Adaptation and compensation in a bacterial gene regulatory network evolving under antibiotic selection

  1. Vishwa Patel
  2. Nishad Matange

  • Curated by eLife

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    Evaluation Summary:

    This paper investigates the evolutionary path of Escherichia coli resistance to the antibiotic trimethoprim. The authors show that adaptive mutations that accumulate early are often not in the drug target itself, but rather mutations that lead to transcriptional up-regulation of the drug target. Higher-level resistance can then evolve due to the addition of mutations in the drug target; however, at lower drug concentrations, cells are more likely to accumulate mutations that reverse the fitness defect associated with the initially acquired mutations. Overall, this study shows that regulatory mutations can play a major role in the evolution of antibiotic resistance in bacterial populations, and that the evolutionary path is influenced by the level of drug exposure.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewers #1 , #2, and #3 agreed to share their names with the authors.)

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Abstract

Gene regulatory networks allow organisms to generate coordinated responses to environmental challenges. In bacteria, regulatory networks are re-wired and re-purposed during evolution, though the relationship between selection pressures and evolutionary change is poorly understood. In this study, we discover that the early evolutionary response of Escherichia coli to the antibiotic trimethoprim involves derepression of PhoPQ signaling, an Mg 2+ -sensitive two-component system, by inactivation of the MgrB feedback-regulatory protein. We report that derepression of PhoPQ confers trimethoprim-tolerance to E. coli by hitherto unrecognized transcriptional upregulation of dihydrofolate reductase (DHFR), target of trimethoprim. As a result, mutations in mgrB precede and facilitate the evolution of drug resistance. Using laboratory evolution, genome sequencing, and mutation re-construction, we show that populations of E. coli challenged with trimethoprim are faced with the evolutionary ‘choice’ of transitioning from tolerant to resistant by mutations in DHFR, or compensating for the fitness costs of PhoPQ derepression by inactivating the RpoS sigma factor, itself a PhoPQ-target. Outcomes at this evolutionary branch-point are determined by the strength of antibiotic selection, such that high pressures favor resistance, while low pressures favor cost compensation. Our results relate evolutionary changes in bacterial gene regulatory networks to strength of selection and provide mechanistic evidence to substantiate this link.

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  1. Version published to 10.7554/elife.70931 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    [...] The approach taken by the authors is very thorough, and the conclusions are well supported by the data. I think this is an important contribution to the field, and I have only a few specific comments:

    – The authors should sequence the mgrB gene and upstream sequence, and the rpoS gene for TMPR6-10. If these strains don't have mutations in mgrB, I think it's important to sequence their genomes to find out why DHFR levels are higher than in wt cells.

    Response: This is an important point that we had overlooked. We have now amplified and Sanger sequenced the mgrB gene and its promoter from all 10 TMPR isolates. As expected, we do indeed find mutations at the mgrB promoter in all 10 isolates. These data have been added to the revised manuscript in the Figure 1A schematic.

    – Presumably the higher number of mutations in mgrB rather than folA reflects the mutational space available, i.e. there are more possible mutations that reduce mgrB expression than there are gain-of-function folA mutations. This is worth mentioning, since it has a big impact on the evolutionary path to resistance.

    Response: We thank the Reviewer for pointing this out. We have discussed this point in the revised version of the manuscript (Page 17, Line 477-480).

    Reviewer #2 (Public Review):

    [...] 1) The authors find that mutations in the mgrB locus precede mutations in folA during E. coli's response to TMP. Why only sequence 5 of the 10 TMPR mutants? Was this subset chosen for sequencing based on any specific criteria? Below are some follow-up comments.

    Response: We thank the reviewer for this comment. Initially TMPR 1-5 were chosen since these isolates encompassed the entire range of drug IC50 values observed by us. We have now amplified and Sanger sequenced the mgrB gene and its promoter from all 10 TMPR isolates. As expected, we do indeed find mutations at the mgrB promoter in all 10 isolates. These data have been added to the revised manuscript in the Figure 1A schematic.

    a. Do any of the mutations cause growth defects relative to the wild-type strain?

    Response: This is an insightful question indeed. We have not measured growth rates of the trimethoprim resistant isolates. However, we have measured fitness of TMPR1-5 relative to wild type in competitive growth assays. In these experiments all 5 isolates have measurable fitness costs (relative fitness for isolates was between 0.7-0.8) when grown in drug-free media. Since mgrB mutations are found in all 5 TMPR isolates, we believe this result to be generally in line with our values of fitness for the mgrB-knock out strain. However, since TMPR1-5 have multiple genetic changes, attributing the measured fitness costs of these isolates to mgrB-deficiency alone is not possible. We are currently in the process of dissecting out the relative contribution of the various mutations in TMPR1-5 towards shaping the final fitness of the isolates. However, these will likely be reported in a later manuscript.

    b. Line 103: What are the mutations in folA promoter region? Only mutations in the coding sequence are listed in table 1 and figure 1A.

    Response: We apologise for this error. Though we have sequenced both promoter and ORF of the folA gene, we only found mutations in the coding sequence. We have made the necessary change in the revised manuscript.

    c. Line 109: The authors speculate that IS-element insertions in the mgrB promoter region reduce its expression, maybe they can provide a reference here from previous studies that have analyzed such mutations. Also, including details of the length/size of these insertion elements within table 1 would be helpful.

    Response: We have added references substantiating our claim that IS-element insertion in the mgrB promoter reduces its expression (Page 4, Line 110, ref 34, 35). The length of the insertions is indicated in Table 1.

    d. Line 111: the phrase "stop-codon readthrough" is misleading. The authors should rephrase to clarify that the single nucleotide deletion leads to a shift in the reading frame leading to an altered protein sequence at the C-terminal end.

    Response: We agree that this phrase is mis-leading. We have modified it in the revised manuscript (Page 4, Line 112).

    1. Based on growth assays including competitions, and measurements of folA gene expression in mgrB-deficient E. coli cells, the authors conclude that tolerance to TMP is caused by PhoP-dependent upregulation of DHFR.

    a. The authors should rewrite the text (lines 143-155) to make the experimental design of the competitions more obvious to the reader. Indicating either within the figure legend or main text what ∆mgrB/total means would definitely make analysis of the figure and results easier for the reader The reader needs to go to the materials section to get a full understanding how exactly this experiment was performed.

    Response: We have re-written this section for greater clarity and also changed Figure 1D accordingly.

    b. In Figure 1C, the IC50 value for ∆phoP is similar to that of wild type. If PhoP-dependent expression of folA important for TMP tolerance/resistance, shouldn't we expect to see a lower IC50, similar to that of ∆mgrB∆phoP? Intriguingly, the data for wild type in Figure 1C appears to be in conflict with the data in Figure 3B, please clarify.

    Response: This is an important issue, and we thank the Reviewer for pointing this out. We think that the reason phoP deletion reverses the phenotype of mgrB-deletion, but has no detectable effect in an mgrB-expressing background is due to the culture media used by us. Our experiments were performed in LB, which is a low magnesium medium. Since magnesium activates the PhoPQ pathway, in LB basal activity of PhoPQ is expected to be very low. Upon deletion of mgrB, we believe that there is an elevation in ‘unstimulated’ PhoPQ activity. This elevation is due to loss of feedback inhibition by MgrB protein. As a result, the effects of PhoP deletion are most pronounced in an mgrB knockout strain. We are, however, unable to explain why the IC50 of ∆mgrB∆phoP is lower than wild type. The possibility that there may be cross-phosphorylation of other response regulators by uninhibited PhoQ cannot be ruled out, however we do not have any data to substantiate this yet.

    The data is Figures 1C and 3B come from independently performed replicates. The mean values of IC50 of Wt in these figures are 26±13 ng/mL and 40±20 ng/mL respectively, which are not statistically significantly different.

    c. In Figure 1D, it is hard to figure out the exact strains and conditions of each competition. For instance, the ratios 10:1, 100:1 and 1000:1 needs to be clearly labeled, "wild type: mgrB" or "wild type: specific mutant" as applicable, the label on the X-axis is misplaced. Does "WmgrB" refer to ∆mgrB? If yes, change to ∆mgrB. Fitness values need a label or put into a table.

    Response: We have re-formatted this figure for better clarity as suggested. ‘w’ refers to calculated value of relative fitness and we have moved these values to the main text (Page 5, Line 149-151).

    d. Line 172: incorrect figure citation, replace Figure 2B with 2A.

    Response: We have made this correction.

    e. Lines 180-181: Only 5 out of the 10 TMPR isolates were sequenced and found to have mutations in the mgrB locus. In the absence of sequencing data confirming such mutations in TMPR 6-10 isolates, the increased levels of DHFR cannot be attributed to loss of mgrB.

    Response: We have now amplified and Sanger sequenced the mgrB gene and its promoter from all 10 TMPR isolates. As expected, we do indeed find mutations at the mgrB promoter in all 10 isolates. These data have been added to the revised manuscript in the Figure 1A schematic.

    f. In Figure 2C, it would be helpful to show the GFP fluorescence data for the single deletions, ΔphoP and ΔrpoS, to further support the claim that TMP tolerance via DHFR upregulation is PhoP dependent. In addition, the X-axis should specify the promoter reporter that was used.

    Response: We have added these data to Figure 2C and also specified the promoter reporter used.

    g. Lines 181-183: reference for the previous work on W30G folA is missing.

    Response: We thank the reviewer for bringing this to our notice. We have added the appropriate reference.

    h. In Figure 2, there is a discrepancy in the level of DHFR observed for both TMPR2 and 3 isolates in panels D and E - the DHFR protein levels are much higher in panel E. Can the authors explain this discrepancy, especially given the W30G mutation in TMPR3 (expected to show reduced levels of DHFR)? Is the same amount of protein loaded in both experiments? If so, why are the levels of protein different (and vastly different for TMPR3)? Better quantification of the western blots depicting the signal for the replicates would be helpful.

    Response: In order to be able to detect the lower levels of DHFR in ΔphoP derivates of TMPR strains, we have had to overexpose the Western blots. This may explain the apparent discrepancy between Figure 2D and E. To enhance clarity and ease of interpretation we have now quantitated all the immunoblots in the manuscript and reported fold changes in expression level.

    1. The data presented here also show that mgrB and folA mutations act in synergy in TMP resistant E. coli.

    a. It would be useful to the reader to include a table listing the MIC values in Figure 3. The plate images showing the E-tests are difficult to read and less helpful in interpreting the MICs and can be moved to the supplement.

    Response: We thank for reviewer for this suggestion. We have removed the E-test images from the figure and have included a table with the MIC values in Figure 3.

    b. In Figure 3E (and lines 234-238), what was the strain background used for DHFR overexpression? The details are missing from the paper.

    Response: The pPRO-DHFR plasmid was transformed into wild type E. coli MG1655. This information has been included in the revised Figure 3E.

    1. To follow the adaptive pathway for TMP resistance, the authors sequenced genomes of TMP-resistant isolates.

    a. Line 283: How many strains were sequenced at each time point? "3 to 5" is confusing.

    Response: The number of strains sequenced by us varied for different time points and lineages. We have rephrased this to ‘upto 5’ strains to prevent confusion. The exact number of isolates sequenced at each timepoint are given in the supplementary tables.

    b. In Figure 4, the data points/symbols and lines are hard to read in both panels A and B. These graphs can be replotted with open symbols or different colors to help the reader analyze the figure much more easily.

    Response: We have used different colours for clearer representation of data in the revised figure.

    c. Overall, it is still unclear how folA expression is regulated by PhoP regulation. An alternate hypothesis is that loss of MgrB may influence folA gene expression in a PhoP independent manner. Have the authors ruled out this possibility?

    Response: We agree that our study has not shed light on the precise molecular mechanism by which PhoP signalling affects folA levels, except that it is unlikely to be a direct effect. The reason we do not think that the effect is PhoP-independent is that phoP-deletion reverses the phenotype of the mgrB knockout, as well as the TMPR1-5 isolates. However, we cannot yet argue that there is no contribution from PhoP-independent mechanisms. Further genetic analyses are underway in our laboratory to determine other molecular players of this pathway.

  3. eLife

    Evaluation Summary:

    This paper investigates the evolutionary path of Escherichia coli resistance to the antibiotic trimethoprim. The authors show that adaptive mutations that accumulate early are often not in the drug target itself, but rather mutations that lead to transcriptional up-regulation of the drug target. Higher-level resistance can then evolve due to the addition of mutations in the drug target; however, at lower drug concentrations, cells are more likely to accumulate mutations that reverse the fitness defect associated with the initially acquired mutations. Overall, this study shows that regulatory mutations can play a major role in the evolution of antibiotic resistance in bacterial populations, and that the evolutionary path is influenced by the level of drug exposure.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewers #1 , #2, and #3 agreed to share their names with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This paper looks at the evolutionary trajectory of E. coli populations exposed to the antibiotic trimethoprim. The authors argue that mutations in mgrB are initially selected since these lead to an increase in folA expression. At high drug concentrations, mutations in folA are seleted, but at lower drug concentrations, mutations in rpoS are selected since these compensate for the growth defect of an mgrB mutant. Overall, the authors conclude that the evolution of resistance to trimethoprim first involves the accumulation of mutations outside of folA that modulate gene expression, and that the evolutionary path to resistance depends on the concentration of drug.

    The approach taken by the authors is very thorough, and the conclusions are well supported by the data. I think this is an important contribution to the field, and I have only a few specific comments:

    – The authors should sequence the mgrB gene and upstream sequence, and the rpoS gene for TMPR6-10. If these strains don't have mutations in mgrB, I think it's important to sequence their genomes to find out why DHFR levels are higher than in wt cells.

    – Presumably the higher number of mutations in mgrB rather than folA reflects the mutational space available, i.e. there are more possible mutations that reduce mgrB expression than there are gain-of-function folA mutations. This is worth mentioning, since it has a big impact on the evolutionary path to resistance.

  5. eLife

    Reviewer #2 (Public Review):

    This paper by Patel and Matange focuses on understanding the evolutionary response of E. coli cells to the antibiotic trimethoprim (TMP). Mutations in the gene folA, which encodes the dihydrofolate reductase (DHFR) enzyme - an established target of TMP, are known to mediate intrinsic resistance to TMP. This work shows that de-repression of the PhoQ/PhoP pathway via inactivation of MgrB, which is a negative feedback inhibitor of PhoQ, leads to TMP tolerance. Further, this response to TMP is due to the upregulation of DHFR expression. Similarly, inactivation of MgrB and a corresponding increase in the PhoQ/PhoP-regulated gene expression is a prominent mechanism for acquired colistin resistance in clinical isolates of Klebsiella pneumoniae. The authors performed adaptive laboratory evolution of E. coli under TMP selection and identified factors contributing to the transition of TMP-tolerant bacterial cells to TMP-resistant cells. At high TMP concentrations, the cells become resistant via mutations in DHFR. Cells evolved under low (sub-MIC) concentrations of TMP develop mutations inactivating the RpoS sigma factor to offset the cost of PhoQ de-repression. The data presented in the paper are clear and the conclusions are mostly valid. However, the authors need to modify parts of the main text and figure presentations for clarity and a better/more straightforward interpretation of the results by the reader. In general, the results obtained in this study explain well the evolutionary consequences of these mutations and the pathway for the acquisition of the mutations.

    1. The authors find that mutations in the mgrB locus precede mutations in folA during E. coli's response to TMP. Why only sequence 5 of the 10 TMPR mutants? Was this subset chosen for sequencing based on any specific criteria? Below are some follow-up comments.

    a. Do any of the mutations cause growth defects relative to the wild-type strain?

    b. Line 103: What are the mutations in folA promoter region? Only mutations in the coding sequence are listed in table 1 and figure 1A.

    c. Line 109: The authors speculate that IS-element insertions in the mgrB promoter region reduce its expression, maybe they can provide a reference here from previous studies that have analyzed such mutations. Also, including details of the length/size of these insertion elements within table 1 would be helpful.

    d. Line 111: the phrase "stop-codon readthrough" is misleading. The authors should rephrase to clarify that the single nucleotide deletion leads to a shift in the reading frame leading to an altered protein sequence at the C-terminal end.

    1. Based on growth assays including competitions, and measurements of folA gene expression in mgrB-deficient E. coli cells, the authors conclude that tolerance to TMP is caused by PhoP-dependent upregulation of DHFR.

    a. The authors should rewrite the text (lines 143-155) to make the experimental design of the competitions more obvious to the reader. Indicating either within the figure legend or main text what ∆mgrB/total means would definitely make analysis of the figure and results easier for the reader The reader needs to go to the materials section to get a full understanding how exactly this experiment was performed.

    b. In Figure 1C, the IC50 value for ∆phoP is similar to that of wild type. If PhoP-dependent expression of folA important for TMP tolerance/resistance, shouldn't we expect to see a lower IC50, similar to that of ∆mgrB∆phoP? Intriguingly, the data for wild type in Figure 1C appears to be in conflict with the data in Figure 3B, please clarify.

    c. In Figure 1D, it is hard to figure out the exact strains and conditions of each competition. For instance, the ratios 10:1, 100:1 and 1000:1 needs to be clearly labeled, "wild type: mgrB" or "wild type: specific mutant" as applicable, the label on the X-axis is misplaced. Does "WmgrB" refer to ∆mgrB? If yes, change to ∆mgrB. Fitness values need a label or put into a table.

    d. Line 172: incorrect figure citation, replace Figure 2B with 2A.

    e. Lines 180-181: Only 5 out of the 10 TMPR isolates were sequenced and found to have mutations in the mgrB locus. In the absence of sequencing data confirming such mutations in TMPR 6-10 isolates, the increased levels of DHFR cannot be attributed to loss of mgrB.

    f. In Figure 2C, it would be helpful to show the GFP fluorescence data for the single deletions, ΔphoP and ΔrpoS, to further support the claim that TMP tolerance via DHFR upregulation is PhoP dependent. In addition, the X-axis should specify the promoter reporter that was used.

    g. Lines 181-183: reference for the previous work on W30G folA is missing.

    h. In Figure 2, there is a discrepancy in the level of DHFR observed for both TMPR2 and 3 isolates in panels D and E - the DHFR protein levels are much higher in panel E. Can the authors explain this discrepancy, especially given the W30G mutation in TMPR3 (expected to show reduced levels of DHFR)? Is the same amount of protein loaded in both experiments? If so, why are the levels of protein different (and vastly different for TMPR3)? Better quantification of the western blots depicting the signal for the replicates would be helpful.

    1. The data presented here also show that mgrB and folA mutations act in synergy in TMP resistant E. coli.

    a. It would be useful to the reader to include a table listing the MIC values in Figure 3. The plate images showing the E-tests are difficult to read and less helpful in interpreting the MICs and can be moved to the supplement.

    b. In Figure 3E (and lines 234-238), what was the strain background used for DHFR overexpression? The details are missing from the paper.

    1. To follow the adaptive pathway for TMP resistance, the authors sequenced genomes of TMP-resistant isolates.

    a. Line 283: How many strains were sequenced at each time point? "3 to 5" is confusing.

    b. In Figure 4, the data points/symbols and lines are hard to read in both panels A and B. These graphs can be replotted with open symbols or different colors to help the reader analyze the figure much more easily.

    c. Overall, it is still unclear how folA expression is regulated by PhoP regulation. An alternate hypothesis is that loss of MgrB may influence folA gene expression in a PhoP independent manner. Have the authors ruled out this possibility?

  6. eLife

    Reviewer #3 (Public Review):

    The study by Patel and Matange explores how trimethoprim resistance evolves in E. coli in vitro, with a focus on the role of gene regulatory networks in the evolution of antibiotic resistance. In vitro antibiotic resistance evolution is often studied through a "one-hit" model, wherein antibiotic pressure selects for mutations in a single target gene. Here, the authors explore how different levels of antibiotic selection result in different evolutionary "choices" made by the bacterial population. They also show that mutations affecting the PhoPQ regulatory system decrease antibiotic susceptibility (i.e. increased IC50) but do not cause overt resistance (i.e. increased MIC), and that these mutations are an early adaptation under antibiotic selection.

    Major strengths of the study include the clearly-planned and executed experiments, the use of multiple replicates to establish the reproducibility of the observed mutations, and the employment of relevant functional assays to confirm hypotheses generated by the comparative genomics analyses performed. Weaknesses are generally minor, and include an occasional lack of justification and sufficient explanation for some experimental design choices, as well as omission of a few easy-to-perform experiments that would further support the authors' conclusions.

    In my view, the authors have achieved their aims and their results largely support the conclusions that are drawn. Overall, this study makes a meaningful contribution to our current understanding of the role of the PhoPQ system in the evolution of antibiotic resistance in E. coli, and with minor modifications the study would be of significant interest to researchers studying in vitro evolution of antibiotic resistance in bacteria.

  7. Version published to 10.1101/2021.08.31.458319v1 on bioRxiv