P40 and P90 from Mpn142 are Targets of Multiple Processing Events on the Surface of Mycoplasma pneumoniae

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.

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

“…For example, 18 cleavage sites clustered between amino acids 1343-1361 in the C-terminus of P1 (Table 1). Sequential cleavage patterns similar to this was also reported in Mpn142 28 and in Mhp493, a paralog of the major adhesin P97 (Mhp183) in M. hyopneumoniae 74 . Surfaceome studies of M. pneumoniae (data not shown) revealed the presence of surface accessible aminopeptidases that may target a neo-N-terminal cleavage event and sequentially clip amino acids subsequent to the initial cleavage event.…”

“…Processing of adhesin molecules is not confined to M. hyopneumoniae but has been described in Mycoplasma gallisepticum 117 , Mycoplasma fermentans [118][119][120] , M. genitalium 121 , and Spiroplasma citri 122 . Here we show that major adhesion molecules in M. pneumoniae, a phylogenetically distinct human pathogen, are processed 12,28 . All these studies suggest that the processing of surface accessible proteins is widespread in Mollicutes.…”

“…We recently showed that Mpn142, a member of the same operon that houses the P1 gene (mpn141), and the surface accessible moonlighting adhesin, elongation factor Tu (Ef-Tu), are cleaved extensively 12,28 . Post-translational processing of adhesins has been well characterised in M. hyopneumoniae where cleavage fragments have been shown to adhere to porcine cilia, porcine kidney epithelial cells, and a range of host molecules such as the glycosaminoglycan mimic heparin [59][60][61][62]64,72,74,[110][111][112][113][114][115] , plasminogen 60,[112][113][114] , actin 116 , and fibronectin 59,72,[112][113][114] .…”

“…Surface proteome analysis of M. pneumoniae. Biotinylation of the M. pneumoniae cells was performed as described previously 28 . The biotinylation reaction was allowed to proceed for 30 seconds on ice.…”

“…as described previously 28 . The bioinformatic tools used were: ProtParam 65 , TMpred 66 , PONDR ® (VSL2 predictor) 67 , and ScanProsite 68 .…”