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Research Article | Molecular Biology and Physiology

Proteomic Study of the Survival and Resuscitation Mechanisms of Filamentous Persisters in an Evolved Escherichia coli Population from Cyclic Ampicillin Treatment

Jordy Evan Sulaiman, Henry Lam
Joshua E. Elias, Editor
Jordy Evan Sulaiman
aDepartment of Chemical and Biological Engineering, The Hong Kong University of Science & Technology, Kowloon, Hong Kong
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Henry Lam
aDepartment of Chemical and Biological Engineering, The Hong Kong University of Science & Technology, Kowloon, Hong Kong
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Joshua E. Elias
Chan Zuckerberg Biohub
Roles: Editor
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DOI: 10.1128/mSystems.00462-20
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  • FIG 1
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    FIG 1

    Evo3A has increased survival toward ampicillin treatment, and the persisters exhibit extensive filamentation. (a) Survival (%) of E. coli K-12 and Evo3A to ampicillin (125 μg/ml), ofloxacin (5 μg/ml), and kanamycin (150 μg/ml) treatment for 3 h (mean ± standard error of the mean [SEM], n = 3). Significance of difference with ancestral population: **, P < 0.01; ns, not significant (two-tailed t test with unequal variances of the log-transformed values). (b) Epifluorescence microscopy of E. coli K-12 and Evo3A during antibiotic treatment. Cells before and after 1, 3, 5, and 7 h of ampicillin treatment (∼15× MIC) were stained with a LIVE/DEAD kit and visualized by epifluorescence microscopy. Green cells are viable cells, and red cells are dead cells. (c) Length of ampicillin-treated Evo3A cells, calculated by averaging 50 distinct individual cells randomly from each time point (data were shown as median with interquartile range, n = 50). (d to f) DNA (d), protein (e), and ATP (f) extracted from Evo3A before and after 1, 3, and 5 h of ampicillin treatment. DNA was extracted using Qiagen’s DNeasy blood and tissue kit, protein concentrations were determined by the bicinchoninic acid (BCA) assay, and ATP measurement was performed using the BacTiter-Glo assay. Data are presented relative to the untreated cells (0 h), as marked by the horizontal dotted lines. Data represent three biological replicates, and each data point is denoted as mean ± SEM.

  • FIG 2
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    FIG 2

    Microscopy of persisters from E. coli K-12 and Evo3A during resuscitation after ampicillin treatment. (a) Cells exposed to ampicillin (∼15× MIC) for 5 h were resuspended in fresh LB medium and observed by epifluorescence microscopy at indicated time points during resuscitation. Green cells are live cells while red cells are dead cells (defined as those with compromised membrane). (b) Time-lapse microscopy of Evo3A during resuscitation on LB pads. Figures are representative phase-contrast images of recovering ampicillin-treated Evo3A cells on LB agar pads, and the panel at t = 180 min shows the incipient colony formation from a filament. Red arrow shows a lysed filament, and blue arrows indicate cell division events that are slower than the other cells. Bars, 50 μm.

  • FIG 3
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    FIG 3

    Time-series analysis of the protein expression profile on Evo3A during antibiotic treatment. (a) Venn diagram for proteome comparison of Evo3A before and after 1 h, 3 h, and 5 h of ampicillin treatment (125 μg/ml). (b) Volcano plots of the protein expression profile on Evo3A from 1 h to 5 h of ampicillin treatment. Fold change values of the proteins after 1 h, 3 h, and 5 h of treatment are fitted to a linear regression model, and the slopes of the fitted curve were shown with the respective P value for each protein. Slopes with a P value below 0.05 or an absolute log2(P value) above 4.32 are considered significant. Proteins with increasing expression profiles are marked by the blue dots, while proteins with decreasing expression profiles are marked by the red dots. (c) Protein-protein interaction network of the proteins with increasing and decreasing expression profiles throughout the antibiotic treatment as predicted by STRING v10.5. The lines represent protein interaction (thicker lines mean higher confidence), and the dots in different colors represent different biological processes involved. Nodes with black outlines are proteins with increasing expression profile, and nodes with no outlines are proteins with decreasing expression profile. Nodes without function enrichment are hidden from the network. (d) Linear model fitting of the 18 proteins with increasing expression profile during antibiotic treatment from 3 biological replicates of Evo3A. Red lines represent the fitted linear models with the 95% confidence bounds shown as red dotted curves.

  • FIG 4
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    FIG 4

    Proteome comparison of Evo3A after 5 h of ampicillin treatment to that before treatment. (a) Venn diagram for proteome comparison of Evo3A before and after 5 h of ampicillin treatment (125 μg/ml). (b) Volcano plot for Evo3A after 5 h of ampicillin treatment compared with Evo3A before ampicillin treatment. Red dots are the downregulated proteins, and blue dots are the upregulated proteins. (c) Pathway enrichment study (KEGG) by DAVID of the differentially expressed proteins and newly detected proteins on Evo3A after 5 h of ampicillin treatment compared to those before treatment. Fold enrichment is defined as the ratio of proportion of the input information to the background information. (d) Functional classification of the differentially expressed and newly detected proteins on Evo3A after 5 h of ampicillin treatment compared to those before treatment by gene ontology (GO) analysis using DAVID, classified by biological process. (e) Total ROS generation in cultures treated with ampicillin (125 μg/ml) or 50 μM FeSO4 and 10 mM H2O2 for 1 h was assayed using H2DCFDA probe and normalized by CFU counts (mean ± SEM, n = 3). (f) Survival of cultures exposed to ampicillin (125 μg/ml) for 5 h in the presence or absence of 5% DMSO (an OH· scavenger). Aliquots were washed and plated to determine survival (mean ± SEM, n = 3). Significance of difference: ***, P < 0.001; **, P < 0.01; ns, not significant (two-tailed t test with unequal variances of the log-transformed values). (g) Frequency of mutagenesis was assayed in cells previously exposed to ampicillin for 1 h. Cultures were washed, grown overnight, and plated on LB agar plates supplemented with rifampin (100 μg/ml). Changes in the frequency of generation of rifampin-resistant mutants were used as an indicator for DNA damage (mean ± SEM, n = 3). Significance of difference: ***, P < 0.001; *, P < 0.05; ns, not significant (two-tailed t test with unequal variances).

  • FIG 5
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    FIG 5

    Separation of filaments from the new progeny after 3 h of resuspension in fresh medium. (a) Workflow for separation of filaments from the new progeny. A mid-exponential culture of Evo3A was treated with ampicillin (∼15× MIC) for 5 h, washed to remove antibiotic, and resuspended in fresh LB medium. After 3 h of resuspension, there was a mixture of filaments and newly divided cells. The mixture was filtered with a PVDF membrane (5-μm pore size, 25-mm diameter) to separate the filaments (residue) and the normal-sized new progeny (filtrate). (b) Epifluorescence microscopy of the cells before and after filtration. The top row shows filtration using one filter for 3 ml culture, and the bottom row shows filtration using 6 filters for 3 ml culture (one filter per 500 μl). (c and d) Venn diagrams for proteome comparison of the enriched filaments to those before resuspension (c) and the enriched new progeny to the enriched filaments (d). (e and f) Volcano plots of the enriched filaments compared to those before resuspension (e) and the enriched new progeny compared to the enriched filaments (f). Differentially expressed proteins are defined as those with Benjamini-Hochberg-corrected P values below 0.1, and fold change higher or lower than ±1.5, corresponding to the colored dots. Red dots are the downregulated proteins, and blue dots are the upregulated proteins. (g and h) Functional classification of the differentially expressed proteins identified in the enriched filaments compared to those before resuspension (g) and the enriched new progeny compared to the enriched filaments (h) by gene ontology (GO) analysis using DAVID, classified by biological process. Dot size is proportional to the y axis. (i) ATP level of Evo3A after 5 h of ampicillin treatment, Evo3A after 3 h of resuscitation, and WT after 3 h of resuscitation, compared to those before treatment, measured using the BacTiter-Glo assay. To calculate the abundance on a per-cell basis, the measured values were normalized by CFU counts by serially diluting and plating washed aliquots. Data are presented relative to the untreated cells (marked by the horizontal dashed line). Data represent three biological replicates, and each data point is denoted as mean ± SEM.

  • FIG 6
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    FIG 6

    Importance of ElaB in WT E. coli. (a) Survival of wild-type E. coli BW25113 (WT) and ΔelaB cells after treatment with ampicillin (125 μg/ml) at the indicated time points (mean ± SEM, n = 3). Significance of difference with ancestral population: ***, P < 0.001; **, P < 0.01 (two-tailed t test with unequal variances of the log-transformed values). (b) Survival upon expression of elaB via pCA24N-elaB in the ΔelaB cells was determined with ampicillin (125 μg/ml) treatment for 3 h (mean ± SEM, n = 3). *, P < 0.05; ns, not significant (two-tailed t test with unequal variances of the log-transformed values). Overnight cultures at 1:1,000 were grown in fresh LB until the turbidity at 600 nm reached 0.3, and 1 mM IPTG was added for 2 h. The cells were then treated with ampicillin (125 μg/ml) for 3 h, and survival was measured. Empty plasmid pCA24N was used as a negative control. (c) Colony morphologies for WT and ΔelaB cells after 3 h of resuscitation. After treatment with ampicillin (125 μg/ml) for 3 h, cells were washed to remove antibiotics and resuspended in fresh LB for 3 h. After 3 h of growth, aliquots of both WT and ΔelaB strains were serially diluted and plated on LB agar (37°C, 20 h of incubation time). Images shown are representative images from 3 biological replicates.

  • FIG 7
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    FIG 7

    Mechanism of the filamentous persisters during antibiotic treatment and resuscitation. Upon ampicillin treatment, the cells downregulate the expression of ribosomal proteins while upregulating the expression of proteins involved in carbohydrate metabolism. The DNA, protein, and ATP contents are also increased. Ampicillin treatment led to an increased ROS and hydroxyl radical production, which is counterbalanced by an oxidative stress response in the filaments to prevent cell lysis. At the same time, the oxidative stress also causes DNA damage which then induces SOS response (marked by the increasing expression of RecA) and, in turn, filamentation. Filamentation likely serves as one of their survival strategies, as it minimizes the need for cell wall synthesis, the target of ampicillin. During resuscitation, the cells upregulate porins, allowing passive diffusion of the trace antibiotics, and also allowing ribosomal proteins to get ready for cell division. Several carbohydrate metabolism processes are downregulated, while the expression of ribosomal proteins is upregulated. The cells also have reduced oxidative stress response, thereby returning the cells to a normal state to enable cell division.

Tables

  • Figures
  • Supplemental Material
  • TABLE 1

    List of differentially expressed proteins of the enriched new progeny (after division) compared to the enriched filaments (before division) after 3 h of resuspension in fresh medium

    TABLE 1

Supplemental Material

  • Figures
  • Tables
  • FIG S1

    Proteomics analysis of Evo3A after resuspension in fresh medium. Download FIG S1, TIF file, 1.2 MB.

    Copyright © 2020 Sulaiman and Lam.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S2

    Localization of ElaB protein and knockout of elaB gene on E. coli. Download FIG S2, JPG file, 1.0 MB.

    Copyright © 2020 Sulaiman and Lam.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S3

    Protein expression of the genes in which deletions are predicted to increase H2O2 levels is either not differential or upregulated in Evo3A during ampicillin treatment. Download FIG S3, TIF file, 0.3 MB.

    Copyright © 2020 Sulaiman and Lam.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S1

    Strains and plasmids used for gene knockout and complementation experiments. Download Table S1, DOCX file, 0.01 MB.

    Copyright © 2020 Sulaiman and Lam.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S2

    Proteins on Evo3A with increasing or decreasing profile throughout 1 h, 3 h, and 5 h of antibiotic treatment. Download Table S2, DOCX file, 0.02 MB.

    Copyright © 2020 Sulaiman and Lam.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S3

    Differentially expressed proteins of Evo3A after 5 h of antibiotic treatment compared to Evo3A before antibiotic treatment. Download Table S3, DOCX file, 0.02 MB.

    Copyright © 2020 Sulaiman and Lam.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S4

    Newly detected proteins during 5 h after ampicillin treatment. Download Table S4, DOCX file, 0.02 MB.

    Copyright © 2020 Sulaiman and Lam.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S5

    Differentially expressed proteins of Evo3A after 1 h, 2 h, and 3 h of resuspension in fresh medium compared to those before resuspension (0 h). Download Table S5, DOCX file, 0.03 MB.

    Copyright © 2020 Sulaiman and Lam.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S6

    Differentially expressed proteins of enriched filaments after 3 h of resuspension in fresh medium compared to those before resuspension. Download Table S6, DOCX file, 0.02 MB.

    Copyright © 2020 Sulaiman and Lam.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S7

    List of genes whose deletions were predicted in silico to increase ROS H2O2 production levels, with the associated protein expression profile from our proteomics data. Download Table S7, DOCX file, 0.01 MB.

    Copyright © 2020 Sulaiman and Lam.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Proteomic Study of the Survival and Resuscitation Mechanisms of Filamentous Persisters in an Evolved Escherichia coli Population from Cyclic Ampicillin Treatment
Jordy Evan Sulaiman, Henry Lam
mSystems Jul 2020, 5 (4) e00462-20; DOI: 10.1128/mSystems.00462-20

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Proteomic Study of the Survival and Resuscitation Mechanisms of Filamentous Persisters in an Evolved Escherichia coli Population from Cyclic Ampicillin Treatment
Jordy Evan Sulaiman, Henry Lam
mSystems Jul 2020, 5 (4) e00462-20; DOI: 10.1128/mSystems.00462-20
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KEYWORDS

antibiotic
ampicillin
evolution
persisters
resuscitation
filaments
proteomics

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