Molecular and functional studies of protein P30 in Mycoplasma pneumoniae colonization

Abstract

Mycoplasma pneumoniae belongs to the taxonomic class Mollicutes. The species in this class are cell wall-less and possess minimal genomes with miniature cell size. Despite the small genome size, M. pneumoniae is a serious respiratory pathogen which is capable of causing atypical pneumonia and tracheobronchitis in young adults and older children, and recently increasing evidence showing strong correlations between M. pneumoniae infections and asthma attacks. M. pneumoniae possesses a specialized, membrane-bound structure called the “terminal organelle,” a multi-protein complex that functions in adherence, cell division, and gliding motility. Protein P30 has been demonstrated to be required for functionality of the terminal organelle through mutant analysis. P30, located at the distal end of the terminal organelle, is an integral membrane protein oriented with the N-terminus in the cell interior and the C-terminus on the cell surface. Through domain-deletion analysis, two functional domains of P30 were identified. The extracellular domain is required for P30 stability and likely involves an association with the protein P65 for functionality, whereas the cytoplasmic domain is not required for P30 stability but is essential for the cytadherence and motility. In addition, the post-translational processing of the N-terminus of P30 was examined by analyzing P30 derivatives with His6 tags engineered at various locations. The results demonstrated that P30 is proteolytically processed, most likely between residues 52 and 53. A processing-defective mutant of P30 was also created to allow further study. The results indicate that processing is required for fully functional maturation of P30. It is hypothesized that the cleavage event may trigger conformational changes in the extracellular domain to allow interaction with a binding partner. Lastly, M. pneumoniae colonization was examined with a novel in vitro air-liquid interface system of normal human bronchial epithelial cells. The efficiency of the pathogen to colonize NHBE cells appears correlated to the levels of both cytadherence and gliding velocity.