Dataset Information

Proteomics analysis reveals a role for E. coli polyphosphate kinase in membrane structure and polymyxin resistance during starvation.

ABSTRACT: Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (∆ppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and ∆ppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of required building blocks. From our dataset, we were particularly interested in Arn and EptA proteins, which were downregulated in ∆ppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins, and provide evidence that this mis-regulation in ∆ppk cells stems from a failure to induce the BasS/BasR two-component system during starvation. We also show that ∆ppk mutants unable to upregulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK.

SUBMITTER: Baijal K

PROVIDER: S-EPMC10350021 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Proteomics analysis reveals a role for E. coli polyphosphate kinase in membrane structure and polymyxin resistance during starvation.

Baijal Kanchi K Abramchuk Iryna I Herrera Carmen M CM Stephen Trent M M Lavallée-Adam Mathieu M Downey Michael M

bioRxiv : the preprint server for biology 20230706

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (∆ppk) during star ...[more]

PMID: 37461725

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Project description:Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1,000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work, we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (Δppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and Δppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of the required building blocks. From our data set, we were particularly interested in Arn and EptA proteins, which were down-regulated in Δppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins in K-12 strains and a uropathogenic isolate, and provide evidence that this mis-regulation in Δppk cells stems from a failure to induce the BasRS two-component system during starvation. We also show that Δppk mutants unable to up-regulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK.

Project description:Polyphosphates (polyP) are chains of inorganic phosphates that can reach 1000s of residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (∆ppk mutants) during nutrient starvation and to identify novel pathways regulated by PPK. In response to nutrient starvation, we found 92 proteins significantly differentially expressed between wild-type and ∆ppkmutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport while Δppkmutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK cells remain inappropriately primed for growth. From our data set, we were particularly interested in Arn and EptA proteins, which were downregulated in ∆ppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins, and provide evidence that this mis-regulation in ∆ppk cells stems from a failure to induce the BasS/BasR two component system during starvation. We also show that ∆ppk mutants unable to upregulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in diverse pathways impacted by PPK.

Dataset Information

Proteomics analysis reveals a role for E. coli polyphosphate kinase in membrane structure and polymyxin resistance during starvation.

Publications

Proteomics analysis reveals a role for <i>E. coli</i> polyphosphate kinase in membrane structure and polymyxin resistance during starvation.

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