Biology subjects

Kent, J. E.

Publications and source records attributed to Kent, J. E..

3 recordsLinked to original sources

bioRxiv2023

Evolution of resistance to a tricyclic pyrimidoindole DNA gyrase/topoisomerase inhibitor in Escherichia coli and Acinetobacter baumannii is driven by upregulation of efflux machinery

Comprehensive knowledge of mechanisms driving the acquisition of antimicrobial resistance is essential for the development of new drugs with minimized resistibility. To gain this knowledge, we combine experimental evolution in a continuous culturing device, the morbidostat, with whole genome sequencing of evolving cultures followed by characterization of drug-resistant isolates. Here, this approach was used to assess evolutionary dynamics of resistance acquisition against DNA gyrase/topoisomerase TriBE inhibitor GP6 in Escherichia coli and Acinetobacter baumannii. The evolution of GP6 resistance in both species was driven by a combination of two classes of mutational events: (i) amino acid substitutions near the ATP-binding site of the GyrB subunit of the DNA gyrase target; and (ii) various mutations and genomic rearrangements leading to upregulation of efflux pumps, species-specific (AcrAB/TolC in E. coli and AdeIJK in A. baumannii) and shared by both species (MdtK). A comparison with the experimental evolution of resistance to ciprofloxacin (CIP), previously performed using the same workflow and strains, revealed fundamental differences between these two distinct classes of compounds. Most notable were non-overlapping spectra of target mutations and distinct evolutionary trajectories that, in the case of GP6, were dominated by upregulation of efflux machinery prior to (or even in lieu) of target modification. Most of efflux-driven GP6-resistant isolates of both species displayed a robust cross-resistance to CIP, while CIP-resistant clones showed no appreciable increase in GP6-resistance. ImportanceThe significance of this work is in assessing the mutational landscape and evolutionary dynamics of resistance acquisition against a novel antibiotic, GP6. This approach showed that, in contrast to ciprofloxacin (CIP), a previously studied a canonical DNA gyrase/topoisomerase-targeting clinical antibiotic, evolution of GP6-resistance is driven largely by early and most prominent mutational events leading to upregulation of efflux machinery. An identified asymmetry in cross-resistance of evolved GP6- vs CIP-resistant clones provides important guidelines for rational selection of potential treatment regimens. This study illustrates the utility of the established morbidostat-based comparative resistomics workflow for the assessment of new drug candidates and clinical antibiotics.

microbiology↗

bioRxiv2023

Dynamic nuclear polarization illuminates key protein-lipid interactions in the native bacterial cell envelope

Elucidating the structure and interactions of proteins in native environments has become a fundamental goal of structural biology. Nuclear magnetic resonance (NMR) spectroscopy is well suited for this task but often suffers from low sensitivity, especially in complex biological settings. Here, we use a sensitivity-enhancement technique called dynamic nuclear polarization (DNP) to overcome this challenge. We apply DNP to capture the membrane interactions of the outer membrane protein Ail, a key component of the host invasion pathway of Yersinia pestis. We show that the DNP-enhanced NMR spectra of Ail in native bacterial cell envelopes are well resolved and enriched in correlations that are invisible in conventional solid-state NMR experiments. Furthermore, we demonstrate the ability of DNP to capture elusive interactions between the protein and the surrounding lipopolysaccharide layer. Our results support a model where the extracellular loop arginine residues remodel the membrane environment, a process that is crucial for host invasion and pathogenesis.

biochemistry↗

bioRxiv2020

Resolving atomic site interactions of the Y. pestis outer membrane protein Ail with human serum in the bacterial cell envelope

Understanding microbe-host interactions at the molecular level is a major goal of fundamental biology and therapeutic drug development. Structural biology strives to capture biomolecular structures in action, but the samples are often highly simplified versions of the complex native environment. Here we present an E. coli model system that allows us to probe the structure and function of Ail, the major surface protein of the deadly pathogen Yersinia pestis. We show that cell surface expression of Ail produces Y. pestis virulence phenotypes in E. coli, including resistance to human serum, co-sedimentation of human vitronectin and pellicle formation. Moreover, isolated bacterial cell envelopes, encompassing inner and outer membranes, yield high-resolution solid-state nuclear magnetic resonance (NMR) spectra that reflect the structure of Ail and reveal Ail sites that are sensitive to the bacterial membrane environment and involved in the interactions with human serum components. The data capture the structure and function of Ail in a bacterial outer membrane and set the stage for probing its interactions with the complex milieu of immune response proteins present in human serum. SIGNIFICANCEAil is a critical virulence factor of Y. pestis, and its interactions with human serum are central for promoting the immune resistance of bacteria to the human host defenses. Here we capture the action of Ail in a functional bacterial environment and set the stage for probing its interactions with the complex milieu of immune response proteins present in human serum. The development of an E. coli model system of Y. pestis for biophysical studies is new and biologically important. Finally, the work extends the range in-situ NMR spectroscopy to include models of microbial infection.

biochemistry↗