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Protein S-mycothiolation and real-time redox imaging in Corynebacterium diphtheriae during ROS stress and infection conditions

Objective

Glutathione serves as the major thiol-redox buffer in the defense against Reactive Oxygen Species (ROS) in eukaryotes. Firmicutes bacteria utilize as thiol redox buffer bacillithiol (Cys-GlcN-Mal, BSH) and Actinomycetes produce the related redox buffer mycothiol (AcCys-GlcN-Ins, MSH). In eukaryotes, proteins are post-translational modified to S-glutathionylated proteins in response to oxidative stress. S-glutathionylation has emerged as major redox-regulatory mechanism and protects cysteine residues against overoxidation to sulfonic acids. Using thiol-redox proteomics and mass spectrometry (MS) we have recently discovered protein S-bacillithiolations as mixed BSH protein disulfides in response to oxidative stress in Firmicutes bacteria. Protein S-bacillithiolation controls the activity of the redox-sensing OhrR repressor and protects active site cysteine residues of metabolic enzymes, antioxidant function proteins and translation factors. However, it is unknown if ROS and infection conditions cause protein S-mycothiolations and affect the cellular MSH redox potential in pathogenic Mycobacteria and Corynebacteria. Here we aim to explore the comprehensive mycothiolome in the major respiratory pathogen Corynebacterium diphtheriae.
We apply gel-based and novel MS-based thiol-redox proteomic approaches for the quantitative analysis of the S-mycothiolome in C. diphtheriae under oxidative stress conditions (e.g. NEM-Biotin-Switch-Assay). Novel genetically encoded redox biosensors (Mrx1-roGFP2 and roGFP2-Orp1) will be developed for real-time imaging of the MSH redox potential and ROS production during infections in C. diphtheriae. The role of S-mycothiolated proteins for redox regulation, fitness, stress resistance and virulence mechanisms will be investigated. Our studies provide leads to understand the physiological role of thiol-redox switches in the defense against the host immune system and in the regulation of virulence mechanisms in Gram-positive pathogens.

Field of science

  • /natural sciences/chemical sciences/analytical chemistry/quantitative analysis
  • /natural sciences/biological sciences/biochemistry/biomolecules/proteins
  • /medical and health sciences/basic medicine/immunology
  • /natural sciences/biological sciences/biochemistry/biomolecules/proteins/proteomics
  • /natural sciences/biological sciences/biochemistry/biomolecules/proteins/enzymes
  • /natural sciences/chemical sciences/analytical chemistry/mass spectrometry
  • /engineering and technology/environmental biotechnology/biosensing

Call for proposal

ERC-2013-CoG
See other projects for this call

Funding Scheme

ERC-CG - ERC Consolidator Grants

Host institution

FREIE UNIVERSITAET BERLIN
Address
Kaiserswerther Strasse 16-18
14195 Berlin
Germany
Activity type
Higher or Secondary Education Establishments
EU contribution
€ 1 525 449,60
Principal investigator
Haike Antelmann (Dr.)
Administrative Contact
Michael Hune (Mr.)

Beneficiaries (2)

FREIE UNIVERSITAET BERLIN
Germany
EU contribution
€ 1 525 449,60
Address
Kaiserswerther Strasse 16-18
14195 Berlin
Activity type
Higher or Secondary Education Establishments
Principal investigator
Haike Antelmann (Dr.)
Administrative Contact
Michael Hune (Mr.)
UNIVERSITAET GREIFSWALD

Participation ended

Germany
EU contribution
€ 432 864,40
Address
Domstrasse 11
17489 Greifswald
Activity type
Higher or Secondary Education Establishments
Administrative Contact
Diana Lode (Ms.)