The cell wall-less prokaryote Mycoplasma pneumoniae causes bronchitis and atypical pneumonia in humans. Mycoplasma attachment and gliding motility are required for colonization of the respiratory epithelium and are mediated largely by a differentiated terminal organelle. P30 is a membrane protein at the distal end of the terminal organelle and is required for cytadherence and gliding motility, but little is known about the functional role of its specific domains. In the current study, domain deletion and substitution derivatives of P30 were engineered and introduced into a P30 null mutant by transposon delivery to assess their ability to rescue P30 function. Domain deletions involving the extracellular region of P30 severely impacted protein stability and adherence and gliding function, as well as the capacity to stabilize terminal organelle protein P65. Amino acid substitutions in the transmembrane domain revealed specific residues uniquely required for P30 stability and function, perhaps to establish correct topography in the membrane for effective alignment with binding partners. Deletions within the predicted cytoplasmic domain did not affect P30 localization or its capacity to stabilize P65 but markedly impaired gliding motility and cytadherence. The larger of two cytoplasmic domain deletions also appeared to remove the P30 signal peptide processing site, suggesting a larger leader peptide than expected. We propose that the P30 cytoplasmic domain may be required to link P30 to the terminal organelle core, to enable the P30 extracellular domain to achieve a functional conformation, or perhaps both.The cell wall-less prokaryote Mycoplasma pneumoniae is a human pathogen causing community-acquired respiratory disease in persons of all ages but especially older children and young adults (32). Although it is perhaps best known as the etiologic agent of atypical, or "walking," pneumonia, accounting for up to 20% of all cases of community-acquired pneumonia (41), infections most commonly manifest as tracheobronchitis. Chronic lung disease and extrapulmonary sequelae due to immunopathology or direct invasion can ensue, including a strong correlation with onset and exacerbation of asthma (3,29,42). Colonization of the epithelial mucosa of the conducting airways is mediated largely by the terminal organelle, a polar, differentiated, membrane-bound extension of the mycoplasma cell (7, 30). The adhesins P30 and P1 and several adherence accessory proteins localize to this structure (23), which is defined by a complex electron-dense core that is a prominent component of the mycoplasma cytoskeleton (2, 17, 18). In addition to its role in adherence, the terminal organelle is the motor for gliding motility (14), and its duplication precedes cell division (15).P30 is a membrane protein oriented with the C terminus (domain III) on the mycoplasma cell surface, based on accessibility to antibodies and protease (10, 25) (Fig. 1A), and the N terminus likely in the cell interior (domain I). Its predicted transmembrane (TM) domain a...

The cell wall-less prokaryote Mycoplasma pneumoniae causes bronchitis and atypical pneumonia in humans. Mycoplasma attachment to the host respiratory epithelium is required for colonization and mediated largely by a differentiated terminal organelle. P30 is an integral membrane protein located at the distal end of the terminal organelle. The P30 null mutant II-3 is unable to attach to host cells and nonmotile and has a branched cellular morphology compared to the wild type, indicating an important role for P30 in M. pneumoniae biology. P30 is predicted to have an N-terminal signal sequence, but the presence of such a motif has not been confirmed experimentally. In the current study we analyzed P30 derivatives having epitope tags engineered at various locations to demonstrate that posttranslational processing occurred in P30. Several potential cleavage sites predicted in silico were examined, and a processing-defective mutant was created to explore P30 maturation further. Our results suggested that signal peptide cleavage occurs between residues 52 and 53 to yield mature P30. The processing-defective mutant exhibited reduced gliding velocity and cytadherence, indicating that processing is required for fully functional maturation of P30. We speculate that P30 processing may trigger a conformational change in the extracellular domain or expose a binding site on the cytoplasmic domain to allow interaction with a binding partner as a part of functional maturation.Mycoplasma pneumoniae is an important etiologic agent of community-acquired tracheobronchitis and pneumonia in humans. This cell wall-less prokaryote causes respiratory disease in persons of all ages but especially older children and young adults, and a strong correlation exists between M. pneumoniae infections and onset and exacerbation of asthma (3,27,29,36). Adherence to the respiratory epithelium is essential for colonization and virulence (21) and is mediated largely by the terminal organelle (5, 28), a polar, differentiated, membranebound extension of the mycoplasma cell having a complex electron-dense core capped by a terminal button at the distal end (2, 15). The terminal organelle is also the motor for gliding motility (11), and its duplication precedes cell division (12). Studies have identified several key terminal organelle components, the loss of which impacts cytadherence, motility, and cell division (11,13,21).P30 is an integral membrane protein that localizes to the distal end of the terminal organelle (1, 6, 7). Loss of P30 due to a frameshift in its corresponding gene (MPN453) results in the inability to cytadhere or glide and a branched cellular morphology (30). Complementation with the corresponding recombinant wild-type allele restores a wild-type phenotype (30). The deduced N terminus of P30 is positively charged with five basic residues, which are followed by a 23-residue hydrophobic region, and is thought to comprise a signal peptide (6, 7) (Fig. 1A). However, deletion of a region downstream of this putative signal peptide (residues 3...

No abstract