MtrR Modulates rpoH Expression and Levels of Antimicrobial Resistance in Neisseria gonorrhoeae

ABSTRACT The MtrR transcriptional-regulatory protein is known to repress transcription of the mtrCDE operon, which encodes a multidrug efflux pump possessed by Neisseria gonorrhoeae that is important in the ability of gonococci to resist certain hydrophobic antibiotics, detergents, dyes, and host-derived antimicrobials. In order to determine whether MtrR can exert regulatory action on other gonococcal genes, we performed a whole-genome microarray analysis using total RNA extracted from actively growing broth cultures of isogenic MtrR-positive and MtrR-negative gonococci. We determined that, at a minimum, 69 genes are directly or indirectly subject to MtrR control, with 47 being MtrR repressed and 22 being MtrR activated. rpoH, which encodes the general stress response sigma factor RpoH (sigma 32), was found by DNA-binding studies to be directly repressed by MtrR, as it was found to bind to a DNA sequence upstream of rpoH that included sites within the rpoH promoter. MtrR also repressed the expression of certain RpoH-regulated genes, but this regulation was likely indirect and a reflection of MtrR control of rpoH expression. Inducible expression of MtrR was found to repress rpoH expression and to increase gonococcal susceptibility to hydrogen peroxide (H2O2) and an antibiotic (erythromycin) recognized by the MtrC-MtrD-MtrE efflux pump system. We propose that, apart from its ability to control the expression of the mtrCDE-encoded efflux pump operon and, as a consequence, levels of gonococcal resistance to host antimicrobials (e.g., antimicrobial peptides) recognized by the efflux pump, the ability of MtrR to regulate the expression levels of rpoH and RpoH-regulated genes also modulates levels of gonococcal susceptibility to H2O2.

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Differential Regulation of ponA and pilMNOPQ Expression by the MtrR Transcriptional Regulatory Protein in Neisseria gonorrhoeae

Journal of Bacteriology ◽

10.1128/jb.00286-07 ◽

2007 ◽

Vol 189 (13) ◽

pp. 4569-4577 ◽

Cited By ~ 23

Author(s):

Jason P. Folster ◽

Vijaya Dhulipala ◽

Robert A. Nicholas ◽

William M. Shafer

Keyword(s):

Neisseria Gonorrhoeae ◽

Efflux Pump ◽

Regulatory Protein ◽

Penicillin G ◽

Penicillin Binding Protein ◽

Pump System ◽

Type Iv ◽

Mucosal Surfaces ◽

Transcriptional Regulatory ◽

Resistance To Penicillin

ABSTRACT Neisseria gonorrhoeae utilizes the mtrCDE-encoded efflux pump system to resist not only host-derived, hydrophobic antimicrobials that bathe mucosal surfaces, which likely aids in its ability to colonize and infect numerous sites within the human host, but also antibiotics that have been used clinically to treat infections. Recently, overexpression of the MtrC-MtrD-MtrE efflux pump was shown to be critically involved in the capacity of gonococci to develop chromosomally mediated resistance to penicillin G, which for over 40 years was used to treat gonococcal infections. Mutations in either the promoter or the coding sequence of the mtrR gene, which encodes a repressor of the efflux pump operon, decrease gonococcal susceptibility to penicillin. We now describe the capacity of MtrR to directly or indirectly influence the expression of two other loci that are involved in gonococcal susceptibility to penicillin: ponA, which encodes penicillin-binding protein 1 (PBP 1), and the pilMNOPQ operon, which encodes components of the type IV pilus secretion system, with PilQ acting as a channel for entry for penicillin. We determined that MtrR increases the expression of ponA directly or indirectly, resulting in increased levels of PBP 1, while repressing the expression of the divergently transcribed pilM gene, the first gene in the pilMNOPQ operon. Taken together with other studies, the results presented herein indicate that transcriptional regulation of gonococcal genes by MtrR is centrally involved in determining levels of gonococcal susceptibility to penicillin and provides a framework for understanding how resistance developed over the years.

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Cryo-EM Structures of a Gonococcal Multidrug Efflux Pump Illuminate a Mechanism of Drug Recognition and Resistance

mBio ◽

10.1128/mbio.00996-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 5

Author(s):

Meinan Lyu ◽

Mitchell A. Moseng ◽

Jennifer L. Reimche ◽

Concerta L. Holley ◽

Vijaya Dhulipala ◽

...

Keyword(s):

Electron Microscopy ◽

Drug Resistance ◽

Neisseria Gonorrhoeae ◽

Antimicrobial Agents ◽

Efflux Pump ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Transmitted Infection ◽

Content Type ◽

Cryo Electron Microscopy

ABSTRACT Neisseria gonorrhoeae is an obligate human pathogen and causative agent of the sexually transmitted infection (STI) gonorrhea. The most predominant and clinically important multidrug efflux system in N. gonorrhoeae is the multiple transferrable resistance (Mtr) pump, which mediates resistance to a number of different classes of structurally diverse antimicrobial agents, including clinically used antibiotics (e.g., β-lactams and macrolides), dyes, detergents and host-derived antimicrobials (e.g., cationic antimicrobial peptides and bile salts). Recently, it has been found that gonococci bearing mosaic-like sequences within the mtrD gene can result in amino acid changes that increase the MtrD multidrug efflux pump activity, probably by influencing antimicrobial recognition and/or extrusion to elevate the level of antibiotic resistance. Here, we report drug-bound solution structures of the MtrD multidrug efflux pump carrying a mosaic-like sequence using single-particle cryo-electron microscopy, with the antibiotics bound deeply inside the periplasmic domain of the pump. Through this structural approach coupled with genetic studies, we identify critical amino acids that are important for drug resistance and propose a mechanism for proton translocation. IMPORTANCE Neisseria gonorrhoeae has become a highly antimicrobial-resistant Gram-negative pathogen. Multidrug efflux is a major mechanism that N. gonorrhoeae uses to counteract the action of multiple classes of antibiotics. It appears that gonococci bearing mosaic-like sequences within the gene mtrD, encoding the most predominant and clinically important transporter of any gonococcal multidrug efflux pump, significantly elevate drug resistance and enhance transport function. Here, we report cryo-electron microscopy (EM) structures of N. gonorrhoeae MtrD carrying a mosaic-like sequence that allow us to understand the mechanism of drug recognition. Our work will ultimately inform structure-guided drug design for inhibiting these critical multidrug efflux pumps.

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MepR, a Repressor of the Staphylococcus aureus MATE Family Multidrug Efflux Pump MepA, Is a Substrate-Responsive Regulatory Protein

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.4.1276-1281.2006 ◽

2006 ◽

Vol 50 (4) ◽

pp. 1276-1281 ◽

Cited By ~ 48

Author(s):

Glenn W. Kaatz ◽

Carmen E. DeMarco ◽

Susan M. Seo

Keyword(s):

Staphylococcus Aureus ◽

Efflux Pump ◽

Regulatory Protein ◽

Inducible Promoter ◽

Multidrug Efflux ◽

Recognition Sequence ◽

Multidrug Efflux Pump ◽

Mobility Shift ◽

Repressive Effect ◽

Operator Interaction

ABSTRACT The mepRAB gene cluster of Staphylococcus aureus encodes a MarR family repressor (MepR; known to repress mepA expression), a MATE family multidrug efflux pump (MepA), and a protein of unknown function (MepB). In this report, we show that MepR also is autoregulatory, repressing the expression of its own gene. Exposure of strains containing a mepR::lacZ fusion with mepR provided in trans under the control of an inducible promoter, or a mepA::lacZ fusion alone, to subinhibitory concentrations of MepA substrates resulted in variably increased expression mainly of mepA. Mobility shift assays revealed that MepR binds upstream of mepR and mepA, with an apparently higher affinity for the mepA binding site. MepA substrates abrogated MepR binding to each site in a differential manner, with the greatest effect observed on the MepR-mepA operator interaction. DNase I footprinting identified precise binding sites which included promoter motifs, inverted repeats, and transcription start sites for mepR and mepA, as well as a conserved GTTAG motif, which may be a signature recognition sequence for MepR. Analogous to other multidrug efflux pump regulatory proteins such as QacR, the substrate-MepR interaction likely results in its dissociation from its mepA, and in a more limited fashion its mepR, operator sites and relief of its repressive effect. The enhanced effect of substrates on mepA compared to mepR expression, and on the MepR-mepA operator interaction, results in significant relief of mepA and relative maintenance of mepR repression, leading to increased MepA protein unimpeded by MepR when the need for detoxification exists.

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Structural, Biochemical, and In Vivo Characterization of MtrR-Mediated Resistance to Innate Antimicrobials by the Human Pathogen Neisseria gonorrhoeae

Journal of Bacteriology ◽

10.1128/jb.00401-19 ◽

2019 ◽

Vol 201 (20) ◽

Author(s):

Grace A. Beggs ◽

Yaramah M. Zalucki ◽

Nicholas Gene Brown ◽

Sheila Rastegari ◽

Rebecca K. Phillips ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Bile Salts ◽

Efflux Pump ◽

Binding Modes ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Content Type ◽

Multiple Drugs

ABSTRACT Neisseria gonorrhoeae responds to host-derived antimicrobials by inducing the expression of the mtrCDE-encoded multidrug efflux pump, which expels microbicides, such as bile salts, fatty acids, and multiple extrinsically administered drugs, from the cell. In the absence of these cytotoxins, the TetR family member MtrR represses the mtrCDE genes. Although antimicrobial-dependent derepression of mtrCDE is clear, the physiological inducers of MtrR are unknown. Here, we report the crystal structure of an induced form of MtrR. In the binding pocket of MtrR, we observed electron density that we hypothesized was N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), a component of the crystallization reagent. Using the MtrR-CAPS structure as an inducer-bound template, we hypothesized that bile salts, which bear significant chemical resemblance to CAPS, are physiologically relevant inducers. Indeed, characterization of MtrR-chenodeoxycholate and MtrR-taurodeoxycholate interactions, both in vitro and in vivo, revealed that these bile salts, but not glyocholate or taurocholate, bind MtrR tightly and can act as bona fide inducers. Furthermore, two residues, W136 and R176, were shown to be important in binding chenodeoxycholate but not taurodeoxycholate, suggesting different binding modes of the bile salts. These data provide insight into a crucial mechanism utilized by the pathogen to overcome innate human defenses. IMPORTANCE Neisseria gonorrhoeae causes a significant disease burden worldwide, and a meteoric rise in its multidrug resistance has reduced the efficacy of antibiotics previously or currently approved for therapy of gonorrheal infections. The multidrug efflux pump MtrCDE transports multiple drugs and host-derived antimicrobials from the bacterial cell and confers survival advantage on the pathogen within the host. Transcription of the pump is repressed by MtrR but relieved by the cytosolic influx of antimicrobials. Here, we describe the structure of induced MtrR and use this structure to identify bile salts as physiological inducers of MtrR. These findings provide a mechanistic basis for antimicrobial sensing and gonococcal protection by MtrR through the derepression of mtrCDE expression after exposure to intrinsic and clinically applied antimicrobials.

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Gene cloning and characteristics of the RND-type multidrug efflux pump MuxABC-OpmB possessing two RND components in Pseudomonas aeruginosa

Microbiology ◽

10.1099/mic.0.031260-0 ◽

2009 ◽

Vol 155 (11) ◽

pp. 3509-3517 ◽

Cited By ~ 32

Author(s):

Takehiko Mima ◽

Naoki Kohira ◽

Yang Li ◽

Hiroshi Sekiya ◽

Wakano Ogawa ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Resistant Mutant ◽

Sequencing Analysis ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Pump System ◽

Single Nucleotide ◽

Nucleotide Insertion ◽

Drug Efflux Pump

muxA-muxB-muxC-opmB (formerly PA2528-PA2527-PA2526-opmB), encoding a putative resistance nodulation cell division (RND)-type multidrug efflux pump system, was cloned from Pseudomonas aeruginosa PAO1. Introduction of muxABC-opmB into P. aeruginosa YM64, a drug-hypersusceptible strain, led to elevated MICs of aztreonam, macrolides, novobiocin and tetracycline. Since muxB and muxC, both of which encode RND components, were essential for function, MuxABC-OpmB is thought to be a drug efflux pump with four components. One novobiocin-resistant mutant, PMX725, isolated from P. aeruginosa PMX7 showed elevated resistance not only to novobiocin but also to aztreonam, macrolides and tetracycline. Increased mRNA expression of muxABC-opmB was observed in the mutant PMX725 compared with the parental strain. Sequencing analysis revealed that a single-nucleotide insertion had occurred in the deduced promoter region for muxABC-opmB in PMX725. In this study, we have characterized the last RND-type multidrug efflux pump predicted from the genome sequence in P. aeruginosa.

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Evidence of Horizontal Gene Transfer of 50S Ribosomal Genes rplB, rplD, and rplY in Neisseria gonorrhoeae

Frontiers in Microbiology ◽

10.3389/fmicb.2021.683901 ◽

2021 ◽

Vol 12 ◽

Author(s):

Sheeba Santhini Manoharan-Basil ◽

Jolein Gyonne Elise Laumen ◽

Christophe Van Dijck ◽

Tessa De Block ◽

Irith De Baetselier ◽

...

Keyword(s):

Gene Transfer ◽

Horizontal Gene Transfer ◽

Neisseria Gonorrhoeae ◽

Ribosomal Proteins ◽

Efflux Pump ◽

Bacterial Species ◽

23S Rrna ◽

Comparative Genomic ◽

Multidrug Efflux ◽

Multidrug Efflux Pump

Horizontal gene transfer (HGT) in the penA and multidrug efflux pump genes has been shown to play a key role in the genesis of antimicrobial resistance in Neisseria gonorrhoeae. In this study, we evaluated if there was evidence of HGT in the genes coding for the ribosomal proteins in the Neisseria genus. We did this in a collection of 11,659 isolates of Neisseria, including N. gonorrhoeae and commensal Neisseria species (N. cinerea, N. elongata, N. flavescens, N. mucosa, N. polysaccharea, and N. subflava). Comparative genomic analyses identified HGT events in three genes: rplB, rplD, and rplY coding for ribosomal proteins L2, L4 and L25, respectively. Recombination events were predicted in N. gonorrhoeae and N. cinerea, N. subflava, and N. lactamica were identified as likely progenitors. In total, 2,337, 2,355, and 1,127 isolates possessed L2, L4, and L25 HGT events. Strong associations were found between HGT in L2/L4 and the C2597T 23S rRNA mutation that confers reduced susceptibility to macrolides. Whilst previous studies have found evidence of HGT of entire genes coding for ribosomal proteins in other bacterial species, this is the first study to find evidence of HGT-mediated chimerization of ribosomal proteins.

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