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Characterization of Posttranslationally Modified Multidrug Efflux Pumps Reveals an Unexpected Link between Glycosylation and Antimicrobial Resistance.

ABSTRACT: The substantial rise in multidrug-resistant bacterial infections is a current global imperative. Cumulative efforts to characterize antimicrobial resistance in bacteria has demonstrated the spread of six families of multidrug efflux pumps, of which resistance-nodulation-cell division (RND) is the major mechanism of multidrug resistance in Gram-negative bacteria. RND is composed of a tripartite protein assembly and confers resistance to a range of unrelated compounds. In the major enteric pathogen Campylobacter jejuni, the three protein components of RND are posttranslationally modified with N-linked glycans. The direct role of N-linked glycans in C. jejuni and other bacteria has long been elusive. Here, we present the first detailed account of the role of N-linked glycans and the link between N-glycosylation and antimicrobial resistance in C. jejuni We demonstrate the multifunctional role of N-linked glycans in enhancing protein thermostability, stabilizing protein complexes and the promotion of protein-protein interaction, thus mediating antimicrobial resistance via enhancing multidrug efflux pump activity. This affirms that glycosylation is critical for multidrug efflux pump assembly. We present a generalized strategy that could be used to investigate general glycosylation system in Campylobacter genus and a potential target to develop antimicrobials against multidrug-resistant pathogens.IMPORTANCE Nearly all bacterial species have at least a single glycosylation system, but the direct effects of these posttranslational protein modifications are unresolved. Glycoproteome-wide analysis of several bacterial pathogens has revealed general glycan modifications of virulence factors and protein assemblies. Using Campylobacter jejuni as a model organism, we have studied the role of general N-linked glycans in the multidrug efflux pump commonly found in Gram-negative bacteria. We show, for the first time, the direct link between N-linked glycans and multidrug efflux pump activity. At the protein level, we demonstrate that N-linked glycans play a role in enhancing protein thermostability and mediating the assembly of the multidrug efflux pump to promote antimicrobial resistance, highlighting the importance of this posttranslational modification in bacterial physiology. Similar roles for glycans are expected to be found in other Gram-negative pathogens that possess general protein glycosylation systems.

SUBMITTER: Abouelhadid S

PROVIDER: S-EPMC7683400 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Characterization of Posttranslationally Modified Multidrug Efflux Pumps Reveals an Unexpected Link between Glycosylation and Antimicrobial Resistance.

Abouelhadid Sherif S Raynes John J Bui Tam T Cuccui Jon J Wren Brendan W BW

mBio 20201117 6

The substantial rise in multidrug-resistant bacterial infections is a current global imperative. Cumulative efforts to characterize antimicrobial resistance in bacteria has demonstrated the spread of six families of multidrug efflux pumps, of which resistance-nodulation-cell division (RND) is the major mechanism of multidrug resistance in Gram-negative bacteria. RND is composed of a tripartite protein assembly and confers resistance to a range of unrelated compounds. In the major enteric pathoge ...[more]

PMID: 33203757

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Project description:RND (Resistance-Nodulation-Division) family transporters are widespread especially among Gram-negative bacteria, and catalyze the active efflux of many antibiotics and chemotherapeutic agents. They have very large periplasmic domains, and form tripartite complexes with outer membrane channels and periplasmic adaptor proteins. AcrAB-TolC complex of Escherichia coli, which pumps out a very wide range of drugs, has been studied most intensively. Early studies showed that the transporter captures even those substrates that cannot permeate across the cytoplasmic membrane, such as dianionic beta-lactams, suggesting that the capture can occur from the periplasm. It was also suggested that the capture occurs from the cytoplasmic membrane/periplasm interface, because most substrates contain a sizable hydrophobic domain; however, this may simply be a reflection of the nature of the binding site within AcrB. Genetic studies of chimeric transporters showed that much of the substrate specificity is determined by their periplasmic domains. Biochemical studies with intact cells recently led to the determination of the kinetic constants of AcrB for some beta-lactams, and the result confirms the old prediction that AcrB is a rather slow pump. Reconstitution of purified AcrB and its relatives showed that the pump is a drug/proton antiporter, that AcrA strongly stimulates the activity of the pump, and that AcrB seems to have a highest affinity for conjugated bile salts. Structural study with mutants of the network of charged residues in the transmembrane domain showed that protonation here produced a far-reaching conformational change, which was found to be present in one of the protomers in the asymmetric crystal structure of the wild-type AcrB. The functional rotatory hypothesis then predicts that the drug bound in the periplasmic domain is extruded through this conformational change initiated by the protonation of one of the residues in the aforementioned network, an idea that was recently supported by disulfide cross-linking as well as by the behavior of linked AcrB protomers.

Dataset Information

Characterization of Posttranslationally Modified Multidrug Efflux Pumps Reveals an Unexpected Link between Glycosylation and Antimicrobial Resistance.

Publications

Characterization of Posttranslationally Modified Multidrug Efflux Pumps Reveals an Unexpected Link between Glycosylation and Antimicrobial Resistance.

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