Deep Mutational Scanning Reveals the Active-Site Sequence Requirements for the Colistin Antibiotic Resistance Enzyme MCR-1

Sun, Zhizeng; Palzkill, Timothy

doi:10.1128/mbio.02776-21

Cited by 7 publications

(5 citation statements)

References 44 publications

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“…Deep mutational scanning is a powerful way to study protein function [1][2][3] , stability 4 , amino acid roles 5 , evolvability 6 , epistasis 7 and more. The power of deep mutational scanning approaches relies on the construction of large mutant libraries that contain a wide variety of gene variants, followed by the selection, evaluation and identification of these variants 8,9 .…”

mentioning

confidence: 99%

Deep mutational scanning of essential bacterial proteins can guide antibiotic development

Dewachter

Brooks²,

Noon³

et al. 2023

Nat Commun

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Deep mutational scanning is a powerful approach to investigate a wide variety of research questions including protein function and stability. Here, we perform deep mutational scanning on three essential E. coli proteins (FabZ, LpxC and MurA) involved in cell envelope synthesis using high-throughput CRISPR genome editing, and study the effect of the mutations in their original genomic context. We use more than 17,000 variants of the proteins to interrogate protein function and the importance of individual amino acids in supporting viability. Additionally, we exploit these libraries to study resistance development against antimicrobial compounds that target the selected proteins. Among the three proteins studied, MurA seems to be the superior antimicrobial target due to its low mutational flexibility, which decreases the chance of acquiring resistance-conferring mutations that simultaneously preserve MurA function. Additionally, we rank anti-LpxC lead compounds for further development, guided by the number of resistance-conferring mutations against each compound. Our results show that deep mutational scanning studies can be used to guide drug development, which we hope will contribute towards the development of novel antimicrobial therapies.

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confidence: 99%

Deep mutational scanning of essential bacterial proteins can guide antibiotic development

Dewachter

Brooks²,

Noon³

et al. 2023

Nat Commun

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“…These modifications are primarily mediated by mutations in the PmrA/PmrB and PhoP/PhoQ two-component regulatory systems, as well as the acquisition of the mcr-1 gene. The mcr-1 gene encodes the phosphoethanolamine transferase MCR-1, which modifies lipid A of LPS [ 16 ]. The synergy of pentamidine with linezolid, rifampicin, erythromycin, and tetracycline was maintained in colistin-resistant GNB with different molecular resistance mechanisms, such as the presence of mcr-1 or PmrA / PmrB and PhoP / PhoQ gene mutations ( Figure 4 and Table S1 ).…”

Section: Resultsmentioning

confidence: 99%

The Properties of Linezolid, Rifampicin, and Vancomycin, as Well as the Mechanism of Action of Pentamidine, Determine Their Synergy against Gram-Negative Bacteria

Tang,

Zhao,

Liu

et al. 2023

IJMS

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Combining pentamidine with Gram-positive-targeting antibiotics has been proven to be a promising strategy for treating infections from Gram-negative bacteria (GNB). However, which antibiotics pentamidine can and cannot synergize with and the reasons for the differences are unclear. This study aimed to identify the possible mechanisms for the differences in the synergy of pentamidine with rifampicin, linezolid, tetracycline, erythromycin, and vancomycin against GNB. Checkerboard assays were used to detect the synergy of pentamidine and the different antibiotics. To determine the mechanism of pentamidine, fluorescent labeling assays were used to measure membrane permeability, membrane potential, efflux pump activity, and reactive oxygen species (ROS); the LPS neutralization assay was used to evaluate the target site; and quantitative PCR was used to measure changes in efflux pump gene expression. Our results revealed that pentamidine strongly synergized with rifampicin, linezolid, and tetracycline and moderately synergized with erythromycin, but did not synergize with vancomycin against E. coli, K. pneumoniae, E. cloacae, and A. baumannii. Pentamidine increased the outer membrane permeability but did not demolish the outer and inner membranes, which exclusively permits the passage of hydrophobic, small-molecule antibiotics while hindering the entry of hydrophilic, large-molecule vancomycin. It dissipated the membrane proton motive force and inactivated the efflux pump, allowing the intracellular accumulation of antimicrobials that function as substrates of the efflux pump, such as linezolid. These processes resulted in metabolic perturbation and ROS production which ultimately was able to destroy the bacteria. These mechanisms of action of pentamidine on GNB indicate that it is prone to potentiating hydrophobic, small-molecule antibiotics, such as rifampicin, linezolid, and tetracycline, but not hydrophilic, large-molecule antibiotics like vancomycin against GNB. Collectively, our results highlight the importance of the physicochemical properties of antibiotics and the specific mechanisms of action of pentamidine for the synergy of pentamidine–antibiotic combinations. Pentamidine engages in various pathways in its interactions with GNB, but these mechanisms determine its specific synergistic effects with certain antibiotics against GNB. Pentamidine is a promising adjuvant, and we can optimize drug compatibility by considering its functional mechanisms.

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“…DMS studies have been performed for identifying the determinants underlying different functional properties, 14 , 15 for protein and antibody engineering, 16 , 17 , 18 for interaction interface and epitope mapping, 19 , 20 and for researching antibiotic resistance. 21 Furthermore, DMS has proven its utility in understanding viral infections, such as in the ongoing COVID-19 pandemic. 17 , 22 , 23 , 24 The technology can provide insights into the evolutionary arms races of host antiviral proteins, 25 expose the genetic variation in host infectivity genes underlying susceptibility, 26 reveal the mutational landscape of viral proteins toward increased infectivity and antibody escape, 19 , 24 and assess the role of viral proteins in replication fitness.…”

Section: Introductionmentioning

confidence: 99%

“…As one can appreciate, the focus of DMS studies has been on disease-related proteins. 36 , 37 , 38 , 39 Exogenous DMS studies additionally focus on proteins involved in host-virus interactions 20 , 21 and therapeutic proteins. 34 , 40 , 41 Of note, DMS studies of viral proteins to understand replication fitness, where viruses expressing protein variants are selected through passaging in tissue culture, 42 are not covered.…”

Section: Introductionmentioning

confidence: 99%

Deep mutational scanning of proteins in mammalian cells

Maes,

Deploey,

Peelman

et al. 2023

Cell Reports Methods

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Deep Mutational Scanning Reveals the Active-Site Sequence Requirements for the Colistin Antibiotic Resistance Enzyme MCR-1

Abstract: Polymyxin antibiotics are used as last-line antibiotics in treating infections caused by multidrug-resistant pathogens. However, widespread use of polymyxins has led to the emergence of resistance.

Cited by 7 publications

References 44 publications

Deep mutational scanning of essential bacterial proteins can guide antibiotic development

Deep mutational scanning of essential bacterial proteins can guide antibiotic development

The Properties of Linezolid, Rifampicin, and Vancomycin, as Well as the Mechanism of Action of Pentamidine, Determine Their Synergy against Gram-Negative Bacteria

Deep mutational scanning of proteins in mammalian cells

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