Unravelling the role of redox modifications Reactive oxygen species (ROS) can cause oxidative stress and protein damage in organisms, but they are also involved in signalling processes and regulate cellular processes. Professor Haike Antelmann tells us about her research into specific protein modifications caused by ROS, molecular switches that play a role as oxidative stress defense mechanisms in bacteria, and are important for the development of effective treatment against specific pathogens The exposure of bacteria to ROS results in oxidative stress responses and cellular damage, but low doses of ROS are also implicated in signalling processes and regulate specific thiol-switches. Based at the Freie Universität in Berlin, Professor Haike Antelmann is the Principal Investigator of the Mycothiolome project, an ERC-backed initiative investigating the topic. “My project is investigating the role of these thiol-switches, protein modifications that play a role under oxidative stress conditions,” she outlines. When bacteria are exposed to ROS during infection, or just in the course of everyday life due to aerobic growth, specific proteins inside the bacteria are modified by ROS. “ROS can damage proteins, and hence bacteria need specific protection and defense mechanisms,” explains Professor Antelmann. “Low molecular weight (LMW) thiols help to protect against protein damage and to keep the redox balance.”
Low molecular weight Thiols in bacteria This forms a core part of Professor Antelmann’s research agenda. Actinomycetes, specific members of Gram-positive bacteria, are an area of particular interest in the project. “Mycothiol is the major LMW thiol in Actinomycetes which can modify proteins under oxidative stress,” says Professor Antelmann. In the case of eukaryotes and most Gram-negative bacteria, the major LMW thiol is glutathione (GSH), an antioxidant that protects cellular components from damage by ROS. There is a large body of research about the role of protein modifications by GSH in humans, which are implicated in many physiological and pathophysiological processes, and hence can be thought of as molecular switches controlling human health or disease. However, Grampositive bacteria, such as Actinomycetes and Firmicutes do not produce GSH, but instead utilize alternative LMW thiols, such as mycothiol (MSH) and bacillithiol (BSH). “We have found that proteins are protected under oxidative stress by MSH and BSH. These protein modifications can lead to changes in the activity of the proteins, meaning that the proteins become inactive or active, which has a regulatory effect,” continues Professor Antelmann.
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We identified 58 proteins with S-mycothiolations in M. smegmatis under HOCl stress that are shown by color codes based on their % oxidation and classified into different functional categories in this Voronoi treemap. The cell size denotes the protein abundance in the total proteome. [Figure 2 published in Hillion et al., Scientific Reports 7: 1195. (2017)].
The project’s primary focus is fundamental research into the function and structure of specific proteins and how they are modified and what is the physiological consequence for the bacteria. Professor Antelmann and her colleagues in the project are using sophisticated techniques, including mass spectrometry and novel thiol-redox proteomics approaches, to analyse the S-mycothiolome under oxidative
to protect proteins from lethal damage in these conditions.” A number of interesting results have been gained, while researchers are also investigating the process by which proteins are switched-on or off between different conformational and functional states. Thiol switches play a central role in this regard. “We aim to find out what the most important thiol switches are with respect
We are investigating which modifications occur under oxidative stress, and the effect on the physiology of the cells. We have found that proteins are protected and redoxcontrolled under oxidative stress by mycothiol, which could be important under infection-related conditions stress conditions. “In this project we have applied new redox proteomics methods and visualization tools to unravel the different kinds of protein modifications in a quantitative manner. More than a thousand proteins are modified in different ways under oxidative stress conditions,” she says. There are several different forms of oxidative stress; one major area of research is hypochlorous acid stress. “Hypochlorous acid (HOCl) is a very potent oxidant,” explains Professor Antelmann. “Mycothiolations, protein modifications by the LMW thiol MSH, can help
to these protein modifications. With these thiol switches, you can almost switch a protein on or off,” explains Professor Antelmann. Many thiol switches have been found, and researchers now aim to develop a deeper understanding of the underlying mechanisms involved and their wider effects on cellular physiology. “Do these modifications play an important role in protecting the proteins against damage or do they change protein activity? We want to understand the physiology behind these protein modifications,” outlines Professor Antelmann.
EU Research
MYCOTHIOLOME
Corynebacterium diphtheriae and Mycobacteria This research is quite fundamental in nature at this stage, yet it also holds real importance to our understanding of the body’s response to certain pathologies. The project aims to explore the comprehensive S-mycothiolome of Corynebacterium diphtheriae, the bacterium that causes diphtheria and in Mycobacterium smegmatis as the model bacterium for pathogenic mycobacteria. Professor Antelmann says this work could help in future drug development. “We have found important regulatory thiol switches that are controlled by MSH and are conserved also in the pathogenic agent of tuberculosis Mycobacterium tuberculosis. If we find that mutants are compromised in its resistance to oxidative stress, this could indicate that this protein could be novel drug target,” she outlines. “We could use these proteins as targets to develop novel antimicrobial drugs. This is something we will investigate in a future study.” Thus, there is significant scope for continued research in this area, to both pursue further fundamental investigations, and to translate new findings into improved treatment of pathogenic bacteria. “Developing drugs to inhibit specific proteins could be an effective method of treating multi-resistant bacteria. This is our major longterm vision, and we are focusing on exploring the role of redox switches that are important for pathogen protection,” continues Professor Antelmann. The development of new drugs that target redox-regulated proteins depends on fundamental knowledge of the functions of target proteins in the pathogen’s defense against the immune system. Professor Antelmann emphasizes the importance of continued basic research to the discovery of new drug targets. “There is not much fundamental research on redox-switches in pathogenic bacteria that are modified by their own LMW thiols and confer
Protein S-mycothiolation and real-time redox imaging in Corynebacterium diphtheriae during ROS stress and infection conditions
Project Objectives
MYCOTHIOLOME Team
resistance to the host defense.” The project already discovered several new redox regulators that control antibiotics resistance and function also as ROS defense mechanisms which may be important also in M. tuberculosis. Moreover, the redox control mechanisms of main metabolic enzymes were studied in great detail. Some of these thiolswitches are conserved in the major pathogen Staphylococcus aureus which is another research focus. Thus, the developed redox tools were applied in other important pathogens, such as S. aureus to study adaptation under infection conditions. With the project being more than half way through its funding period, Professor Antelmann now concentrates her research on the molecular details and mechanisms of the thiol-switches using genetics, molecular biology, biochemistry and microbial physiology. “We focus on characterising the most important redox switches to understand the role of the thiol-switches in microbial physiology and pathogenicity,” she says. Translating this fundamental knowledge into the development of new drugs would be a task for a future project “We have found several interesting new drug targets and redox regulators, and we could potentially map out a research path for the next 10 years, for detailed investigation of these thiolswitches,” continues Professor Antelmann. “We work closely with other research groups, and have established many national and international collaborations at the Freie Universität Berlin.”
The ERC Consolidator Grant MYCOTHIOLOME aims to elucidate the physiological role of mycothiol (MSH) for post-translational thiol-modification of proteins in Corynebacterium diphtheriae and Mycobacterium smegmatis and to monitor in real-time the changes in the MSH redox potential under oxidative stress using genetically encoded redox biosensors.
Project Funding
European Research Council (ERC) Consolidator Grant MYCOTHIOLOME / GA No. 615585
Project Partners
• Brussels Center for Redox biology, Vrije Universiteit Brussels, Belgium • Department of Structural Biology, Freie Universität Berlin, Germany • Helmholtz-Zentrum für Umweltforschung, Leipzig, Germany • Institute for Microbiology, University of Greifswald, Germany • Centre for Biotechnology, University of Bielefeld, Germany • Center for Organismal Studies, Heidelberg, Germany
Contact Details
Project Coordinator, Professor Haike Antelmann Freie Universität Berlin Institut für Biologie-Mikrobiologie Königin-Luise-Straße 12-16 14195 Berlin T: +49-30-838-51221 E: haike.antelmann@fu-berlin.de W: http://www.bcp.fu-berlin.de/en/ biologie/arbeitsgruppen/mikrobiologie/ ag_antelmann/index.html Professor Haike Antelmann
Since 2015, Haike Antelmann has been full Professor of Molecular Microbiology at the Institute for Biology at the Freie Universität Berlin. Her research is focused on the physiological role of thiol-switches and the low molecular weight thiols bacillithiol and mycothiol in redox-regulation, cellular metabolism and virulence mechanisms in pathogenic Gram-positive bacteria.
MD simulations of BSH docked into the active site Cys of GapDH of S. aureus indicates that S-bacillithiolation does not require major structural changes. The figure was generated in collaboration with Frauke Gräter (Heidelberg) and Agnieszka Pietrzyk-Brzezinska (Lodz).
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