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 paradigm chaperonin, EcGroEL, and its cochaperonin, EcGroES, assist in the folding of proteins via an ATP-dependent mechanism. In addition to the presence of and homologs, paralogs are found in many bacteria, including pathogens, and have evolved poorly understood species-specific functions. spp., which are obligate intracellular bacteria, have reduced genomes that nonetheless contain three genes, (), , and We hypothesized that ChGroEL is the bona fide chaperonin and that the paralogs perform novel -specific functions. To test our hypothesis, we investigated the biochemical properties of ChGroEL and its cochaperonin, ChGroES, and queried the essentiality of the three genes through targeted mutagenesis in 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 , 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. is 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 from and, which might indicate a divergent evolution of GroEL during the transition from a free-living organism to an obligate intracellular lifestyle.

Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each

“…trachomatis propagation and detection of Clp proteins. DFCT28, a green fluorescent protein (GFP)-expressing C. trachomatis 434/Bu clone (62), was routinely grown in and the titer was determined (using the IFU assay) on HeLa cells (63). Briefly, cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and grown at 37°C with 5% CO 2 .…”

When Bacteria and Viruses Collide: A Tale of Chlamydia trachomatis and Sexually Transmitted Viruses

“…Under unfavorable growth conditions characterized by the presence of antibiotic or host immune responses, a state of persistence within the inclusion begins in which CT remains viable but atypical, with an enlarged, aberrant form and quiescent metabolism [ 65 , 66 ]. In this persistent form, CT substantially upregulates the synthesis and release of its chaperonins (Hsp60s) known as GroEL from the host cells [ 71 , 72 , 73 ]. GroEL acts as a virulence factor that promotes the survival of the bacterium by modulating the host immune responses and suppressing the activation of pro-inflammatory responses, inhibiting apoptosis, and assisting in nutrient acquisition and utilization.…”

LA PREDICCIÓN IN SILICO DETERMINA LA PRESENCIA DE EPÍTOPOS INMUNOGÉNICOS ALTAMENTE ESPECÍFICOS EN FRAGMENTOS DE LA PROTEÍNA DE LA MEMBRANA POLIMÓRFICA (PMP17G) DE Chlamydia abortus

“…Para el proceso de transformación celular, se empleó la cepa bacteriana de E. coli DH5α químicamente competente empleando el método de CaCl2 (Illingworth et al, 2017), las células y los plásmidos recombinantes (pGEM-T-POMP90-3 y pGEM-T-POMP90-4) se cotransformaron y se sembraron en agar selectivo Luria Bertani (LB) (500 µL) suplementado con ampicilina (100 µg/mL), Isopropilβ-D-1-tiogalactopiranósido (IPTG)/X-Gal, con concentraciones finales de 96 µg/mL y 40 µg/mL respectivamente, las placas fueron incubadas a 37°C por 24 hrs., finalmente las colonias resultantes se seleccionaron a través del método de selección de colonias azul-blancas (Nabi et al, 2016).…”