Dataset Information

Genetic basis of persister tolerance to aminoglycosides in Escherichia coli.

ABSTRACT: UNLABELLED:Persisters are dormant variants that form a subpopulation of drug-tolerant cells largely responsible for the recalcitrance of chronic infections. However, our understanding of the genetic basis of antibiotic tolerance remains incomplete. In this study, we applied transposon sequencing (Tn-Seq) to systematically investigate the mechanism of aminoglycoside tolerance in Escherichia coli. We constructed a highly saturated transposon library that covered the majority of E. coli genes and promoter regions and exposed a stationary-phase culture to a lethal dose of gentamicin. Tn-Seq was performed to evaluate the survival of each mutant to gentamicin exposure. We found that the disruption of several distinct pathways affected gentamicin tolerance. We identified 105 disrupted gene/promoter regions with a more than 5-fold reduction in gentamicin tolerance and 37 genes with a more than 5-fold increased tolerance. Functional cluster analysis suggests that deficiency in motility and amino acid synthesis significantly diminished persisters tolerant to gentamicin, without changing the MIC. Amino acid auxotrophs, including serine, threonine, glutamine, and tryptophan auxotrophs, exhibit strongly decreased tolerance to gentamicin, which cannot be restored by supplying the corresponding amino acids to the culture. Interestingly, supplying these amino acids to wild-type E. coli sensitizes stationary-phase cells to gentamicin, possibly through the inhibition of amino acid synthesis. In addition, we found that the deletion of amino acid synthesis genes significantly increases gentamicin uptake in stationary phase, while the deletion of flagellar genes does not affect gentamicin uptake. We conclude that activation of motility and amino acid biosynthesis contributes to the formation of persisters tolerant to gentamicin. IMPORTANCE:Persisters are responsible for the recalcitrance of chronic infections to antibiotics. The pathways of persister formation in E. coli are redundant, and our understanding of the mechanism of persister formation is incomplete. Using a highly saturated transposon insertion library, we systematically analyzed the contribution of different cellular processes to the formation of persisters tolerant to aminoglycosides. Unexpectedly, we found that activation of amino acid synthesis and motility strongly contributes to persister formation. The approach used in this study leads to an understanding of aminoglycoside tolerance and provides a general method to identify genes affecting persister formation.

SUBMITTER: Shan Y

PROVIDER: S-EPMC4453570 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Genetic basis of persister tolerance to aminoglycosides in Escherichia coli.

Shan Yue Y Lazinski David D Rowe Sarah S Camilli Andrew A Lewis Kim K

mBio 20150407 2

<h4>Unlabelled</h4>Persisters are dormant variants that form a subpopulation of drug-tolerant cells largely responsible for the recalcitrance of chronic infections. However, our understanding of the genetic basis of antibiotic tolerance remains incomplete. In this study, we applied transposon sequencing (Tn-Seq) to systematically investigate the mechanism of aminoglycoside tolerance in Escherichia coli. We constructed a highly saturated transposon library that covered the majority of E. coli gen ...[more]

PMID: 25852159

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Project description:To cope with fluctuations in their environment, bacteria have evolved multiple adaptive stress responses. One such response is the nitrogen regulation stress response, which allows bacteria, such as Escherichia coli, to cope with and overcome conditions of nitrogen limitation. This response is directed by the two-component system NtrBC, where NtrC acts as the major transcriptional regulator to activate the expression of genes to mount the response. Recently, my colleagues and I showed that NtrC directly regulates the expression of the relA gene, the major (p)ppGpp synthetase in E. coli, coupling the nitrogen regulation stress and stringent responses. As elevated levels of (p)ppGpp have been implicated in the formation of persister cells, here, I investigated whether nitrogen starvation promotes their formation and whether the NtrC-RelA regulatory cascade plays a role. The results reveal that nitrogen-starved E. coli synthesizes (p)ppGpp and forms a higher percentage of persister cells than nonstarved cells and that both NtrC and RelA are important for these processes. This study provides novel insights into how the formation of persisters can be promoted in response to a nutritional stress.IMPORTANCE Bacteria often reside in environments where nutrient availability is scarce; therefore, they have evolved adaptive responses to rapidly cope with conditions of feast and famine. Understanding the mechanisms that underpin the regulation of how bacteria cope with this stress is a fundamentally important question in the wider context of understanding the biology of the bacterial cell and bacterial pathogenesis. Two major adaptive mechanisms to cope with starvation are the nitrogen regulation (ntr) stress and stringent responses. Here, I describe how these bacterial stress responses are coordinated under conditions of nitrogen starvation to promote the formation of antibiotic-tolerant persister cells by elevating levels of the secondary messenger (p)ppGpp.

Project description:Despite the identification of many genes and pathways involved in the persistence phenomenon in bacteria, the mechanisms of persistence are not well understood. Here, using Escherichia coli, we identified polynucleotide phosphorylase (PNPase) as a key regulator of persister formation. We constructed the pnp knockout strain (Δpnp) and its complemented strain and exposed them to antibiotics and stress conditions. The results showed that, compared with the wild-type strain W3110, the Δpnp strain had significant defects in persistence to antibiotics and stresses, and the persistence phenotype was restored upon complementation with the pnp gene. Transcriptome sequencing (RNA-seq) analysis revealed that 242 (166 upregulated and 76 downregulated) genes were differentially expressed in the Δpnp strain compared with the W3110 strain. KEGG analysis of the upregulated genes showed that these genes were mostly mapped to metabolism and virulence pathways, of which most are positively regulated by the global regulator cyclic AMP receptor protein (CRP). Correspondingly, the transcription level of the crp gene in the Δpnp strain increased 3.22-fold in the early stationary phase. We further explored the indicators of cellular metabolism of the Δpnp strain, the phenotype of the pnp and crp double-deletion mutant, and the transcriptional activity of the crp gene. Our results indicate that PNPase controls cellular metabolism by negatively regulating the crp operon via targeting the 5'-untranslated region of the crp transcript. This study reveals a persister mechanism and provides novel targets for the development of drugs against persisters for more effective treatment. IMPORTANCE Persisters pose significant challenges for a more effective treatment of persistent infections. An improved understanding of mechanisms of persistence will provide therapeutic targets important for the development of better treatments. Since recent studies with the key tuberculosis persister drug pyrazinamide have implicated polynucleotide phosphorylase (PNPase) as a drug target, in this study, we addressed the possibility that PNPase might be involved in persistence in Escherichia coli. Our study demonstrates PNPase indeed being involved in persistence, provides a mechanism by which PNPase controls persister formation, and suggests a new therapeutic target for treating persistent bacterial infections.

Dataset Information

Genetic basis of persister tolerance to aminoglycosides in Escherichia coli.

Publications

Genetic basis of persister tolerance to aminoglycosides in Escherichia coli.

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