Adaptive laboratory evolution of antimicrobial resistance in bacteria for genetic and phenotypic analyses

Vinchhi, Rhea; Jena, Chinmaya; Matange, Nishad

doi:10.1016/j.xpro.2022.102005

Cited by 4 publications

(7 citation statements)

References 17 publications

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“…The detailed methodology for laboratory evolution of trimethoprim resistance is described in Patel and Matange (2021) [1] and Vinchhi et al (2023) [76]. Trimethoprim was used at a concentration of 100 ng/mL which corresponds to ~MIC/9.…”

Section: Laboratory Evolution Of Trimethoprim Resistancementioning

confidence: 99%

“…Genome sequencing and variant calling for evolved bacteria was carried out as described in Patel and Matange (2021) [1] and Vinchhi et al (2023) [76]. Sequencing services were provided by Eurofins, India.…”

Section: Genome Sequencing Of Laboratory-evolved Trimethoprim Resista...mentioning

confidence: 99%

“…MgSO 4 was added to growth media at a concentration of 10 mM wherever appropriate. Genome sequencing of laboratory-evolved trimethoprim resistant isolates Genome sequencing and variant calling for evolved bacteria was carried out as described in Patel and Matange (2021) [1] and Vinchhi et al (2023) [76]. Sequencing services were provided by Eurofins, India.…”

Section: Laboratory Evolution Of Trimethoprim Resistancementioning

confidence: 99%

“…The detailed methodology for laboratory evolution of trimethoprim resistance is described in Patel and Matange (2021) [33] and Vinchhi et al ( 2023) [76]. Briefly, bacterial populations were grown in trimethoprim supplemented LB (low Mg 2+ ) or LB + 10 mM MgSO4 as required for 15-18 hours before passaging (1%) into fresh media.…”

Section: Laboratory Evolution Of Trimethoprim Resistancementioning

confidence: 99%

“…Growth was monitored after 18-20 hours of growth and IC50 values were determined by fitting experimental data to a variable-slope, 4 parameter model using Graphpad Prism (version 9.1.4). Detailed methodology is described in Patel and Matange (2021) [33] and Vinchhi et al ( 2023) [76].…”

Section: Measuring Trimethoprim Resistancementioning

confidence: 99%

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Adaptive and maladaptive consequences of deregulation in a bacterial gene regulatory network

Vinchhi

Yelpure

Balachandran

et al. 2023

Preprint

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The archetypal PhoQP two-component system from Enterobacteria regulates crucial pathways like magnesium homeostasis in Escherichia coli and virulence factor expression in Salmonella enterica. Previously we had reported that a laboratory strain of E. coli rapidly accumulated loss-of-function mutations in the mgrB gene, a negative feedback regulator of PhoQP, when evolved in the presence of the antibiotic trimethoprim (Patel and Matange, eLife 2021). Hyperactive PhoQP enhanced the expression of dihydrofolate reductase, target of trimethoprim, resulting in antibiotic tolerance. Here we ask, firstly, how important are mutations in mgrB for the evolution of trimethoprim resistance? We report that MgrB loss, though itself only mildly beneficial, facilitates the evolution of resistance by enhancing the fixation probability of trimethoprim-resistant bacteria under selection. Secondly, we investigate why negative feedback may be needed in the PhoQP system. We show that under drug-free conditions MgrB is required to mitigate the fitness costs of pervasive gene dysregulation by hyperactive PhoQP. Using RNA-seq transcriptomics and genetic analyses, we demonstrate that PhoQP-hyperactivation perturbs the balance of RpoS and RpoD regulated transcriptional programs, and spontaneous mutations in rpoS rectify this imbalance. We propose that relative adaptive and maladaptive consequences of deregulation explain the evolution of negative feedback in bacterial gene regulatory networks.

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Section: Laboratory Evolution Of Trimethoprim Resistancementioning

confidence: 99%

Section: Genome Sequencing Of Laboratory-evolved Trimethoprim Resista...mentioning

confidence: 99%

Section: Laboratory Evolution Of Trimethoprim Resistancementioning

confidence: 99%

Section: Laboratory Evolution Of Trimethoprim Resistancementioning

confidence: 99%

Section: Measuring Trimethoprim Resistancementioning

confidence: 99%

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Adaptive and maladaptive consequences of deregulation in a bacterial gene regulatory network

Vinchhi

Yelpure

Balachandran

et al. 2023

Preprint

Self Cite

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Antimicrobial peptide-conjugated phage-mimicking nanoparticles exhibit potent bactericidal action against Streptococcus pyogenes in murine wound infection models

Olesk,

Donahue,

Ross

et al. 2024

Nanoscale Adv.

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Pervasive gene deregulation underlies adaptation and maladaptation in trimethoprim-resistant E. coli

Vinchhi,

Yelpure,

Balachandran

et al. 2023

mBio

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The archetypal PhoQP two-component system from Enterobacteria regulates pathways like magnesium homeostasis in Escherichia coli and virulence factor expression in Salmonella enterica . We had previously reported that E. coli rapidly accumulated mutations in the mgrB gene, a negative feedback regulator of PhoQP, when evolved in the antibiotic trimethoprim. Here, we first show that trimethoprim-selected mutations in mgrB either lower its expression or alter the C-terminus of the MgrB protein and prevent interaction with PhoQ. Both mechanisms compromise MgrB activity, leading to PhoQP hyperactivation and overexpression of dihydrofolate reductase ( folA ), which is the target of trimethoprim. We then investigate the consequences of deregulating PhoQP for the fitness of E. coli and elucidate the underlying mechanisms. Using laboratory evolution, we demonstrate that mgrB mutations facilitate rapid fixation of resistant bacteria in populations evolving in trimethoprim, even though their independent effect on drug IC50 is nominal. This effect is explained by a pervasive transcriptional response to deregulated PhoQP, specifically on the downstream RstA-regulon, in addition to activating folA transcription. Pervasive gene deregulation also explained the fitness costs of mgrB mutations, although involving different molecular players. PhoQP hyperactivation perturbed the balance of RpoS- and RpoD-regulated transcriptional programs and mutations that reset this balance-restored bacterial fitness in antibiotic-free conditions. Our study shows that the deregulation of a single signaling pathway permeates the wider gene expression network and leads to adaptation or maladaptation depending on the environmental context. The implications of our findings for the evolution of feedback mechanisms in two-component signaling are discussed. IMPORTANCE Bacteria employ a number of mechanisms to adapt to antibiotics. Mutations in transcriptional regulators alter the expression levels of genes that can change the susceptibility of bacteria to antibiotics. Two-component signaling proteins are a major class of signaling molecule used by bacteria to regulate transcription. In previous work, we found that mutations in MgrB, a feedback regulator of the PhoQP two-component system, conferred trimethoprim tolerance to Escherichia coli . Here, we elucidate how mutations in MgrB have a domino-like effect on the gene regulatory network of E. coli . As a result, pervasive perturbation of gene regulation ensues. Depending on the environmental context, this pervasive deregulation is either adaptive or maladaptive. Our study sheds light on how deregulation of gene expression can be beneficial for bacteria when challenged with antibiotics, and why regulators like MgrB may have evolved in the first place.

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Adaptive laboratory evolution of antimicrobial resistance in bacteria for genetic and phenotypic analyses

Cited by 4 publications

References 17 publications

Adaptive and maladaptive consequences of deregulation in a bacterial gene regulatory network

Adaptive and maladaptive consequences of deregulation in a bacterial gene regulatory network

Antimicrobial peptide-conjugated phage-mimicking nanoparticles exhibit potent bactericidal action against Streptococcus pyogenes in murine wound infection models

Pervasive gene deregulation underlies adaptation and maladaptation in trimethoprim-resistant E. coli

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