname: Ruan
authors.affiliation: Department of Microbiology, Southern Illinois University, Carbondale, Illinois, USA fisher@micro.siu.edu linchen@indiana.edu.
authors.forename: Derek J
authors.identifier: http://orcid.org/0000-0002-1663-8389
authors.initials: DJ
authors.lastname: Fisher
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA fisher@micro.siu.edu linchen@indiana.edu.|Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China.
authors.forename: Lingling
authors.initials: L
authors.lastname: Chen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D018835:Chaperonin 10 / Q000235:genetics* / Q000378:metabolism*; D018834:Chaperonin 60 / Q000235:genetics* / Q000378:metabolism*; D002692:Chlamydia trachomatis / Q000235:genetics* / Q000378:metabolism*; D055786:Gene Knockout Techniques; D020043:Genes, Essential; D006868:Hydrolysis; D008291:Malate Dehydrogenase / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding
chemical_list: D018835:Chaperonin 10; D018834:Chaperonin 60; D000255:Adenosine Triphosphate; D008291:Malate Dehydrogenase
keywords: Chlamydia; GroEL; GroES; Hsp60; chaperonin