Biochemistry and structural biology: Redox protection of pathogens by low-molecular weight thiols

Maintaining the redox homeostasis is an important aspect of bacterial survival. During infection pathogens encounter human neutrophils and macrophages capable of generating reactive species. To limit the extent of damage and to increase survival, bacteria rely on various strategies to combat these toxic and reactive species. Low-molecular weight (LMW) thiols are important redox molecules used to balance the intracellular redox environment. This master project focuses on deciphering important enzymes in these bacterial redox defense networks, which are potential antimicrobial attack points. The master project is in the field of biochemistry and structural biology.

LMW thiols are a class of antioxidant molecules involved in many important cellular processes in all organisms, including a critical protective role in cells by maintaining cytosolic proteins in their reduced state to maintain the redox homeostasis. They also function as thiol cofactors of many enzymes in the protection against reactive oxygen, electrophilic, nitrogen, chlorine, and sulfur species (ROS, RES, RNS, RCS and RSS), as well as detoxification of toxins and antibiotics. LMW thiols are also involved in protection against heavy metals and metal storage.

The growing antimicrobial resistance evolving among pathogens increases the need to search for new antimicrobial targets, including enzymatic networks involved in the maintenance of a pathogen´s redox homeostasis. In humans the main LMW thiol is gluthatione, while bacteria also use unique bacterial LMW thiols. Firmicute bacteria, such as the clinically important pathogens Staphylococcus aureus and Bacillus cereus, use the unique bacterial LMW thiol bacillithiol (BSH) as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections. In this defense BSH is scarified and oxidised to BSSB (bacillithiol disulfide). We have recently solved the first structure of the enzyme that reduces BSSB back to BSH, bacillithiol disulfide reductase Bdr, but the reaction mechanism and the diversity among related organisms are missing. In Chlorobiaceae bacteria it has been suggested that a methylated BSH is the main thiol antioxidant, however, the biochemical proof of this is missing as well as knowledge about the potential methyltransferase that methylates BSH.

Another important role of BSH is thiol-protection under oxidative stress, through the redox post-translational modifications (PTM) termed protein S-bacillithiolation of Cys residues avoiding overoxidation and inactivation. To regenerate the active enzymes after stress, de-bacillithiolation is needed. This is catalyzed by the bacilliredoxin (Brx) redox pathway. Brxs attack the active site Cys on the BSH-mixed protein disulfide on S-bacillithiolated substrates, resulting in the transfer of BSH to the Brx active site Cys. Currently, three Brxs have been identified, which seem to have different specificity towards different target enzymes. However, the diversity in protein substrates among these enzymes is not established. The Brx-SSB intermediate can then be reduced by BSH, leading to the oxidised product BSSB, which again is reduced by Bdr.

Research projects will focus on understanding the enzymes involved in these LMW thiol-based defense systems in bacteria on a molecular level. To be able to answer these questions you will need to express and purify the proteins, solve the structure of them and/or do biochemical and biophysical characterisation. There are several different possibilities in master projects within these topics. Below are some possibilities listed:

  • Deciphering the mechanism for regeneration of BSH by investigating mutants of Bdr
  • Understanding the diversity among Bdrs from different organisms
  • Understanding the diversity among the Brx and de-bacillithiolation
  • Deciphering the methylation of BSH by methyltransferase NmbA
  • Investigation other LMW thiol systems in bacteria

Examples of methods you normally will use and learn:

  • Protein expression
  • Protein purification
  • Protein crystallisation
  • Solving the crystal structure of proteins
  • Activity studies
  • Enzyme kinetics
  • Anaerobic work
  • Spectroscopic charaterisation
  • Binding studies

Data collection for solving the structures will involve travelling abroad to synchrotrons. The biophysical and biochemical methods used in this master project will to a large extend be covered in BIOS4020, taught by the supervisors.

Through the master project, you will also learn to

  • Present your work orally through group meetings
  • Present your work through posters presentations at scientific conferences.
  • Learn to plan and perform scientific work
  • Learn to write up your work as a thesis

Supervision: The master project will be performed in the Structural Redox Biochemistry - Hersleth Lab (Section for Biochemistry and Molecular Biology) and supervised by Hans-Petter Hersleth and Marta Hammerstad.

Contact: Hans-Petter Hersleth room 2313, e-mail: h.p.hersleth@ibv.uio.no or Marta Hammerstad room 2313, e-mail: marta.hammerstad@ibv.uio.no

To read more see our group homepage: http://hersleth.org/

Some relevant references:

M. Hammerstad, I. Gudim & H.-P. Hersleth
The Crystal Structures of Bacillithiol Disulfide Reductase Bdr (YpdA) Provide Structural and Functional Insight into a New Type of FAD-Containing NADPH-Dependent Oxidoreductase.
Biochemistry (2020), 59, 4793-4798. [Link

M. Hammerstad & H.-P. Hersleth
Overview of structurally homologous flavoprotein oxidoreductases containing the low Mr thioredoxin reductase-like fold – A functionally diverse group.
Arch. Biochem. Biophys. (2021), 702, 108826. [Link

M. Shoor, I. Gudim, H.-P. Hersleth & M. Hammerstad
Thioredoxin reductase from Bacillus cereus exhibits distinct reduction and NADPH-binding properties.
FEBS Open Bio (2021), 11, 3019-3031. [Link

 

 

 

Publisert 13. apr. 2022 12:17 - Sist endret 13. apr. 2022 12:56

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