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NsrR: a key regulator circumventing Salmonella enterica serovar Typhimurium oxidative and nitrosative stress in vitro and in IFN-gamma-stimulated J774.2 macrophages

NsrR: a key regulator circumventing Salmonella enterica serovar Typhimurium oxidative and nitrosative stress in vitro and in IFN-gamma-stimulated J774.2 macrophages
NsrR: a key regulator circumventing Salmonella enterica serovar Typhimurium oxidative and nitrosative stress in vitro and in IFN-gamma-stimulated J774.2 macrophages

Over the past decade, the flavohaemoglobin Hmp has emerged as the most significant nitric oxide (NO)-detoxifying protein in many diverse micro-organisms, particularly pathogenic bacteria. Its expression in enterobacteria is dramatically increased on exposure to NO and other agents of nitrosative stress as a result of transcriptional regulation of hmp gene expression, mediated by (at least) four regulators. One such regulator, NsrR, has recently been shown to be responsible for repression of hmp transcription in the absence of NO in Escherichia coli and Salmonella, but the roles of other members of this regulon in Salmonella, particularly in surviving nitrosative stresses in vitro and in vivo, have not been elucidated. This paper demonstrates that an nsrR mutant of Salmonella enterica Serovar Typhimurium expresses high levels of Hmp both aerobically and anaerobically, exceeding those that can be elicited in vitro by supplementing media with S-nitrosoglutathione (GSNO). Elevated transcription of ytfE, ygbA, hcp and hcp is also observed, but no evidence was obtained for tehAB upregulation. The hyper-resistance to GSNO of an nsrR mutant is attributable solely to Hmp, since an nsrR hmp double mutant has a wild-type phenotype. However, overexpression of NsrR-regulated genes other than hmp confers some resistance of respiratory oxygen consumption to NO. The ability to enhance, by mutating NsrR, Hmp levels without recourse to exposure to nitrosative stress was used to test the hypothesis that control of Hmp levels is required to avoid oxidative stress, Hmp being a potent generator of superoxide. Within IFN-gamma-stimulated J774.2 macrophages, in which high levels of nitrite accumulated (indicative of NO production) an hmp mutant was severely compromised in survival. Surprisingly, under these conditions, an nsrR mutant (as well as an nsrR hmp double mutant) was also disadvantaged relative to the wild-type bacteria, attributable to the combined oxidative effect of the macrophage oxidative burst and Hmp-generated superoxide. This explanation is supported by the sensitivity in vitro of an nsrR mutant to superoxide and peroxide. Fur has recently been confirmed as a weak repressor of hmp transcription, and a fur mutant was also compromised for survival within macrophages even in the absence of elevated NO levels in non-stimulated macrophages. The results indicate the critical role of Hmp in protection of Salmonella from nitrosative stress within and outside macrophages, but also the key role of transcriptional regulation in tuning Hmp levels to prevent exacerbation of the oxidative stress encountered in macrophages.

Aerobiosis, Anaerobiosis, Animals, Anti-Bacterial Agents, Bacterial Proteins, Cell Line, Gene Deletion, Gene Expression Regulation, Bacterial, Hydrogen Peroxide, Interferon-gamma, Macrophages, Mice, Microbial Viability, Mutagenesis, Insertional, Nitric Oxide, Oxidative Stress, RNA, Bacterial, RNA, Messenger, Repressor Proteins, S-Nitrosoglutathione, Salmonella typhimurium, Superoxides, Transcription, Genetic, Journal Article, Research Support, Non-U.S. Gov't
1350-0872
1756-1771
Gilberthorpe, Nicola J.
681333af-29b2-4e56-ba25-fb1d8de16c1c
Lee, Margaret E.
a87d108c-c8e3-420c-be05-e7e23ee0619e
Stevanin, Tania M.
f1504019-7365-4b69-8404-e9df63a5348a
Read, Robert C.
b5caca7b-0063-438a-b703-7ecbb6fc2b51
Poole, Robert K.
5f1f3b79-cf45-4ae6-89cd-e4259c03ae56
Gilberthorpe, Nicola J.
681333af-29b2-4e56-ba25-fb1d8de16c1c
Lee, Margaret E.
a87d108c-c8e3-420c-be05-e7e23ee0619e
Stevanin, Tania M.
f1504019-7365-4b69-8404-e9df63a5348a
Read, Robert C.
b5caca7b-0063-438a-b703-7ecbb6fc2b51
Poole, Robert K.
5f1f3b79-cf45-4ae6-89cd-e4259c03ae56

Gilberthorpe, Nicola J., Lee, Margaret E., Stevanin, Tania M., Read, Robert C. and Poole, Robert K. (2007) NsrR: a key regulator circumventing Salmonella enterica serovar Typhimurium oxidative and nitrosative stress in vitro and in IFN-gamma-stimulated J774.2 macrophages. Microbiology (United Kingdom), 153 (Pt 6), 1756-1771. (doi:10.1099/mic.0.2006/003731-0).

Record type: Article

Abstract

Over the past decade, the flavohaemoglobin Hmp has emerged as the most significant nitric oxide (NO)-detoxifying protein in many diverse micro-organisms, particularly pathogenic bacteria. Its expression in enterobacteria is dramatically increased on exposure to NO and other agents of nitrosative stress as a result of transcriptional regulation of hmp gene expression, mediated by (at least) four regulators. One such regulator, NsrR, has recently been shown to be responsible for repression of hmp transcription in the absence of NO in Escherichia coli and Salmonella, but the roles of other members of this regulon in Salmonella, particularly in surviving nitrosative stresses in vitro and in vivo, have not been elucidated. This paper demonstrates that an nsrR mutant of Salmonella enterica Serovar Typhimurium expresses high levels of Hmp both aerobically and anaerobically, exceeding those that can be elicited in vitro by supplementing media with S-nitrosoglutathione (GSNO). Elevated transcription of ytfE, ygbA, hcp and hcp is also observed, but no evidence was obtained for tehAB upregulation. The hyper-resistance to GSNO of an nsrR mutant is attributable solely to Hmp, since an nsrR hmp double mutant has a wild-type phenotype. However, overexpression of NsrR-regulated genes other than hmp confers some resistance of respiratory oxygen consumption to NO. The ability to enhance, by mutating NsrR, Hmp levels without recourse to exposure to nitrosative stress was used to test the hypothesis that control of Hmp levels is required to avoid oxidative stress, Hmp being a potent generator of superoxide. Within IFN-gamma-stimulated J774.2 macrophages, in which high levels of nitrite accumulated (indicative of NO production) an hmp mutant was severely compromised in survival. Surprisingly, under these conditions, an nsrR mutant (as well as an nsrR hmp double mutant) was also disadvantaged relative to the wild-type bacteria, attributable to the combined oxidative effect of the macrophage oxidative burst and Hmp-generated superoxide. This explanation is supported by the sensitivity in vitro of an nsrR mutant to superoxide and peroxide. Fur has recently been confirmed as a weak repressor of hmp transcription, and a fur mutant was also compromised for survival within macrophages even in the absence of elevated NO levels in non-stimulated macrophages. The results indicate the critical role of Hmp in protection of Salmonella from nitrosative stress within and outside macrophages, but also the key role of transcriptional regulation in tuning Hmp levels to prevent exacerbation of the oxidative stress encountered in macrophages.

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More information

Accepted/In Press date: 6 February 2007
e-pub ahead of print date: 1 June 2007
Published date: June 2007
Keywords: Aerobiosis, Anaerobiosis, Animals, Anti-Bacterial Agents, Bacterial Proteins, Cell Line, Gene Deletion, Gene Expression Regulation, Bacterial, Hydrogen Peroxide, Interferon-gamma, Macrophages, Mice, Microbial Viability, Mutagenesis, Insertional, Nitric Oxide, Oxidative Stress, RNA, Bacterial, RNA, Messenger, Repressor Proteins, S-Nitrosoglutathione, Salmonella typhimurium, Superoxides, Transcription, Genetic, Journal Article, Research Support, Non-U.S. Gov't

Identifiers

Local EPrints ID: 416320
URI: https://eprints.soton.ac.uk/id/eprint/416320
ISSN: 1350-0872
PURE UUID: e4eb4721-dbdf-4d23-a252-c592c13521ac
ORCID for Robert C. Read: ORCID iD orcid.org/0000-0002-4297-6728

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Date deposited: 12 Dec 2017 17:30
Last modified: 14 Mar 2019 01:36

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Contributors

Author: Nicola J. Gilberthorpe
Author: Margaret E. Lee
Author: Tania M. Stevanin
Author: Robert C. Read ORCID iD
Author: Robert K. Poole

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