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Chest Infections |

Surface Expressed Proteins Are Identified in Mycobacterium tuberculosis With a Proteomic Approach

Megan Rees, MBBS; Alexander Smith, PhD; Ross Coppel, PhD
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Monash University, Melbourne, VIC, Australia


Chest. 2015;148(4_MeetingAbstracts):145A. doi:10.1378/chest.2221452
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Abstract

SESSION TITLE: Chest Infections Posters: Tuberculosis

SESSION TYPE: Original Investigation Poster

PRESENTED ON: Wednesday, October 28, 2015 at 01:30 PM - 02:30 PM

PURPOSE: Proteins expressed on the surface of mycobacteria are the first to come into contact with the host and therefore likely to have a significant role in interactions with the host. Cell wall or surface proteins make up the majority of novel anti-TB agents in current development and clinical trials. Describing the full repertoire of surface expressed proteins in M. tuberculosis could identify novel therapeutic targets.

METHODS: A proteomic approach was used to characterise surface expressed proteins. A protease was employed to directly cleave peptide fragments from proteins on the surface of intact and viable bacteria. These liberated peptide fragments were identified by HPLC coupled to tandem mass spectrometry, and matched to the predicted proteome derived from the complete genome sequence of M. tuberculosis. The laboratory reference strain of M. tuberculosis H37Rv was used in of six biological replicates, to generate a list of 752 surface expressed proteins confidently identified in all replicates. This dataset was verified and refined by performing membrane shaving on 6 recent clinical isolates of M. tuberculosis from active cases of tuberculosis. Contaminating proteins, resulting from either cell lysis or spontaneous secretion from these live bacteria, were excluded from the final dataset by a label free qualitative proteomic analysis of control samples of the same bacterial strains treated with either mechanical lysis or trypsin free incubation respectively.

RESULTS: An initial set of 752 surface expressed proteins were confidently identified by mass spectrometry in the H37Rv reference strain of M. tuberculosis. Five hundred and sixty two of these surface expressed proteins were also identified for each six clinical strains. After exclusion of possible contaminating proteins released by secretion or spontaneous cell lysis, a total of 259 proteins were determined to be surface expressed, of which 83% were predicted to be cell wall or surface proteins by TBpred, a bioinformatic algorithm.

CONCLUSIONS: A high throughput proteomics approach was used to identify the complete repertoire of surface expressed proteins in M. tuberculosis with verification on recently isolated clinical strains.

CLINICAL IMPLICATIONS: This comprehensive characterisation of surface expressed proteins in live mycobacteria documents the repertoire of bacterial proteins initially presented to the host, this allows insights into host pathogen interactions and provides potential novel therapeutic targets.

DISCLOSURE: The following authors have nothing to disclose: Megan Rees, Alexander Smith, Ross Coppel

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