Abstract
Mycoplasma pneumoniae is a significant cause of community acquired pneumonia globally. Despite having a genome less than 1 Mb in size, M. pneumoniae presents a structurally sophisticated attachment organelle that (i) provides cell polarity, (ii) directs adherence to receptors presented on respiratory epithelium, and (iii) plays a major role in cell motility. The major adhesins, P1 (Mpn141) and P30 (Mpn453), are localised to the tip of the attachment organelle by the surface accessible cleavage fragments P90 and P40 derived from Mpn142. Two events play a defining role in the formation of P90 and P40; removal of a leader peptide at position 26 (23SLA↓NTY28) during secretion to the cell surface and cleavage at amino acid 455 (452GPL↓RAG457) generating P40 and P90. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) analysis of tryptic peptides generated by digesting size-fractionated cell lysates of M. pneumoniae identified 15 cleavage fragments of Mpn142 ranging in mass from 9–84 kDa. Further evidence for the existence of cleavage fragments of Mpn142 was generated by mapping tryptic peptides to proteins recovered from size fractionated eluents from affinity columns loaded with heparin, fibronectin, fetuin, actin, plasminogen and A549 surface proteins as bait. To define the sites of cleavage in Mpn142, neo-N-termini in cell lysates of M. pneumoniae were dimethyl-labelled and characterised by LC-MS/MS. Our data suggests that Mpn142 is cleaved to generate adhesins that are auxiliary to P1 and P30.
Highlights
Mycoplasma pneumoniae (M. pneumoniae) is a respiratory pathogen estimated to cause 100,000 hospitalisations annually in the USA [1] and up to 40% of cases of community-acquired pneumonia globally [2]
Peptides spanning fragments of Mpn142 obtained from 1D/2D SDS Polyacrylamide Gel Electrophoresis (PAGE) of whole cell lysates; recovery of biotinyled proteins; or A549, fetuin, fibronectin, actin, heparin or plasminogen based affinity chromatography were identified by LC-MS/MS
A growing body of evidence exists to suggest that molecules that reside on the cell surface of mycoplasmal pathogens are processed into discrete functional domains via a process known as ectodomain shedding [58,59,60,61,62,63,64,65,66,67,68,69,70,71,72]
Summary
Mycoplasma pneumoniae (M. pneumoniae) is a respiratory pathogen estimated to cause 100,000 hospitalisations annually in the USA [1] and up to 40% of cases of community-acquired pneumonia globally [2]. P65 and HMW1 are unusual because they reside intracellularly as part of the cytoskeletal core and on the extracellular side of the attachment organelle, suggesting the existence of different proteoforms [25] It is not known how HMW1 traffics to the cell surface, because it lacks evidence of transmembrane spanning domains and a secretion signal. While most studies have focussed on characterising interactions between P40 and P90 with other proteins in the attachment organelle, their presence on the surface of M. pneumoniae in close association with P1 indicates that they may have roles in adherence to host molecules. These studies provide new insights in the structural and functional capabilities of P90 and P40 and identified regions within these two molecules worthy of further study
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