Role of Glutathione in Buffering Excess Intracellular Copper in <italic toggle="yes">Streptococcus pyogenes</italic>

ABSTRACT Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally responsive system for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (group A Streptococcus [GA...

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Autores principales:	Louisa J. Stewart, Cheryl-lynn Y. Ong, May M. Zhang, Stephan Brouwer, Liam McIntyre, Mark R. Davies, Mark J. Walker, Alastair G. McEwan, Kevin J. Waldron, Karrera Y. Djoko
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	copper homeostasis copper export metal buffer glutathione group A Streptococcus copper stress Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/54f175ae2c47421291edd930a86b688d
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spelling	oai:doaj.org-article:54f175ae2c47421291edd930a86b688d2021-11-15T15:55:43ZRole of Glutathione in Buffering Excess Intracellular Copper in <italic toggle="yes">Streptococcus pyogenes</italic>10.1128/mBio.02804-202150-7511https://doaj.org/article/54f175ae2c47421291edd930a86b688d2020-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02804-20https://doaj.org/toc/2150-7511ABSTRACT Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally responsive system for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]), this system is encoded by the copYAZ operon. This study demonstrates that although the site of GAS infection represents a Cu-rich environment, inactivation of the copA Cu efflux gene does not reduce virulence in a mouse model of invasive disease. In vitro, Cu treatment leads to multiple observable phenotypes, including defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde-3-phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as a consequence of mismetalation of noncognate metal-binding sites by Cu. Surprisingly, the onset of these effects is delayed by ∼4 h even though expression of copZ is upregulated immediately upon exposure to Cu. Further biochemical investigations show that the onset of all phenotypes coincides with depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu treatment. These results indicate that GSH buffers excess intracellular Cu when the transcriptionally responsive Cu export system is overwhelmed. Thus, while the copYAZ operon is responsible for Cu homeostasis, GSH has a role in Cu tolerance and allows bacteria to maintain metabolism even in the presence of an excess of this metal ion. IMPORTANCE The control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it has been established that rising intracellular Cu levels eventually fill the metal-sensing site of the endogenous Cu-sensing transcriptional regulator, which in turn induces transcription of a copper export pump. This response caps intracellular Cu availability below a well-defined threshold and prevents Cu toxicity. Glutathione, abundant in many bacteria, is known to bind Cu and has long been assumed to contribute to bacterial Cu handling. However, there is some ambiguity since neither its biosynthesis nor uptake is Cu-regulated. Furthermore, there is little experimental support for this physiological role of glutathione beyond measuring growth of glutathione-deficient mutants in the presence of Cu. Our work with group A Streptococcus provides new evidence that glutathione increases the threshold of intracellular Cu availability that can be tolerated by bacteria and thus advances fundamental understanding of bacterial Cu handling.Louisa J. StewartCheryl-lynn Y. OngMay M. ZhangStephan BrouwerLiam McIntyreMark R. DaviesMark J. WalkerAlastair G. McEwanKevin J. WaldronKarrera Y. DjokoAmerican Society for Microbiologyarticlecopper homeostasiscopper exportmetal bufferglutathionegroup A Streptococcuscopper stressMicrobiologyQR1-502ENmBio, Vol 11, Iss 6 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	copper homeostasis copper export metal buffer glutathione group A Streptococcus copper stress Microbiology QR1-502
spellingShingle	copper homeostasis copper export metal buffer glutathione group A Streptococcus copper stress Microbiology QR1-502 Louisa J. Stewart Cheryl-lynn Y. Ong May M. Zhang Stephan Brouwer Liam McIntyre Mark R. Davies Mark J. Walker Alastair G. McEwan Kevin J. Waldron Karrera Y. Djoko Role of Glutathione in Buffering Excess Intracellular Copper in <italic toggle="yes">Streptococcus pyogenes</italic>
description	ABSTRACT Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally responsive system for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]), this system is encoded by the copYAZ operon. This study demonstrates that although the site of GAS infection represents a Cu-rich environment, inactivation of the copA Cu efflux gene does not reduce virulence in a mouse model of invasive disease. In vitro, Cu treatment leads to multiple observable phenotypes, including defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde-3-phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as a consequence of mismetalation of noncognate metal-binding sites by Cu. Surprisingly, the onset of these effects is delayed by ∼4 h even though expression of copZ is upregulated immediately upon exposure to Cu. Further biochemical investigations show that the onset of all phenotypes coincides with depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu treatment. These results indicate that GSH buffers excess intracellular Cu when the transcriptionally responsive Cu export system is overwhelmed. Thus, while the copYAZ operon is responsible for Cu homeostasis, GSH has a role in Cu tolerance and allows bacteria to maintain metabolism even in the presence of an excess of this metal ion. IMPORTANCE The control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it has been established that rising intracellular Cu levels eventually fill the metal-sensing site of the endogenous Cu-sensing transcriptional regulator, which in turn induces transcription of a copper export pump. This response caps intracellular Cu availability below a well-defined threshold and prevents Cu toxicity. Glutathione, abundant in many bacteria, is known to bind Cu and has long been assumed to contribute to bacterial Cu handling. However, there is some ambiguity since neither its biosynthesis nor uptake is Cu-regulated. Furthermore, there is little experimental support for this physiological role of glutathione beyond measuring growth of glutathione-deficient mutants in the presence of Cu. Our work with group A Streptococcus provides new evidence that glutathione increases the threshold of intracellular Cu availability that can be tolerated by bacteria and thus advances fundamental understanding of bacterial Cu handling.
format	article
author	Louisa J. Stewart Cheryl-lynn Y. Ong May M. Zhang Stephan Brouwer Liam McIntyre Mark R. Davies Mark J. Walker Alastair G. McEwan Kevin J. Waldron Karrera Y. Djoko
author_facet	Louisa J. Stewart Cheryl-lynn Y. Ong May M. Zhang Stephan Brouwer Liam McIntyre Mark R. Davies Mark J. Walker Alastair G. McEwan Kevin J. Waldron Karrera Y. Djoko
author_sort	Louisa J. Stewart
title	Role of Glutathione in Buffering Excess Intracellular Copper in <italic toggle="yes">Streptococcus pyogenes</italic>
title_short	Role of Glutathione in Buffering Excess Intracellular Copper in <italic toggle="yes">Streptococcus pyogenes</italic>
title_full	Role of Glutathione in Buffering Excess Intracellular Copper in <italic toggle="yes">Streptococcus pyogenes</italic>
title_fullStr	Role of Glutathione in Buffering Excess Intracellular Copper in <italic toggle="yes">Streptococcus pyogenes</italic>
title_full_unstemmed	Role of Glutathione in Buffering Excess Intracellular Copper in <italic toggle="yes">Streptococcus pyogenes</italic>
title_sort	role of glutathione in buffering excess intracellular copper in <italic toggle="yes">streptococcus pyogenes</italic>
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/54f175ae2c47421291edd930a86b688d
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Role of Glutathione in Buffering Excess Intracellular Copper in <italic toggle="yes">Streptococcus pyogenes</italic>

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