Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in <italic toggle="yes">Pseudomonas aeruginosa</italic> Biofilms

ABSTRACT Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanoni...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Brett M. Babin, Lydia Atangcho, Mark B. van Eldijk, Michael J. Sweredoski, Annie Moradian, Sonja Hess, Tim Tolker-Nielsen, Dianne K. Newman, David A. Tirrell
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	BONCAT Pseudomonas aeruginosa antibiotic resistance biofilms proteomics Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/304e4d5b63f845a58cf8ae5331b0818c
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:304e4d5b63f845a58cf8ae5331b0818c
record_format	dspace
spelling	oai:doaj.org-article:304e4d5b63f845a58cf8ae5331b0818c2021-11-15T15:51:51ZSelective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in <italic toggle="yes">Pseudomonas aeruginosa</italic> Biofilms10.1128/mBio.01593-172150-7511https://doaj.org/article/304e4d5b63f845a58cf8ae5331b0818c2017-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01593-17https://doaj.org/toc/2150-7511ABSTRACT Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance. IMPORTANCE Bacterial growth is frequently characterized by behavioral heterogeneity at the single-cell level. Heterogeneity is especially evident in the physiology of biofilms, in which distinct cellular subpopulations can respond differently to stresses, including subpopulations of pathogenic biofilms that are more tolerant to antibiotics. Global proteomic analysis affords insights into cellular physiology but cannot identify proteins expressed in a particular subpopulation of interest. Here, we report a chemical biology method to selectively label, enrich, and identify proteins expressed by cells within distinct regions of biofilm microcolonies. We used this approach to study changes in protein synthesis by the subpopulation of antibiotic-tolerant cells throughout a course of treatment. We found substantial differences between the initial response and the long-term adaptive strategy that biofilm cells use to cope with antibiotic stress. The method we describe is readily applicable to investigations of bacterial heterogeneity in diverse contexts.Brett M. BabinLydia AtangchoMark B. van EldijkMichael J. SweredoskiAnnie MoradianSonja HessTim Tolker-NielsenDianne K. NewmanDavid A. TirrellAmerican Society for MicrobiologyarticleBONCATPseudomonas aeruginosaantibiotic resistancebiofilmsproteomicsMicrobiologyQR1-502ENmBio, Vol 8, Iss 5 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	BONCAT Pseudomonas aeruginosa antibiotic resistance biofilms proteomics Microbiology QR1-502
spellingShingle	BONCAT Pseudomonas aeruginosa antibiotic resistance biofilms proteomics Microbiology QR1-502 Brett M. Babin Lydia Atangcho Mark B. van Eldijk Michael J. Sweredoski Annie Moradian Sonja Hess Tim Tolker-Nielsen Dianne K. Newman David A. Tirrell Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in <italic toggle="yes">Pseudomonas aeruginosa</italic> Biofilms
description	ABSTRACT Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance. IMPORTANCE Bacterial growth is frequently characterized by behavioral heterogeneity at the single-cell level. Heterogeneity is especially evident in the physiology of biofilms, in which distinct cellular subpopulations can respond differently to stresses, including subpopulations of pathogenic biofilms that are more tolerant to antibiotics. Global proteomic analysis affords insights into cellular physiology but cannot identify proteins expressed in a particular subpopulation of interest. Here, we report a chemical biology method to selectively label, enrich, and identify proteins expressed by cells within distinct regions of biofilm microcolonies. We used this approach to study changes in protein synthesis by the subpopulation of antibiotic-tolerant cells throughout a course of treatment. We found substantial differences between the initial response and the long-term adaptive strategy that biofilm cells use to cope with antibiotic stress. The method we describe is readily applicable to investigations of bacterial heterogeneity in diverse contexts.
format	article
author	Brett M. Babin Lydia Atangcho Mark B. van Eldijk Michael J. Sweredoski Annie Moradian Sonja Hess Tim Tolker-Nielsen Dianne K. Newman David A. Tirrell
author_facet	Brett M. Babin Lydia Atangcho Mark B. van Eldijk Michael J. Sweredoski Annie Moradian Sonja Hess Tim Tolker-Nielsen Dianne K. Newman David A. Tirrell
author_sort	Brett M. Babin
title	Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in <italic toggle="yes">Pseudomonas aeruginosa</italic> Biofilms
title_short	Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in <italic toggle="yes">Pseudomonas aeruginosa</italic> Biofilms
title_full	Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in <italic toggle="yes">Pseudomonas aeruginosa</italic> Biofilms
title_fullStr	Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in <italic toggle="yes">Pseudomonas aeruginosa</italic> Biofilms
title_full_unstemmed	Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in <italic toggle="yes">Pseudomonas aeruginosa</italic> Biofilms
title_sort	selective proteomic analysis of antibiotic-tolerant cellular subpopulations in <italic toggle="yes">pseudomonas aeruginosa</italic> biofilms
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/304e4d5b63f845a58cf8ae5331b0818c
work_keys_str_mv	AT brettmbabin selectiveproteomicanalysisofantibiotictolerantcellularsubpopulationsinitalictoggleyespseudomonasaeruginosaitalicbiofilms AT lydiaatangcho selectiveproteomicanalysisofantibiotictolerantcellularsubpopulationsinitalictoggleyespseudomonasaeruginosaitalicbiofilms AT markbvaneldijk selectiveproteomicanalysisofantibiotictolerantcellularsubpopulationsinitalictoggleyespseudomonasaeruginosaitalicbiofilms AT michaeljsweredoski selectiveproteomicanalysisofantibiotictolerantcellularsubpopulationsinitalictoggleyespseudomonasaeruginosaitalicbiofilms AT anniemoradian selectiveproteomicanalysisofantibiotictolerantcellularsubpopulationsinitalictoggleyespseudomonasaeruginosaitalicbiofilms AT sonjahess selectiveproteomicanalysisofantibiotictolerantcellularsubpopulationsinitalictoggleyespseudomonasaeruginosaitalicbiofilms AT timtolkernielsen selectiveproteomicanalysisofantibiotictolerantcellularsubpopulationsinitalictoggleyespseudomonasaeruginosaitalicbiofilms AT dianneknewman selectiveproteomicanalysisofantibiotictolerantcellularsubpopulationsinitalictoggleyespseudomonasaeruginosaitalicbiofilms AT davidatirrell selectiveproteomicanalysisofantibiotictolerantcellularsubpopulationsinitalictoggleyespseudomonasaeruginosaitalicbiofilms
_version_	1718427326458363904

Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in <italic toggle="yes">Pseudomonas aeruginosa</italic> Biofilms

Ejemplares similares