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Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry
Published on: March 24, 2012
Partial proteome of Mycobacterium avium subsp. paratuberculosis under oxidative and nitrosative stress
Satoko Kawaji1, Ling Zhong, Richard J Whittington
1Faculty of Veterinary Science, The University of Sydney, 425 Werombi Road, Camden, New South Wales 2570, Australia.
Abstract:
The growth pattern and protein expression profiles of sheep (S) and cattle (C) strains of Mycobacterium avium subsp. paratuberculosis (MAP) under oxidative and nitrosative stress were characterised. Oxidative stress was induced using 0.05% (v/v) H(2)O(2) in BACTEC medium, and was lethal for an inoculum of 10(4) cells. However, an inoculum of 10(7) cells survived and proteomic changes were observed at 7 days. Nitrosative stress was induced using 1mM NaNO(2); it slowed the growth of an inoculum of 10(4) cells, but both strains recovered quickly when resuscitated in fresh media. Silver staining showed higher sensitivity for detection of 2D spots compared to SYPRO Ruby staining. A total of 18 proteins were regulated under oxidative and/or nitrosative stress. The expression of four antioxidant enzymes (AhpC, AhpD, OxcA and SodA) and four proteins involved in fatty acid metabolism (DesA2, FadA6_3, FabG and FadE19) was altered, together with a range of other proteins. Only one protein, AhpC was differentially regulated in both strains of MAP. Seven proteins (DesA2, AhpC, AhpD, Ppa, FabG, and hypothetical proteins MAP2411 and MAP 1885c) were identified in previous in vitro studies with temperature, hypoxia and/or nutrient starvation stressors and may be general stress response proteins of MAP. Prior studies have identified immune responses directed against AhpC and Ppa in animals with Johne's disease, expression of sodA and ppa within macrophages, and reduced virulence of impA mutants in mice, highlighting the relevance of proteomic studies using these in vitro stress models for pathogenesis studies.
Insights
Mycobacterium avium subsp. paratuberculosis (MAP) strains showed altered protein expression under oxidative and nitrosative stress. Key antioxidant and fatty acid metabolism proteins were affected, with AhpC being differentially regulated in both sheep and cattle strains.
Area of Science:
- Microbiology
- Proteomics
- Bacterial Stress Response
Background:
- Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease.
- Understanding MAP's response to environmental stressors is crucial for disease control.
- Oxidative and nitrosative stress are relevant conditions encountered by MAP in host environments.
Purpose of the Study:
- To characterize the growth and protein expression profiles of sheep (S) and cattle (C) strains of MAP under oxidative and nitrosative stress.
- To identify specific proteins involved in the stress response of MAP.
- To compare the stress response between MAP strains from different hosts.
Main Methods:
- Induction of oxidative stress using hydrogen peroxide (H(2)O(2)) and nitrosative stress using sodium nitrite (NaNO(2)).
- Cultivation of MAP strains in BACTEC medium with varying inoculum sizes.
- Proteomic analysis using 2D gel electrophoresis and silver staining for protein detection.
- Identification of differentially expressed proteins using mass spectrometry.
Main Results:
- Oxidative stress was lethal for low inoculums (10(4) cells) but allowed survival and proteomic changes at higher inoculums (10(7) cells).
- Nitrosative stress slowed growth but allowed recovery upon resuscitation.
- Eighteen proteins were regulated under stress, including antioxidant enzymes (AhpC, AhpD, OxcA, SodA) and fatty acid metabolism proteins (DesA2, FadA6_3, FabG, FadE19).
- AhpC was the only protein differentially regulated in both sheep and cattle MAP strains.
- Seven identified proteins, including AhpC and Ppa, are potentially general stress response proteins.
Conclusions:
- MAP exhibits distinct proteomic changes in response to oxidative and nitrosative stress.
- Antioxidant and fatty acid metabolism pathways are significantly impacted by these stressors.
- The identified stress response proteins, particularly AhpC, are potential targets for understanding MAP pathogenesis and developing control strategies.
- Proteomic studies under in vitro stress conditions provide valuable insights into MAP pathogenesis relevant to Johne's disease.
