Title: Constitutive Activation of RpoH and the Addition of L-arabinose Influence Antibiotic Sensitivity of PHL628 E. coli
Antibiotics are used to combat the ever-present threat of infectious diseases, but bacteria are continually evolving an assortment of defenses that enable their survival against even the most potent treatments. While the demand for novel antibiotic agents is high, the discovery of a new agent is exceedingly rare. We chose to focus on understanding how different signal transduction pathways in the gram-negative bacterium Escherichia coli (E. coli) influence the sensitivity of the organism to antibiotics from three different classes: tetracycline, chloramphenicol, and levofloxacin. Using the PHL628 strain of E. coli, we exogenously overexpressed two transcription factors, FliA and RpoH.I54N (a constitutively active mutant), to determine their influence on the minimum inhibitory concentration (MIC) and minimum duration of killing (MDK) concentration for each of the studied antibiotics. We hypothesized that activating these pathways, which upregulate genes that respond to specific stressors, could mitigate bacterial response to antibiotic treatment. We also compared the exogenous overexpression of the constitutively active RpoH mutant to thermal heat shock that has feedback loops maintained. While FliA overexpression had no impact on MIC or antibiotic tolerance, RpoH.I54N overexpression reduced the MIC for tetracycline and chloramphenicol but had no independent impact on antibiotic tolerance. Thermal heat shock alone also did not affect MIC or antibiotic tolerance. L-arabinose, the small molecule used to induce expression in our system, unexpectedly independently increased the MICs for tetracycline (>2-fold) and levofloxacin (3-fold). Additionally, the combination of thermal heat shock and arabinose provided a synergistic, 5-fold increase in MIC for chloramphenicol. Arabinose increased the tolerance, as assessed by MDK99, for chloramphenicol (2-fold) and levofloxacin (4-fold). These experiments highlight the potential of the RpoH pathway to modulate antibiotic sensitivity and the emerging implication of arabinose in enhanced MIC and antibiotic tolerance. more »« less
Patel, Mohit B.; Meisel, Joseph W.; Negin, Saeedeh; Gokel, Michael R.; Garrad, Evan; Gokel, George W.
(, Annals of pharmacology and pharmaceutics)
null
(Ed.)
Hydraphiles are synthetic amphiphiles that form pores in bilayer membranes. A study was undertaken to determine if the formation of pores could assist the penetration of antibiotics into bacteria. The disruption of ion homeostasis by the pore-formers leads to microbial toxicity. Co-administration of hydraphiles at concentration ≤ ½ MIC and antimicrobials to E. coli or P. aeruginosa showed potency enhancements of up to 30-fold. A possible mechanism is the enhancement of antibiotic influx owing to membrane disruption and/or altering the ion balance within the bacterial cells.
There is an urgent need to find novel treatments for combating multidrug-resistant bacteria. Multidrug efflux pumps that expel antibiotics out of cells are major contributors to this problem. Therefore, using efflux pump inhibitors (EPIs) is a promising strategy to increase antibiotic efficacy. However, there are no EPIs currently approved for clinical use especially because of their toxicity. This study investigates sodium malonate, a natural, non-hazardous, small molecule, for its use as a novel EPI of AcrAB-TolC, the main multidrug efflux pump of the Enterobacteriaceae family. Using ethidium bromide accumulation experiments, we found that 25 mM sodium malonate inhibited efflux by the AcrAB-TolC and other MDR pumps of Escherichia coli to a similar degree than 50 μΜ phenylalanine-arginine-β-naphthylamide, a well-known EPI. Using minimum inhibitory concentration assays and molecular docking to study AcrB-ligand interactions, we found that sodium malonate increased the efficacy of ethidium bromide and the antibiotics minocycline, chloramphenicol, and ciprofloxacin, possibly via binding to multiple AcrB locations, including the AcrB proximal binding pocket. In conclusion, sodium malonate is a newly discovered EPI that increases antibiotic efficacy. Our findings support the development of malonic acid/sodium malonate and its derivatives as promising EPIs for augmenting antibiotic efficacy when treating multidrug-resistant bacterial infections.
ABSTRACT There is an urgent need for strategies to discover secondary drugs to prevent or disrupt antimicrobial resistance (AMR), which is causing >700,000 deaths annually. Here, we demonstrate that tetracycline-resistant (Tet R ) Escherichia coli undergoes global transcriptional and metabolic remodeling, including downregulation of tricarboxylic acid cycle and disruption of redox homeostasis, to support consumption of the proton motive force for tetracycline efflux. Using a pooled genome-wide library of single-gene deletion strains, at least 308 genes, including four transcriptional regulators identified by our network analysis, were confirmed as essential for restoring the fitness of Tet R E. coli during treatment with tetracycline. Targeted knockout of ArcA, identified by network analysis as a master regulator of this new compensatory physiological state, significantly compromised fitness of Tet R E. coli during tetracycline treatment. A drug, sertraline, which generated a similar metabolome profile as the arcA knockout strain, also resensitized Tet R E. coli to tetracycline. We discovered that the potentiating effect of sertraline was eliminated upon knocking out arcA , demonstrating that the mechanism of potential synergy was through action of sertraline on the tetracycline-induced ArcA network in the Tet R strain. Our findings demonstrate that therapies that target mechanistic drivers of compensatory physiological states could resensitize AMR pathogens to lost antibiotics. IMPORTANCE Antimicrobial resistance (AMR) is projected to be the cause of >10 million deaths annually by 2050. While efforts to find new potent antibiotics are effective, they are expensive and outpaced by the rate at which new resistant strains emerge. There is desperate need for a rational approach to accelerate the discovery of drugs and drug combinations that effectively clear AMR pathogens and even prevent the emergence of new resistant strains. Using tetracycline-resistant (Tet R ) Escherichia coli , we demonstrate that gaining resistance is accompanied by loss of fitness, which is restored by compensatory physiological changes. We demonstrate that transcriptional regulators of the compensatory physiologic state are promising drug targets because their disruption increases the susceptibility of Tet R E. coli to tetracycline. Thus, we describe a generalizable systems biology approach to identify new vulnerabilities within AMR strains to rationally accelerate the discovery of therapeutics that extend the life span of existing antibiotics.
Kumar, Lokender; Brenner, Nathanael; Brice, John; Klein-Seetharaman, Judith; Sarkar, Susanta K.
(, Frontiers in Microbiology)
null
(Ed.)
Pseudomonas aeruginosa utilizes the quorum sensing (QS) system to strategically coordinate virulence and biofilm formation. Targeting QS pathways may be a potential anti-infective approach to treat P. aeruginosa infections. In the present study, we define cephalosporins’ anti-QS activity using Chromobacterium violaceum CV026 for screening and QS-regulated mutants of P. aeruginosa for validation. We quantified the effects of three cephalosporins, cefepime, ceftazidime, and ceftriaxone, on (1) pyocyanin production using spectrophotometric assay, (2) bacterial motility using agar plate assay, and (3) biofilm formation using scanning electron microscopy. We also studied isogenic QS mutant strains of PAO1 (Δ lasR ,Δ rhlR ,Δ pqsA , and Δ pqsR) to compare and distinguish QS-mediated effects on the motility phenotypes and bacterial growth with and without sub-MIC concentrations of antibiotics. Results showed that cephalosporins have anti-QS activity and reduce bacterial motility, pyocyanin production, and biofilm formation for CV026 and PAO1. Also, sub-MICs of cefepime increased aminoglycosides’ antimicrobial activity against P. aeruginosa PAO1, suggesting the advantage of combined anti-QS and antibacterial treatment. To correlate experimentally observed anti-QS effects with the interactions between cephalosporins and QS receptors, we performed molecular docking with ligand binding sites of quorum sensing receptors using Autodock Vina. Molecular docking predicted cephalosporins’ binding affinities to the ligand-binding pocket of QS receptors (CviR, LasR, and PqsR). To validate our results using an infection model, we quantified the survival rate of C aenorhabditis elegans following P. aeruginosa PAO1 challenge at concentrations less than the minimum inhibitory concentration (MIC) of antibiotics. C. elegans infected with PAO1 without antibiotics showed 0% survivability after 72 h. In contrast, PAO1-infected C. elegans showed 65 ± 5%, 58 ± 4%, and 49 ± 8% survivability after treatment with cefepime, ceftazidime, and ceftriaxone, respectively. We determined the survival rates of C. elegans infected by QS mutant strains Δ lasR (32 ± 11%), Δ rhlR (27 ± 8%), Δ pqsA (27 ± 10%), and Δ pqsR (37 ± 6%), which suggest essential role of QS system in virulence. In summary, cephalosporins at sub-MIC concentrations show anti-QS activity and enhance the antibacterial efficacy of aminoglycosides, a different class of antibiotics. Thus, cephalosporins at sub-MIC concentrations in combination with other antibiotics are potential candidates for developing therapies to combat infections caused by P. aeruginosa.
Skalnik, Christopher J; Cheah, Sean Y; Yang, Mica Y; Wolff, Mattheus B; Spangler, Ryan K; Talman, Lee; Morrison, Jerry H; Peirce, Shayn M; Agmon, Eran; Covert, Markus W
(, PLOS Computational Biology)
Beard, Daniel A
(Ed.)
Antibiotic resistance poses mounting risks to human health, as current antibiotics are losing efficacy against increasingly resistant pathogenic bacteria. Of particular concern is the emergence of multidrug-resistant strains, which has been rapid among Gram-negative bacteria such asEscherichia coli. A large body of work has established that antibiotic resistance mechanisms depend on phenotypic heterogeneity, which may be mediated by stochastic expression of antibiotic resistance genes. The link between such molecular-level expression and the population levels that result is complex and multi-scale. Therefore, to better understand antibiotic resistance, what is needed are new mechanistic models that reflect single-cell phenotypic dynamics together with population-level heterogeneity, as an integrated whole. In this work, we sought to bridge single-cell and population-scale modeling by building upon our previous experience in “whole-cell” modeling, an approach which integrates mathematical and mechanistic descriptions of biological processes to recapitulate the experimentally observed behaviors of entire cells. To extend whole-cell modeling to the “whole-colony” scale, we embedded multiple instances of a whole-cellE.colimodel within a model of a dynamic spatial environment, allowing us to run large, parallelized simulations on the cloud that contained all the molecular detail of the previous whole-cell model and many interactive effects of a colony growing in a shared environment. The resulting simulations were used to explore the response ofE.colito two antibiotics with different mechanisms of action, tetracycline and ampicillin, enabling us to identify sub-generationally-expressed genes, such as the beta-lactamase ampC, which contributed greatly to dramatic cellular differences in steady-state periplasmic ampicillin and was a significant factor in determining cell survival.
Frizzell, Jenna K, Taylor, Ryan L, and Ryno, Lisa M. Constitutive Activation of RpoH and the Addition of L-arabinose Influence Antibiotic Sensitivity of PHL628 E. coli. Retrieved from https://par.nsf.gov/biblio/10526480. Antibiotics 13.2 Web. doi:10.3390/antibiotics13020143.
Frizzell, Jenna K, Taylor, Ryan L, & Ryno, Lisa M. Constitutive Activation of RpoH and the Addition of L-arabinose Influence Antibiotic Sensitivity of PHL628 E. coli. Antibiotics, 13 (2). Retrieved from https://par.nsf.gov/biblio/10526480. https://doi.org/10.3390/antibiotics13020143
@article{osti_10526480,
place = {Country unknown/Code not available},
title = {Constitutive Activation of RpoH and the Addition of L-arabinose Influence Antibiotic Sensitivity of PHL628 E. coli},
url = {https://par.nsf.gov/biblio/10526480},
DOI = {10.3390/antibiotics13020143},
abstractNote = {Antibiotics are used to combat the ever-present threat of infectious diseases, but bacteria are continually evolving an assortment of defenses that enable their survival against even the most potent treatments. While the demand for novel antibiotic agents is high, the discovery of a new agent is exceedingly rare. We chose to focus on understanding how different signal transduction pathways in the gram-negative bacterium Escherichia coli (E. coli) influence the sensitivity of the organism to antibiotics from three different classes: tetracycline, chloramphenicol, and levofloxacin. Using the PHL628 strain of E. coli, we exogenously overexpressed two transcription factors, FliA and RpoH.I54N (a constitutively active mutant), to determine their influence on the minimum inhibitory concentration (MIC) and minimum duration of killing (MDK) concentration for each of the studied antibiotics. We hypothesized that activating these pathways, which upregulate genes that respond to specific stressors, could mitigate bacterial response to antibiotic treatment. We also compared the exogenous overexpression of the constitutively active RpoH mutant to thermal heat shock that has feedback loops maintained. While FliA overexpression had no impact on MIC or antibiotic tolerance, RpoH.I54N overexpression reduced the MIC for tetracycline and chloramphenicol but had no independent impact on antibiotic tolerance. Thermal heat shock alone also did not affect MIC or antibiotic tolerance. L-arabinose, the small molecule used to induce expression in our system, unexpectedly independently increased the MICs for tetracycline (>2-fold) and levofloxacin (3-fold). Additionally, the combination of thermal heat shock and arabinose provided a synergistic, 5-fold increase in MIC for chloramphenicol. Arabinose increased the tolerance, as assessed by MDK99, for chloramphenicol (2-fold) and levofloxacin (4-fold). These experiments highlight the potential of the RpoH pathway to modulate antibiotic sensitivity and the emerging implication of arabinose in enhanced MIC and antibiotic tolerance.},
journal = {Antibiotics},
volume = {13},
number = {2},
publisher = {MDPI},
author = {Frizzell, Jenna K and Taylor, Ryan L and Ryno, Lisa M},
}
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