Structure/function analysis of HMW2 and proteomic analysis of the electron-dense core in Mycoplasma pneumoniae
Bose, Stephanie Ross
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Mycoplasma pneumoniae, a cell wall-less prokaryote, causes atypical or walking pneumonia and tracheobronchitis in humans. Attachment to host respiratory epithelium is mediated by a polar differentiated organelle that has an electron-dense core, which contains a thin and thick rod. The non-cytadhering mutant I-2 lacks the cytadherence-associated protein HMW2 due to a frameshift mutation, is non-motile, has morphological abnormalities, reduced levels of HMW1, HMW3, and P65, and fails to cluster P1. Another cytadherence-associated protein HMW1 exhibits a reciprocal dependency with HMW2. Mutants lacking HMW1 or HMW2, designated M6 and I-2, respectively, have no obvious attachment organelle or electron-dense core. Thus, both proteins are thought to have an early role in attachment organelle assembly. Although components of the Triton X-100 insoluble fraction of M. pneumoniae have been identified by mass spectrometry studies, proteins specific to the core were not assigned. HMW2 was identified as a component of that fraction. Based this and other evidence, a model was suggested where HMW2 is a major component of the electron-dense core and interacts longitudinally with an internal translation product of the hmw2 transcript, P28, to form the core. Structure and function analysis of HMW2 derivatives allowed for identification of distinct regions, including the C-terminus, coiled-coil domains, and leucine repeats of HMW2, required for normal cellular function and core formation. An HMW2-GFP fusion localized this protein to the attachment organelle. However, high resolution immuno-localization studies allowed for precise localization of HMW2 to the periphery of the electron-dense core and a new model for core formation is described, where HMW2 and P28 comprise the thin rod of the core, which acts as a scaffold for formation of the thick rod. The central portion of HMW2 may act as the spokes, a structural feature of the attachment organelle in the electron-lucent space between the core and the membrane. Liquid chromatography/mass spectrometry analysis allowed for identification of the components of a core-enriched fraction. These 89 proteins included cytadherence-accessory proteins, along with those involved in transcription/translation, cell division, and metabolism.