Dataset Information

Mycoplasma pneumoniae processes functionally diverse proteins (N-Terminiome & Surfaceome)

ABSTRACT: 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.

INSTRUMENT(S): TripleTOF 5600, Q Exactive Plus, QSTAR

ORGANISM(S): Mycoplasma Pneumoniae (strain Atcc 29342 / M129) Mycoplasma Pneumoniae

SUBMITTER: Joel Steele

LAB HEAD: Professor Steven Djordjevic

PROVIDER: PXD022356 | Pride | 2021-05-01

REPOSITORIES: Pride

ACCESS DATA

Publications

Protein cleavage influences surface protein presentation in Mycoplasma pneumoniae.

Berry Iain J IJ Widjaja Michael M Jarocki Veronica M VM Steele Joel R JR Padula Matthew P MP Djordjevic Steven P SP

Scientific reports 20210324 1

Mycoplasma pneumoniae is a significant cause of pneumonia and post infection sequelae affecting organ sites distant to the respiratory tract are common. It is also a model organism where extensive 'omics' studies have been conducted to gain insight into how minimal genome self-replicating organisms function. An N-terminome study undertaken here identified 4898 unique N-terminal peptides that mapped to 391 (56%) predicted M. pneumoniae proteins. True N-terminal sequences beginning with the initia ...[more]

PMID: 33762641

Similar Datasets

Project description:The screening of a cDNA derived expression library of Klebsiella pneumoniae MGH 78578 expressed in E.coli using a fusion construct and specific HaloTag interaction to a modified surface is shown. Thus, 1536 different clones were screened including positive (ompA, mdh) and negative (pyrC, gapA) reference proteins. The goal of the screening was to identify potential novel immunogenic proteins from K. pneumoniae by selecting clones showing a high signal intensity in comparison to the known antigens used as positve markers. Afterwards, the most promising clones were sequenced to identify the gene and corresponding protein and these proteins were then investigated further. Consequently, 14 novel immunogenic proteins could be identified. In total 1536 (4 x 384) different lysates were spotted on different microarray slides. Each slides contained 3600 distinct spots, seperated into two compartments of 1800 spots each. Each compartment comprised quadruplicate replicates of each sample lysate with controls being analysed with more replicates. As controls we used: ompA1, ompA2 and mdh (3 x 40 replicates) as positive reference proteins, as they have been described as immunogenic before. gapA and pyrC (2 x 40 replicates) as negative reference proteins, additionally two sets of E.coli cell lysates without fusionprotein expressed, namely Acella electrocompetent cells (40 replicates) and KRX (32 replicates). Last but not least a buffer control (24 replicates) was included. Therefore, each set of replicate slides contained 376 different samples and 8 controls. Each screening was performed with three replicate slides. Consequently, a total number of 12 slides were screened (4 sets of samples x 3 replicates). For identification rabbit polyclonal antibody to K. pneumoniae (Acris AP00792PU-N) as primary and Goat polyclonal to Rabbit IgG conjugated with ChromeoTM-546 (Abcam ab60317) as secondary antibody were used. The top compartment was incubated first with primary antibody, while the bottom compartment was incubated with PBS at the same time. Afterwards both compartments were incubated with secondary antibody.

Project description:Infection with Chlamydia pneumoniae, a human respiratory pathogen, has been associated with various chronic diseases such as asthma, coronary heart disease and importantly atherosclerosis. Possibly because the pathogen can exist in a persistent form. TNF-a has been reported to induce chlamydial persitence in epithelial cell lines, however the mechanism of TNF-a-induced persistence has not been reported. Moreover, C. pneumoniae persistently infect human dendritic cells (DCs) and activate DCs to produce cytokines including TNF-a. Induction of chlamydial persistence by other cytokines such as IFN-g is known to be due to indoleamine 2,3-dioxygenase (IDO) activity. The present study therefore, investigated whether C. pneumoniae infection can induce IDO activity in dendritic cells, and whether the restriction of chlamydial growth in the DCs by TNF-a is IDO-dependent. Our data indicate that infection of DCs with C. pneumoniae resulted in the induction of IDO expression. Reporting on our use of anti-TNF-a antibody adalimumab and varying concentrations of TNF-a, we further demonstrate that IDO induction following infection of DCs with C. pneumoniae is TNF-a-dependent. The anti-chlamydial activity induced by TNF-a and the expression of chlamydial 16S rRNA gene, euo, groEL1, ftsk and tal genes was correlated with the induction of IDO. Addition of excess amounts of tryptophan to the DC cultures resulted in abrogation of the TNF-a-mediated chlamydial growth restriction. These findings suggest that infection of DCs by C. pneumoniae induces production of functional IDO, which subsequently causes depletion of tryptophan. This may represent a potential mechanism for DCs to restrict bacterial growth in chlamydial infections. Experiment Overall Design: Human monocyte-derived DCs from five heathy donors were infected with C. pneumoniae at multiplicity of infection of 5 for 72 h. Mock-infected (uninfected) DCs were used as a control for the infection. Total RNA from 72 h chlamydiae-infected or mock-infected DCs was isolated with RNeasy Mini Kit (Qiagen, Hilden, Germany) and DNA contamination was removed with RQ1 DNase (Promega). Total RNA from all donors was pooled and the samples were processed and analyzed at the array facility of Helmholtz center for infection biology, Braunschweig. The integrity of the RNA was verified on the Agilent Bioanalyzer system. We used the Affymetrix Gene Chip Human Genome U133 2.0 Plus. Data were processed using Affymetrix software and signal intensity measured was normalized by the Affymetrix MAS5 algorithm. Data were filtered to exclude genes at background levels (non-expressors, signal intensity at 100).

Dataset Information

Mycoplasma pneumoniae processes functionally diverse proteins (N-Terminiome & Surfaceome)

Publications

Protein cleavage influences surface protein presentation in Mycoplasma pneumoniae.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure