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Copper toxicity has been a long-term selection pressure on bacteria due to its presence in the environment and its use as an antimicrobial agent by grazing protozoa, by phagocytic cells of the immune system, and in man-made medical and commercial products. There is recent evidence that exposure to increased copper stress may have been a key driver in the evolution and spread of methicillin-resistant Staphylococcus aureus , a globally important pathogen that causes significant mortality and morbidity worldwide. Yet it is unclear how S. aureus physiology is affected by copper stress or how it adapts in order to be able to grow in the presence of excess copper. Here, we have determined quantitatively how S. aureus alters its proteome during growth under copper stress conditions, comparing this adaptive response in two different types of growth regime. We found that the adaptive response involves induction of the conserved copper detoxification system as well as induction of enzymes of central carbon metabolism, with only limited induction of proteins involved in the oxidative stress response. Further, we identified a protein that binds copper inside S. aureus cells when stressed by copper excess. This copper-binding enzyme, a glyceraldehyde-3-phosphate dehydrogenase essential for glycolysis, is inhibited by copper in vitro and inside S. aureus cells. Together, our data demonstrate that copper stress leads to the inhibition of glycolysis in S. aureus , and that the bacterium adapts to this stress by altering its central carbon utilisation pathways.
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Characterization of the cyclic dipeptide cyclo(His-Pro) in Arabidopsis
Abstract Diketopiperazines (DKPs) are chemically and functionally diverse cyclic dipeptides associated primarily with microbes. Few DKPs have been reported from plants and animals; the best characterized is cyclo(His-Pro), found in the mammalian central nervous system, where it arises from the proteolytic cleavage of a thyrotropin-releasing tripeptide hormone. Herein, we report the identification of cyclo(His-Pro) in Arabidopsis (Arabidopsis thaliana), where its levels increase upon abiotic stress conditions, including high salt, heat, and cold. To screen for potential protein targets, we used isothermal shift assays, which examine changes in protein-melting stability upon ligand binding. Among the identified proteins, we focused on the glycolytic enzyme, cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC1). Binding between the GAPC1 protein and cyclo(His-Pro) was validated using nano-differential scanning fluorimetry and microscale thermophoresis, and we could further demonstrate that cyclo(His-Pro) inhibits GAPC1 activity with an IC50 of ∼200 μm. This inhibition was conserved in human GAPDH. Inhibition of glyceraldehyde-3-phosphate dehydrogenase activity has previously been reported to reroute carbon from glycolysis toward the pentose phosphate pathway. Accordingly, cyclo(His-Pro) supplementation augmented NADPH levels, increasing the NADPH/NADP+ ratio. Phenotypic screening revealed that plants supplemented with cyclo(His-Pro) were more tolerant to high-salt stress, as manifested by higher biomass, which we show is dependent on GAPC1/2. Our work reports the identification and functional characterization of cyclo(His-Pro) as a modulator of glyceraldehyde-3-phosphate dehydrogenase in plants.
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- PAR ID:
- 10591858
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Plant Physiology
- Volume:
- 198
- Issue:
- 1
- ISSN:
- 0032-0889
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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