Development of a Global Strategy for the Analysis of Protein Cleavage and its Relationship to Pathogen Virulence — The Association Specialists
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Development of a Global Strategy for the Analysis of Protein Cleavage and its Relationship to Pathogen Virulence (354)

Iain J Berry 1 2 , Jessica L Tacchi 1 2 , Veronica M Jarocki 1 2 , Benjamin B A Raymond 1 2 , Matthew P Padula 1 2 , Steven P Djordjevic 1 2
  1. Proteomics Core Facility, University of Technology, Sydney , NSW
  2. The ithree Institute, University of Technology, Sydney, NSW

Proteolytic cleavage is one of the most ubiquitous post-translational modification to proteins, responsible for protein signalling, activation, localisation and ultimately degradation. Due to a variety of experimental difficulties and technical limitations, this important physiological process has been largely overlooked, particularly in prokaryotes and archaea. In order to explore the underlying mechanisms in the production of mature, cleaved proteins and the function of cleavage products, including in pathogenesis, a high-throughput “whole organism” (global) approach is needed to determine the multitude of processing events that occur.

The primary focus of this project is to optimise a protocol to identify N-terminal sequences of mature protein cleavage products, for application in the characterisation of prokaryotic pathogens. For this purpose, a model organism, Mycoplasma hyopneumoniae, was selected for analysis due to the large body of literature demonstrating the extensive proteolytic processing of many highly expressed surface proteins of this organism, which are critical for pathogenesis. Additionally, M. hyopneumoniae has enormous agricultural impact as it is an economically important pathogen and a major reason for the use of antibiotics to control respiratory disease in swine production worldwide. This organism is a chronic respiratory pathogen of swine which evades host immune responses.

Using an adapted high-throughput methodology called Terminal Amine Isotopic Labelling of Substrates (TAILS), the data obtained from the analysis of M. hyopneumoniae has provided: i) new data pinpointing the precise sites of cleavage in more than 40 proteins; ii) complementary, orthogonal evidence for the previously reported cleavage products of surface proteins that were determined by Edman degradation and other more costly and tedious protocols; iii) provided insight into further modifications to the protocol that will enable characterisation of processing events in a genome-wide manner. Finally, the existing datasets obtained for this organism have validated this methodology for further application in other pathogenic organisms.