Breaching the Barrier: Genome-Wide Investigation into the Role of a Primary Amine in Promoting E. coli Outer-Membrane Passage and Growth Inhibition by Ampicillin

Maher, Claire; Maharjan, Ram; Sullivan, Geraldine J.; Cain, Amy K.; Hassan, Karl A.

doi:10.1128/spectrum.03593-22

Cited by 5 publications

(7 citation statements)

References 26 publications

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“…This data set was generated using E. coli K12 BW25113, the parental strain of the Keio collection ( 15 ). A TraDIS library of 350,000 unique Tn 5 mutants ( 16 ) was challenged with a subinhibitory concentration of ciprofloxacin (1/2 MIC) with growth overnight. After analysis with the TraDIS toolkit ( 17 ), we confirmed ciprofloxacin as an inducer of exaggerated CLB as it displayed a distinctive peak of increased reads around the origin of replication ( Fig.…”

Section: Resultsmentioning

confidence: 99%

A method to correct for local alterations in DNA copy number that bias functional genomics assays applied to antibiotic-treated bacteria

Sullivan,

Barquist,

Cain

2024

mSystems

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Functional genomics techniques, such as transposon insertion sequencing and RNA-sequencing, are key to studying relative differences in bacterial mutant fitness or gene expression under selective conditions. However, certain stress conditions, mutations, or antibiotics can directly interfere with DNA synthesis, resulting in systematic changes in local DNA copy numbers along the chromosome. This can lead to artifacts in sequencing-based functional genomics data when comparing antibiotic treatment to an unstressed control. Further, relative differences in gene-wise read counts may result from alterations in chromosomal replication dynamics, rather than selection or direct gene regulation. We term this artifact “chromosomal location bias” and implement a principled statistical approach to correct it by calculating local normalization factors along the chromosome. These normalization factors are then directly incorporated into statistical analyses using standard RNA-sequencing analysis methods without modifying the read counts themselves, preserving important information about the mean-variance relationship in the data. We illustrate the utility of this approach by generating and analyzing a ciprofloxacin-treated transposon insertion sequencing data set in Escherichia coli as a case study. We show that ciprofloxacin treatment generates chromosomal location bias in the resulting data, and we further demonstrate that failing to correct for this bias leads to false predictions of mutant drug sensitivity as measured by minimum inhibitory concentrations. We have developed an R package and user-friendly graphical Shiny application, ChromoCorrect, that detects and corrects for chromosomal bias in read count data, enabling the application of functional genomics technologies to the study of antibiotic stress. IMPORTANCE Altered gene dosage due to changes in DNA replication has been observed under a variety of stresses with a variety of experimental techniques. However, the implications of changes in gene dosage for sequencing-based functional genomics assays are rarely considered. We present a statistically principled approach to correcting for the effect of changes in gene dosage, enabling testing for differences in the fitness effects or regulation of individual genes in the presence of confounding differences in DNA copy number. We show that failing to correct for these effects can lead to incorrect predictions of resistance phenotype when applying functional genomics assays to investigate antibiotic stress, and we provide a user-friendly application to detect and correct for changes in DNA copy number.

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Section: Resultsmentioning

confidence: 99%

A method to correct for local alterations in DNA copy number that bias functional genomics assays applied to antibiotic-treated bacteria

Sullivan,

Barquist,

Cain

2024

mSystems

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“…The spatial characteristics of rigidity and globularity are important for traversing the narrow porin channel constriction zone, which ranges from approximately 7 to 11 angstroms 14 . The critical presence of an ionizable nitrogen has been traced to its ability to engage in favourable electrostatic interactions with negatively‐charged amino acids that line the porin constriction zone 12,15–17 . The P. aeruginosa permeation guidelines 9 —dubbed here the PASsagE rules ( P seudomonas a eruginosa S elf‐promoted E ntry)—in contrast provide guidance for tailoring small molecules to productively access the self‐promoted uptake pathway for entry into the cell, consistent with recent work from the Bumann lab suggesting that porins are not important for antibiotic uptake in P. aeruginosa 18 .…”

Section: Figurementioning

confidence: 99%

“…14 The critical presence of an ionizable nitrogen has been traced to its ability to engage in favourable electrostatic interactions with negatively-charged amino acids that line the porin constriction zone. 12,[15][16][17] The P. aeruginosa permeation guidelines 9 -dubbed here the PASsagE rules (Pseudomonas aeruginosa Self-promoted Entry)-in contrast provide guidance for tailoring small molecules…”

mentioning

confidence: 99%

Using permeation guidelines to design new antibiotics—A PASsagE into Pseudomonas aeruginosa

Cain,

Hergenrother

2024

Clinical & Translational Med

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The ability to employ small molecules to kill pathogenic bacteria and prevent serious illness or death has been one of mankind's greatest advances, as before the advent of the first antibiotics (salvarsan, penicillin) approximately 30% of deaths in the United States and United Kingdom were due to bacterial infections. 1 This shield against disease, however, is weakening as resistance to approved antibiotics rises. Worryingly, no new class of antibiotics with activity against gram-negative bacteria has been approved by the FDA in over 55 years, 2 with Pseudomonas aeruginosa being of particular concern. Multidrug-resistant P. aeruginosa is classified as a "serious threat" by the CDC and there are only four antibiotic classes regularly used to treat P. aeruginosa infections (β-lactams, aminoglycosides, fluoroquinolones, and polymyxins). 3,4 This dearth of therapeutic options makes the occurrence of drug-resistant P. aeruginosa even more troubling, and three of these drug classes (aminoglycosides, fluoroquinolones, and polymyxins) have serious toxicity concerns. As it stands approximately 85 000 deaths globally were attributed to antibioticresistant P. aeruginosa in 2019 alone, placing it among the six most deadly pathogens. 5 Gram-negative pathogens are notorious for being difficult to penetrate with small molecules, and P. aeruginosa is particularly problematic in this regard, as it does not possess general diffusion porins and has many general and promiscuous efflux pumps. 6,7 Accordingly, a primary challenge in the development of new therapeutics for P. aeruginosa is achieving effective drug concentrations inside the cell, where many valuable antibacterial targets exist. 8 To address this challenge, we have recently disclosed general permeation guidelines for P. aeruginosa, facilitating the design of small molecules that hijack the bacterium's self-promoted uptake pathway and gain intracellular access. 9 For this work, the intracellular accumulation of individual members from a large and diverse compound collection was determined using an LC-MS/MS assay. 10This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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“…This dataset was generated using E. coli K12 BW25113, the parental strain of the Keio collection (37). A TraDIS library of 350,000 unique Tn5 mutants (38) was challenged with a subinhibitory concentration of ciprofloxacin (1/2 MIC) with growth overnight. After analysis with the TraDIS toolkit (4), we confirmed ciprofloxacin as an inducer of exaggerated CLB as it displayed a distinctive peak of increased reads around the origin of replication (Figure 4A).…”

Section: Transposon Insertion Sequencing Case Study: Ciprofloxacinmentioning

confidence: 99%

“…An E. coli K12 TraDIS library was generated as previously described (38) and challenged with subinhibitory ciprofloxacin (40 µg/mL) in 10 mL of Mueller Hinton cation adjusted media.…”

Section: Tradis Library Ciprofloxacin Challenging and Sequencingmentioning

confidence: 99%

A method to correct for local alterations in DNA copy number that bias functional genomics assays applied to antibiotic-treated bacteria

Sullivan

Barquist

Cain

2023

Preprint

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Functional genomics techniques, such as transposon insertion sequencing and RNA sequencing, are key to studying relative differences in mutant fitness or gene expression under selective conditions. However, certain stress conditions, mutations, or antibiotics can directly interfere with DNA synthesis, resulting in systematic changes in local DNA copy number along the chromosome. This can lead to artefacts in sequencing-based functional genomics data when comparing antibiotic treatment to an unstressed control, with relative differences in gene-wise read counts being the result of alterations in chromosomal replication dynamics rather than selection or direct gene regulation. We term this artefact ‘chromosomal location bias’ and implement a principled statistical approach to correct for it by calculating local normalization factors along the chromosome. These normalization factors are then directly incorporated in statistical analyses using standard RNA-sequencing analysis methods without modifying the read counts themselves, preserving important information about the mean-variance relationship in the data. We illustrate the utility of this approach by generating and analysing a ciprofloxacin-treated transposon insertion sequencing dataset inEscherichia colias a case study. We show that ciprofloxacin treatment generates chromosomal location bias in the resulting data, and we further demonstrate that failing to correct for this bias leads to false predictions of mutant drug sensitivity as measured by minimum inhibitory concentrations. We have developed an R package and user-friendly graphical Shiny application, ChromoCorrect, that detects and corrects for chromosomal bias in read count data, enabling the application of functional genomics technologies to the study of antibiotic stress.IMPORTANCEAltered gene dosage due to changes in DNA replication has been observed under a variety of stresses with a variety of experimental techniques. However, the implications of changes in gene dosage for sequencing-based functional genomics assays are rarely considered. We present a statistically principled approach to correcting for the effect of changes in gene dosage, enabling testing for differences in the fitness effects or regulation of individual genes in the presence of confounding differences in DNA copy number. We show that failing to correct for these effects can lead to incorrect predictions of resistance phenotype when applying functional genomics assays to investigate antibiotic stress, and we provide a user-friendly application to detect and correct for changes in DNA copy number.

show abstract

Breaching the Barrier: Genome-Wide Investigation into the Role of a Primary Amine in Promoting E. coli Outer-Membrane Passage and Growth Inhibition by Ampicillin

Cited by 5 publications

References 26 publications

A method to correct for local alterations in DNA copy number that bias functional genomics assays applied to antibiotic-treated bacteria

A method to correct for local alterations in DNA copy number that bias functional genomics assays applied to antibiotic-treated bacteria

Using permeation guidelines to design new antibiotics—A PASsagE into Pseudomonas aeruginosa

A method to correct for local alterations in DNA copy number that bias functional genomics assays applied to antibiotic-treated bacteria

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