Domain Analysis of Protein P30 in Mycoplasma pneumoniae Cytadherence and Gliding Motility

Abstract

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...

Processing Is Required for a Fully Functional Protein P30 in Mycoplasma pneumoniae Gliding and Cytadherence

“…1A), but this has not previously been tested directly, and some algorithms predict two membranespanning domains and no precursor processing for P30 (data not shown). Recent domain deletion studies suggest that P30 processing indeed occurs, but at a site well downstream of the original prediction (4). Given its unusual compartmentalization to the distal end of the terminal organelle and the potential role a large leader peptide might play in trafficking, we examined P30 processing here in more detail.…”

“…Wild-type M. pneumoniae strain M129 (17th broth passage) (25) and the spontaneously arising noncytadherent P30 null mutant II-3 (21) were grown in SP4 medium (4,21), with gentamicin (18 g/ml) included as appropriate for culture of transformants. Plasmids used in the current study are described in Table 1.…”

“…Plasmids used in the current study are described in Table 1. The recombinant transposon containing the wild-type P30 allele and the promoter region upstream was described previously (pKV75) (4,14,30). The P30His-C, P30His-N, P30His-49, and P30 cleavage site derivatives were engineered by PCR mutagenesis using pKV75 as a template (4) and primers indicated in Table 2, cloning the PCR products for each into pKV74 (14) or pMT85.…”

“…Mycoplasmas were examined by immunofluorescence microscopy with mouse monoclonal anti-P30 antibody at 1:50 and rabbit polyclonal anti-HMW1 antiserum at 1:1,000 (4). Images were captured with OpenLab Imaging Software version 5.01 (v5.01) (Improvision, Lexington, MA) and processed with Adobe Photoshop CS3 v10 (Adobe Systems, Inc., San Jose, CA) (14).…”

“…However, deletion of a region downstream of this putative signal peptide (residues 38 to 68) generates a P30 derivative (P30⌬I) which actually migrates higher than wildtype P30 ( Fig. 2A), suggesting that processing for P30 maturation occurs further downstream than originally thought, within the deleted region in P30⌬I (4). Given its distinctive trafficking to the distal end of the terminal organelle, we sought to explore whether P30 processing plays a role in localization and functional maturation.…”