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Project Area 3 - Engineered thiol switches

 

 

 

 

Antelmann Projektbild

Haike Antelmann, Berlin

 

Functional charcterization of NaOCl-sensitive thiol-switches and their impact on the bacillithiol redox potential in Staphylococcus aureus

Staphylococcus aureus is a major human pathogen that can cause local skin or soft tissue infections, but also life-threatening diseases. During infections, S. aureus has to cope with reactive oxygen species (ROS) and hypochloric acid (HOCl) that are produced by activated macrophages and neutrophils as major killing mechanism. S. aureus uses the low molecular weight thiol bacillithiol (BSH) as protection mechanism against the host immune defense. BSH contributes to virulence of S. aureus and functions in detoxification of ROS, HOCl, toxins, electrophiles and antibiotics. Under hypochlorite stress, BSH forms mixed disulfides with proteins, termed as S‑bacillithiolations as a widespread thiol-protection and redox-switch mechanism. Using the quantitative thiol-redox proteomics approach OxICAT, we recently identified 58 NaOCl-sensitive proteins in S. aureus that could play protective roles against the host immune defense. Among these are five S-bacillithiolated proteins including the glycolytic Gap as the major target. S-bacillithiolation of Gap functions in thiol-protection against overoxidation to irreversible sulfonic acids and redox-regulation under H2O2 and NaOCl stress. The bacilliredoxins BrxA/B were shown to catalyze the reduction of S-bacillithiolated OhrR, MetE in B. subtilis and Gap in S. aureus in vitro. However, the complete Brx redox pathway is unknown. We have further constructed the first genetically encoded bacilliredoxin-fused redox biosensor (Brx-roGFP2) to monitor dynamic changes in the BSH redox potential in S. aureus.

In this project, we will characterize the functions of the interesting NaOCl-sensitive thiol-switches in the defense against oxidative stress. These thiol-switches include redox regulators, bacilliredoxins and other thiol-disulfide reductases, the nitric oxide synthase, the virulence factor SsaA2 and the metabolic enzymes Gap and AldA. In addition, novel Brx-roGFP2 and Tpx-roGFP2 biosensors will be applied to monitor the changes in the BSH redox potential and intracellular H2O2 generation in the S. aureus thiol-switch mutant backgrounds.

 

 

 

Dick

Tobias Dick, Heidelberg


Exploiting peroxidatic thiol switches as ultra-sensitive real-time probes to investigate cellular H2O2 homeostasis

 

 

We aim to establish genetically encoded H2O2 probes that are as sensitive as the most sensitive 2-Cys-peroxiredoxins. We want to be able to monitor in real-time the small changes in H2O2 levels that are sensed by those peroxiredoxins exhibiting second order rate constants on the order of ~107 M-1s-1. We estimate that this sensitivity corresponds to the sensing of intracellular H2O2 concentration changes taking place between the picomolar and lower nanomolar range.

 

By characterizing these probes in mechanistic detail we aim to refine our knowledge about structure-function relationships in the Prx family.

We aim to establish how these probes work in mechanistic detail and how they can be further manipulated to change their kinetic properties, e.g. the rate of probe reduction which determines overall H2O2 consumption by the probe. For example, we want to learn if and how subunit cooperativity and the environment of the CP-SOH influence the in vivo real-time dynamics of oxidation and reduction of peroxiredoxins.

 

We aim to understand how H2O2 probes and endogenous H2O2 consumers influence each other in the cellular context.

We want to understand if and how the ectopically expressed H2O2 probes influence other H2O2 consuming systems in the cells and - if necessary - find ways to minimize the impact of the probe on other processes. More generally, we want to know how H2O2 consumers kinetically compete with each other and with the introduced probe, i.e. how the H2O2 flux is distributed in vivo. Another aspect to be addressed is the in vivo occurrence and influence of hyperoxidation on probe responses.

 

 

We aim to use new probes to obtain insight into metabolic H2O2 homeostasis.

Making use of real-time probes with unprecedented H2O2 sensitivity, we aim to investigate how cytosolic H2O2 levels respond to changes in metabolic parameters including carbon source and O2 levels and to identify the important factors mediating this regulation. Another important aspect will be the study of population diversity with respect to H2O2 levels.

 

Schwarzländer

Markus Schwarzländer, Münster

 

Seeds are our main food source. For plants, seeds allow propagation over long distances and time periods, and provide protection of progeny from hostile environments, such as cold or drought. The remarkable ability of a seed to preserve a preformed embryo in a quiescent state and to rapidly re-activate it when the conditions are favourable is unique to higher plants. During germination the embryo relies on the energy stores within the seed. Their rapid mobilization to provide the ATP that the cells require for efficient germination is critically dependent on the mitochondria. While mitochondrial metabolism has to be largely inactive during quiescence to preserve resources, activation of germination at imbibition necessitates a sharp and rapid kick-start. As part of this wakeup call, numerous protein thiol switches are rapidly operated. We will dissect the specificity and regulatory significance of different mitochondrial thiol switches in controlling and supporting germination of Arabidopsis thaliana. A tailored in situ sensing setup for subcellular redox and energy physiology in intact seeds, embryos and purified mitochondria allows to monitor the rapid transition in thiol redox status and its crosstalk with cellular energy physiology. Sensing will inform state-of-the-art quantitative thiol redox proteomics to elucidate the identity and specificity of the individual target protein thiol switches, which are operated under the endogenous thermodynamic and kinetic constraints. Their individual regulatory significance and specificity will be dissected using targeted cysteine mutagenesis, in silico interaction modelling, as well as respiratory, metabolic and protein biochemical analyses. As such this work will elucidate the significance of mitochondrial thiol switching as an upstream mechanism of germination control. Yet, the investigated principles and the employed techniques will be significant across this SPP into which this project is well-embedded by several ongoing collaborations.