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Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae.

ABSTRACT: Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity.

SUBMITTER: Tacchi JL

PROVIDER: S-EPMC4772806 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae.

Tacchi Jessica L JL Raymond Benjamin B A BB Haynes Paul A PA Berry Iain J IJ Widjaja Michael M Bogema Daniel R DR Woolley Lauren K LK Jenkins Cheryl C Minion F Chris FC Padula Matthew P MP Djordjevic Steven P SP

Open biology 20160201 2

Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on g ...[more]

PMID: 26865024

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Project description:Mycoplasma pneumoniae is a significant cause of pneumonia, particularly in children, the elderly and the immunocompromised, and post infection sequelae affecting organ sites distant to the respiratory tract are common. It is also a model pathogen where extensive ‘omics’ studies have been conducted to gain insight into how minimal genome self-replicating organisms function. Here we conducted an N-terminome study to determine which proteins are targets of processing events in M. pneumoniae. We identified 6,973 unique N-terminal peptides that mapped to 391 (56%) predicted M. pneumoniae proteins. True N-terminal sequences beginning with the initiating methionine (iMet) residue from the predicted Open Reading Frame (ORF) were identified for 163 proteins. Nearly half (317; 46%) of the ORFS derived from M. pneumoniae strain M129 are post-translationally modified, presumably by proteolytic processing, because dimethyl labelled neo-N-termini were characterised that mapped beyond the predicted N-terminus. An analysis of the N-terminome describes endoproteolytic processing events which predominately target negatively charged residues in P1′ (D and E) with lysine or serine/alanine in P2′ and P3′ positions. Surfaceome studies identified 160 proteins (23% of the proteome) to be exposed on the extracellular surface of M. pneumoniae. The two orthogonal methodologies used to characterise the surfaceome each identified the same 116 proteins, a 72% (116/160) overlap. Apart from lipoproteins, transporters, and adhesins, 93/160 (58%) of the surface proteins lack signal peptides and have well characterised, canonical functions in the cell. Mycoplasma pneumoniae culture conditions The M. pneumoniae (strain M129) cells were cultured in modified Hayflick’s medium at 37 °C in tissue culture flasks using established culture conditions63. Modified Hayflick’s medium contained 21 g PPLO broth base without crystal violet, 5 g of D-glucose, 4 mL of 0.5% phenol red, 100 mL of liquid yeast extract (150 g/L), 200 mL heat-inactivated horse serum (56 °C, 30 min) supplemented with 1 g ampicillin (Sigma, A5354, St. Louis, MO, USA) per litre. M. pneumoniae cells were harvested at mid-log phase by washing adherant cells 3 times with PBS and collected using cell scaping.

Dataset Information

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae.

Publications

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae.

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