Mycoplasma pneumoniae is the second most common cause of community acquired pneumonia. Infections are localised to the respiratory tract but can be systemic and affect multiple tissues and organs. M. pneumoniae is a strict parasite and must adhere to the respiratory epithelium to survive. Little is known of the mechanism of pathogenesis and attempts to develop efficacious vaccines have been unsuccessful.

M. pneumoniae possesses an unusually large repertoire of lipoproteins and their functions are largely unknown. Our proteome studies identified a number of lipoproteins in M. pneumoniae. One of these, MPN_284, was unusual in that it was highly expressed, proteolytically processed and surface accessible. N-terminomics of mature proteoforms and 2D SDS-PAGE followed by LC-MS/MS were used to characterize the cleavage sites of MPN_284. Two recombinant fragments were expressed that span the N-terminal (MPN_284 N-terminus) and C-terminal thirds (MPN_284 C-terminus) of MPN_284. Polyclonal antiserum to each fragment was generated to determine the cellular location of the lipoprotein fragments using 3D-Structured Illumination Microscopy and to confirm the identities of the cleavage fragments of MPN_284 by western blotting. Image analysis suggests that the C-terminal region of MPN_284 is accessible and distributed on the surface of M. pneumoniae, while the N-terminal region produced sporadic straining patterns. To investigate the adhesive characteristics of the N- and C-terminal cleavage fragments of MPN_284, inert latex beads were coated separately with MPN_284 fragments and incubated with respiratory epithelial cells (A549). Beads coated with MPN_284 fragments were found to bind to A549 cell monolayers while beads coated with bovine serum albumin did not.

Our data confirms that MPN_284 resides on the extracellular surface of M. pneumoniae and is proteolytically processed, generating multiple surface accessible fragments with putative binding functions for host molecules. The bead experiments were consistent with these observations. This work provides new avenues to explore the identities of binding domains in MPN_284 and the development of an efficacious vaccine for M. pneumoniae.