Structures of Neisseria gonorrhoeae MtrR-operator complexes reveal molecular mechanisms of DNA recognition and antibiotic resistance-conferring clinical mutations

Abstract

Mutations within the mtrR gene are commonly found amongst multidrug resistant clinical isolates of Neisseria gonorrhoeae, which has been labelled a superbug by the Centers for Disease Control and Prevention. These mutations appear to contribute to antibiotic resistance by interfering with the ability of MtrR to bind to and repress expression of its target genes, which include the mtrCDE multidrug efflux transporter genes and the rpoH oxidative stress response sigma factor gene. However, the DNA-recognition mechanism of MtrR and the consensus sequence within these operators to which MtrR binds has remained unknown. In this work, we report the crystal structures of MtrR bound to the mtrCDE and rpoH operators, which reveal a conserved, but degenerate, DNA consensus binding site 5′-MCRTRCRN4YGYAYGK-3′. We complement our structural data with a comprehensive mutational analysis of key MtrR-DNA contacts to reveal their importance for MtrR-DNA binding both in vitro and in vivo. Furthermore, we model and generate common clinical mutations of MtrR to provide plausible biochemical explanations for the contribution of these mutations to multidrug resistance in N. gonorrhoeae. Collectively, our findings unveil key biological mechanisms underlying the global stress responses of N. gonorrhoeae.

Genetic characteristics of azithromycin-resistant Neisseria gonorrhoeae collected in Hyogo, Japan during 2015–2019

“…In addition, R44 is important for DNA recognition of mtrCDE . Transformation experiments in which plasmids with mtrR point mutations in R44A, G45A and Y48F were introduced into the antibiotic-sensitive strain FA19 reported increased resistance to macrolides [33]. In this study as well, it was considered that these mutations may be involved in resistance to AZM in N.…”

A Unique Sequence Is Essential for Efficient Multidrug Efflux Function of the MtrD Protein of Neisseria gonorrhoeae

“…Extensive genetic, biochemical, biophysical, and computational studies have hugely advanced our knowledge of efflux transporter systems and proteins, including the MtrCDE system of N. gonorrhoeae . A large body of knowledge already available with respect to the genetic aspects of the mtrCDE system ( 18 ), as well as the availability of crystal and cryo-EM structures of the MtrD protein in apo form ( 23 ) and in complex with drugs ( 24 ), has provided insights regarding the mechanisms used by this efflux pump to confer resistance against important antibiotics and other antimicrobials. Limited new antibiotics with activity against multidrug-resistant strains of Gram-negative pathogens have been discovered in the past 2 decades, even though they are desperately needed as this important public health problem continues to grow.…”

“…The bacterial strains and plasmids used in this study are listed in Table 2 . The background N. gonorrhoeae strain is based on a FA19 derivative, KH15 that overexpresses the MtrCDE complex due to a single-base-pair deletion in the promoter of the mtrR gene, whose product, the MtrR protein, negatively controls expression of the mtrCDE operon ( 8 , 18 ). KH15Δ mtrD Δ norM is a derivative of KH15 ( 8 ) with the mtrD and norM genes removed and is a highly discriminating strain for antimicrobial resistance assays ( 38 ).…”

“…Overexpression of MtrCDE imparts clinically significant levels of resistance to many important antibiotics used for treatment, such as β-lactams, and the currently used combined frontline treatment for gonorrhea of the third-generation cephalosporin ceftriaxone and macrolides such as azithromycin ( 4 , 8 – 10 ). This can be mediated by mtrR , the transcriptional repressor of the mtrCDE operon ( 11 , 12 ), through mutations in either mtrR or its promoter region ( 13 – 17 ) with the structural mechanism recently described ( 18 ). Furthermore, MtrCDE enables N. gonorrhoeae to persist in the presence of host-derived compounds, such as long-chain fatty acids, bile salts, hormones (e.g., progesterone) and cationic antimicrobial peptides that are found in human body sites frequently infected by N. gonorrhoeae , enhancing the ability of this pathogen to establish an infection or proliferate at mucosal surfaces ( 7 , 19 ).…”

Transcriptional regulation of the mtrCDE efflux pump operon: importance for Neisseria gonorrhoeae antimicrobial resistance

“…4a). A crystal structure of MtrR has been determined (at 2.0-2.7 Å), which illuminated both the HTH in the context of the DNA-binding site as well as critical residues in the C-terminal section that bind inducers such as certain bile salts [45,47]. MtrR serves as a global regulator of Ng gene expression and can control multiple genes outside of the mtr locus [28].…”