Identification and characterization of a Mycoplasma pneumoniae J-domain co-chaperone required for terminal organelle maturation
Cloward, Jason Michael
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While lacking a peptidoglycan layer, typical biosynthetic capabilities, and conventional modes of transcription regulation, Mycoplasma pneumoniae exhibits extraordinary complexity by forming an elaborate cytoskeleton anchored to a polar terminal organelle, distinguished by a central electron-dense core, with adhesin-related proteins clustered at the distal end. The terminal organelle, also implicated in gliding motility and cell division, intimately associates with host cells (cytadherence) as the pivotal event for pathogenesis of human respiratory epithelium, leading to tracheobronchitis and atypical or “walking” pneumonia. Characterization of isolated cytadherence-deficient mutants revealed the major protein components and assembly order leading to a functional terminal organelle, while immunolocalization and nearest-neighbor analysis allowed spatial positioning of terminal organelle proteins. Significantly, those studies identified proteins complexed with the primary adhesin P1, which included DnaK, a chaperone that modulates protein conformation and stability through regulation by the nucleotide exchange factor GrpE and a J-domain co-chaperone. This volume of work confirms the requirement for chaperone assistance in terminal organelle adhesin maturation through characterization of TopJ, a J-domain protein also having domains specific to mycoplasma terminal organelle proteins. Loss of TopJ results in cytadherence-deficient, non-motile cells with defective terminal organelle positioning, despite wild-type terminal organelle protein localization. Furthermore, P24, a cell division initiation protein, requires TopJ for stability. Ultrastructural analysis of cell morphology and electron-dense core positioning in the topJ mutant identified a defect in terminal organelle anchoring or maturation, also manifested as delayed terminal organelle formation or migration during cell division when observed by fluorescence microcinematography. Localization by immunofluorescence microscopy suggested that terminal organelle presence, or specifically the P1-trafficking protein HMW1, was required for TopJ localization. Development of an assay measuring P1 accessibility to protease demonstrated defective terminal organelle protein translocation or failure to attain a functional conformation at the cell surface. Finally, domain deletion analysis confirmed the TopJ J-domain as a functional domain for DnaK activation, with other TopJ domains involved in localization at the terminal organelle and stabilization of P24.