Cell surface processing of the P1 adhesin of Mycoplasma pneumoniae identifies novel domains that bind host molecules

Abstract

Mycoplasma pneumoniae is a genome reduced pathogen and causative agent of community acquired pneumonia. The major cellular adhesin, P1, localises to the tip of the attachment organelle forming a complex with P40 and P90, two cleavage fragments derived by processing Mpn142, and other molecules with adhesive and mobility functions. LC-MS/MS analysis of M. pneumoniae M129 proteins derived from whole cell lysates and eluents from affinity matrices coupled with chemically diverse host molecules identified 22 proteoforms of P1. Terminomics was used to characterise 17 cleavage events many of which were independently verified by the identification of semitryptic peptides in our proteome studies and by immunoblotting. One cleavage event released 1597 tSAAKpGApRppVppKpGApKppVQppKKpA 1627 from the C-terminus of P1 and this peptide was shown to bind to a range of host molecules. A smaller synthetic peptide comprising the C-terminal 15 amino acids, 1613 pGApKppVQppKKpA 1627 , selectively bound cytoskeletal intermediate filament proteins cytokeratin 7, cytokeratin 8, cytokeratin 18, and vimentin from a native A549 cell lysate. Collectively, our data suggests that ectodomain shedding occurs on the surface of M. pneumoniae where it may alter the functional diversity of P1, Mpn142 and other surface proteins such as elongation factor Tu via a mechanism similar to that described in Mycoplasma hyopneumoniae. The attachment organelle is a structurally and functionally sophisticated component of the M. pneumoniae cell that is responsible for the assembly of proteins essential for motility and adherence 1-8. An extensive list of host molecules including fibronectin 9-13 , fibrinogen 10-14 , plasminogen 11-13,15-17 , lactoferrin 10-12 , laminin 10-12 , and vitronectin 10-13 interact with surface accessible adhesins in M. pneumoniae. Other less well-defined host molecules include sialylated molecules 18 , oligosaccharides 19 , glycolipids 20 , and glycoproteins 21. The gene mpn141 encoding the major adhesin P1 is located in the same operon along with mpn140 and mpn142 and these three genes constitute a polycistronic transcriptional unit 22,23. mpn140 encodes for a 28 kDa putative phosphoesterase 24 and while it has been shown to degrade nanoRNA and dephosphorylate 3′-phosphoadenosine 5′-phosphate to AMP 25 , no role in adherence has been assigned for this protein. mpn142 generates a 130 kDa product (Mpn142) that is cleaved into two fragments of 40 kDa (P40) and 90 kDa (P90) immediately after or concurrent with translation 26,27. The cleavage event in Mpn142, first described over 25 years ago, was the first in what is now known to be a highly processed molecule on the surface of M. pneumoniae 28. P1 is a remarkably versatile molecule and the subject of numerous studies over the past 30 years. The only cleavage event that has been accurately assigned to P1 is the removal of the N-terminal 59 amino acids as a leader peptide 29. Molecular cross-linking and immunogold-labelling studies indicated that P1 forms a complex with P30, P40, and P90 ...

Mycoplasma pneumoniae Infections: Pathogenesis and Vaccine Development

“…P1 adhesin of M. pneumoniae M129 is subject to extensive post-translational processing forming 22 proteo-forms, which are specific molecular forms of a protein product arising from a specific gene. Each of the proteo-forms retain the ability to bind to host molecules or their structural mimics and are surface accessible [ 31 ]. There are many issues that require further study, such as whether the antigen variations caused by post-translational modifications can affect the pathogenicity of M. pneumoniae .…”

Infection strategies of mycoplasmas: Unraveling the panoply of virulence factors

“…The most predominant adhesins in M. pneumoniae are P1 and P30 [17]. P1 is a remarkably versatile molecule that forms a complex with P30, P40, and P90; these colocalize to the tip of the terminal organelle to perform different functions, such as receptor recognition and gliding motility [4,18]. Schmitt et al has found that the P1 complex also contains P65, DnaK, C-terminal truncated forms of DnaK and P1, pyruvate dehydrogenase E1 α subunit (PDHA), HMW1, and HMW3 to coordinate the adherence of M. pneumoniae to host cells [4,19].…”

“…P1 is a remarkably versatile molecule that forms a complex with P30, P40, and P90; these colocalize to the tip of the terminal organelle to perform different functions, such as receptor recognition and gliding motility [4,18]. Schmitt et al has found that the P1 complex also contains P65, DnaK, C-terminal truncated forms of DnaK and P1, pyruvate dehydrogenase E1 α subunit (PDHA), HMW1, and HMW3 to coordinate the adherence of M. pneumoniae to host cells [4,19]. The P30 adhesin, originally regarded as an accessory protein, plays a role in localizing P1 to the terminal organelle and is involved in cell development [18]; the amino acid sequence of P30 holds substantial homology with some eukaryotic proteins in humans, indicating that P30 could be associated with the occurrence of certain autoimmune diseases [18].…”

“…To accomplish this pathogenic process, mycoplasmas must first overcome several obstacles to successfully invade the host's defense and reach full lifecycles. The invasiveness of mycoplasmas is mediated by surface adhesins and their accessory proteins, capsular polysaccharides, invasive enzymes, and biofilms, all of which ensure their establishment, reproduction, and spread in vivo [4][5][6][7]. During the parasitism cycle, mycoplasmas absorb nutrients from host cells and release a large number of metabolites, such as hydrogen peroxide (H 2 O 2 ), ammonia (NH 3 ) and hydrogen sulfide (H 2 S), causing localized tissue damage [8,9].…”

Pathogenicity & virulence ofMycoplasma hyopneumoniae

“…Direct contact of M. hyopneumoniae cells with each other and with host cells or ECM molecules may render the pathogen capable of forming biofilms. Biofilm formation is a strategy used by many bacteria, including other mycoplasma species [112][113][114], to cope with host immune response or with antimicrobial effects, thereby rendering bacteria extremely adaptive and thus contributing to virulence. Recent studies have shown that at least some M. hyopneumoniae strains are indeed capable of forming biofilms, in experimental in vitro conditions, on abiotic surfaces or on host cell monolayers, and within the respiratory tract of experimentally infected swine [115,116].…”