Attachment Organelle Formation Represented by Localization of Cytadherence Proteins and Formation of the Electron-Dense Core in Wild-Type and Mutant Strains of Mycoplasma pneumoniae

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Abstract
Cytadherence proteins of Mycoplasma pneumoniae are localized at the attachment organelle, which is involved in adhesion, gliding motility, and cell division. The localization of these proteins in cytadherence-deficient mutants was examined by immunofluorescence microscopy. In the class I-2 mutant, which has a frameshift mutation in the hmw2 gene, fluorescent foci for HMW1 and HMW3 were found with reduced intensity, and P1 adhesin showed reduced focusing. However, foci for P90, P40, P30, and P65 were not observed in this mutant. In the class IV-22 mutant, which lacks expression of P1, P90, and P40, the other cytadherence proteins (HMW1, HMW3, P30, and P65) were focused. In a mutant lacking HMW1, signals for HMW3, P90, P40, P30, and P65 were not found, and P1 was distributed throughout the cell. These results suggest that HMW1 is essential for the localization of all other cytadherence proteins, while HMW2 is essential for the localization of P90, P40, P30, and P65. The electron-dense core in cytadherence mutants was observed by thin-section electron microscopy, suggesting that its formation depends on HMW1 and HMW2 and that P1 localization occurs independent of the formation of the electron-dense core. Doubly stained preparations visualized by immunofluorescence microscopy showed that the P1 adhesin, P90, and P40 colocalized to a subregion of the attachment organelle in the wild-type strain. HMW1 and HMW3 also colocalized to a different subregion of the attachment organelle, while P30 and P65 localized at more distal ends of cell poles than HMW1 and HMW3. These differences were more pronounced in cytadherence mutants. These results suggest that there are three distinct subcellular protein localization sites in the attachment organelle, which were represented by HMW1-HMW3, P1-P90-P40, and P30-P65.