Persister <named-content content-type="genus-species">Escherichia coli</named-content> Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment

ABSTRACT Persister and viable but non-culturable (VBNC) cells are two clonal subpopulations that can survive multidrug exposure via a plethora of putative molecular mechanisms. Here, we combine microfluidics, time-lapse microscopy, and a plasmid-encoded fluorescent pH reporter to measure the dynamic...

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Autores principales:	Olivia Goode, Ashley Smith, Ashraf Zarkan, Jehangir Cama, Brandon M. Invergo, Daaniyah Belgami, Santiago Caño-Muñiz, Jeremy Metz, Paul O’Neill, Aaron Jeffries, Isobel H. Norville, Jonathan David, David Summers, Stefano Pagliara
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2021
Materias:	indole intracellular pH microfluidics single-cell analysis antibiotic resistance antibiotics Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/6d1f31061d5a4954937de418453ed324
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spelling	oai:doaj.org-article:6d1f31061d5a4954937de418453ed3242021-11-10T18:37:50ZPersister <named-content content-type="genus-species">Escherichia coli</named-content> Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment10.1128/mBio.00909-212150-7511https://doaj.org/article/6d1f31061d5a4954937de418453ed3242021-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00909-21https://doaj.org/toc/2150-7511ABSTRACT Persister and viable but non-culturable (VBNC) cells are two clonal subpopulations that can survive multidrug exposure via a plethora of putative molecular mechanisms. Here, we combine microfluidics, time-lapse microscopy, and a plasmid-encoded fluorescent pH reporter to measure the dynamics of the intracellular pH of individual persister, VBNC, and susceptible Escherichia coli cells in response to ampicillin treatment. We found that even before antibiotic exposure, persisters have a lower intracellular pH than those of VBNC and susceptible cells. We then investigated the molecular mechanisms underlying the observed differential pH regulation in persister E. coli cells and found that this is linked to the activity of the enzyme tryptophanase, which is encoded by tnaA. In fact, in a ΔtnaA strain, we found no difference in intracellular pH between persister, VBNC, and susceptible E. coli cells. Whole-genome transcriptomic analysis revealed that, besides downregulating tryptophan metabolism, the ΔtnaA strain downregulated key pH homeostasis pathways, including the response to pH, oxidation reduction, and several carboxylic acid catabolism processes, compared to levels of expression in the parental strain. Our study sheds light on pH homeostasis, proving that the regulation of intracellular pH is not homogeneous within a clonal population, with a subset of cells displaying a differential pH regulation to perform dedicated functions, including survival after antibiotic treatment. IMPORTANCE Persister and VBNC cells can phenotypically survive environmental stressors, such as antibiotic treatment, limitation of nutrients, and acid stress, and have been linked to chronic infections and antimicrobial resistance. It has recently been suggested that pH regulation might play a role in an organism’s phenotypic survival to antibiotics; however, this hypothesis remains to be tested. Here, we demonstrate that even before antibiotic treatment, cells that will become persisters have a more acidic intracellular pH than clonal cells that will be either susceptible or VBNC upon antibiotic treatment. Moreover, after antibiotic treatment, persisters become more alkaline than VBNC and susceptible E. coli cells. This newly found phenotypic feature is remarkable because it distinguishes persister and VBNC cells that have often been thought to display the same dormant phenotype. We then show that this differential pH regulation is abolished in the absence of the enzyme tryptophanase via a major remodeling of bacterial metabolism and pH homeostasis. These new whole-genome transcriptome data should be taken into account when modeling bacterial metabolism at the crucial transition from exponential to stationary phase. Overall, our findings indicate that the manipulation of the intracellular pH represents a bacterial strategy for surviving antibiotic treatment. In turn, this suggests a strategy for developing persister-targeting antibiotics by interfering with cellular components, such as tryptophanase, that play a major role in pH homeostasis.Olivia GoodeAshley SmithAshraf ZarkanJehangir CamaBrandon M. InvergoDaaniyah BelgamiSantiago Caño-MuñizJeremy MetzPaul O’NeillAaron JeffriesIsobel H. NorvilleJonathan DavidDavid SummersStefano PagliaraAmerican Society for Microbiologyarticleindoleintracellular pHmicrofluidicssingle-cell analysisantibiotic resistanceantibioticsMicrobiologyQR1-502ENmBio, Vol 12, Iss 4 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	indole intracellular pH microfluidics single-cell analysis antibiotic resistance antibiotics Microbiology QR1-502
spellingShingle	indole intracellular pH microfluidics single-cell analysis antibiotic resistance antibiotics Microbiology QR1-502 Olivia Goode Ashley Smith Ashraf Zarkan Jehangir Cama Brandon M. Invergo Daaniyah Belgami Santiago Caño-Muñiz Jeremy Metz Paul O’Neill Aaron Jeffries Isobel H. Norville Jonathan David David Summers Stefano Pagliara Persister <named-content content-type="genus-species">Escherichia coli</named-content> Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment
description	ABSTRACT Persister and viable but non-culturable (VBNC) cells are two clonal subpopulations that can survive multidrug exposure via a plethora of putative molecular mechanisms. Here, we combine microfluidics, time-lapse microscopy, and a plasmid-encoded fluorescent pH reporter to measure the dynamics of the intracellular pH of individual persister, VBNC, and susceptible Escherichia coli cells in response to ampicillin treatment. We found that even before antibiotic exposure, persisters have a lower intracellular pH than those of VBNC and susceptible cells. We then investigated the molecular mechanisms underlying the observed differential pH regulation in persister E. coli cells and found that this is linked to the activity of the enzyme tryptophanase, which is encoded by tnaA. In fact, in a ΔtnaA strain, we found no difference in intracellular pH between persister, VBNC, and susceptible E. coli cells. Whole-genome transcriptomic analysis revealed that, besides downregulating tryptophan metabolism, the ΔtnaA strain downregulated key pH homeostasis pathways, including the response to pH, oxidation reduction, and several carboxylic acid catabolism processes, compared to levels of expression in the parental strain. Our study sheds light on pH homeostasis, proving that the regulation of intracellular pH is not homogeneous within a clonal population, with a subset of cells displaying a differential pH regulation to perform dedicated functions, including survival after antibiotic treatment. IMPORTANCE Persister and VBNC cells can phenotypically survive environmental stressors, such as antibiotic treatment, limitation of nutrients, and acid stress, and have been linked to chronic infections and antimicrobial resistance. It has recently been suggested that pH regulation might play a role in an organism’s phenotypic survival to antibiotics; however, this hypothesis remains to be tested. Here, we demonstrate that even before antibiotic treatment, cells that will become persisters have a more acidic intracellular pH than clonal cells that will be either susceptible or VBNC upon antibiotic treatment. Moreover, after antibiotic treatment, persisters become more alkaline than VBNC and susceptible E. coli cells. This newly found phenotypic feature is remarkable because it distinguishes persister and VBNC cells that have often been thought to display the same dormant phenotype. We then show that this differential pH regulation is abolished in the absence of the enzyme tryptophanase via a major remodeling of bacterial metabolism and pH homeostasis. These new whole-genome transcriptome data should be taken into account when modeling bacterial metabolism at the crucial transition from exponential to stationary phase. Overall, our findings indicate that the manipulation of the intracellular pH represents a bacterial strategy for surviving antibiotic treatment. In turn, this suggests a strategy for developing persister-targeting antibiotics by interfering with cellular components, such as tryptophanase, that play a major role in pH homeostasis.
format	article
author	Olivia Goode Ashley Smith Ashraf Zarkan Jehangir Cama Brandon M. Invergo Daaniyah Belgami Santiago Caño-Muñiz Jeremy Metz Paul O’Neill Aaron Jeffries Isobel H. Norville Jonathan David David Summers Stefano Pagliara
author_facet	Olivia Goode Ashley Smith Ashraf Zarkan Jehangir Cama Brandon M. Invergo Daaniyah Belgami Santiago Caño-Muñiz Jeremy Metz Paul O’Neill Aaron Jeffries Isobel H. Norville Jonathan David David Summers Stefano Pagliara
author_sort	Olivia Goode
title	Persister <named-content content-type="genus-species">Escherichia coli</named-content> Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment
title_short	Persister <named-content content-type="genus-species">Escherichia coli</named-content> Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment
title_full	Persister <named-content content-type="genus-species">Escherichia coli</named-content> Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment
title_fullStr	Persister <named-content content-type="genus-species">Escherichia coli</named-content> Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment
title_full_unstemmed	Persister <named-content content-type="genus-species">Escherichia coli</named-content> Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment
title_sort	persister <named-content content-type="genus-species">escherichia coli</named-content> cells have a lower intracellular ph than susceptible cells but maintain their ph in response to antibiotic treatment
publisher	American Society for Microbiology
publishDate	2021
url	https://doaj.org/article/6d1f31061d5a4954937de418453ed324
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Persister <named-content content-type="genus-species">Escherichia coli</named-content> Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment

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