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

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.

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

This review focuses on the mechanisms of transcriptional control of an important multidrug efflux pump system (MtrCDE) possessed by Neisseria gonorrhoeae, the aetiological agent of the sexually transmitted infection termed gonorrhoea. The mtrCDE operon that encodes this tripartite protein efflux pump is subject to both cis- and trans-acting transcriptional factors that negatively or positively influence expression. Critically, levels of MtrCDE can influence levels of gonococcal susceptibility to classical antibiotics, host-derived antimicrobials and various biocides. The regulatory systems that control mtrCDE can have profound influences on the capacity of gonococci to resist current and past antibiotic therapy regimens as well as virulence. The emergence, mechanisms of action and clinical significance of the transcriptional regulatory systems that impact mtrCDE expression in gonococci are reviewed here with the aim of linking bacterial antimicrobial resistance with multidrug efflux capability.

Transcriptional regulation of a gonococcal gene encoding a virulence factor (L-lactate permease).

GdhR is a GntR-type regulator of Neisseria gonorrhoeae encoded by a gene (gdhR) belonging to the MtrR regulon, which comprises multiple genes required for antibiotic resistance such as the mtrCDE efflux pump genes. In previous work we showed that loss of gdhR results in enhanced gonococcal fitness in a female mouse model of lower genital tract infection. Here, we used RNA-Seq to perform a transcriptional profiling study to determine the GdhR regulon. GdhR was found to regulate the expression of 2.3% of all the genes in gonococcal strain FA19, of which 39 were activated and 11 were repressed. Within the GdhR regulon we found that lctP, which encodes a unique L-lactate transporter and has been associated with gonococcal pathogenesis, was the highest of GdhR-repressed genes. By using in vitro transcription and DNase I footpriting assays we mapped the lctP transcriptional start site (TSS) and determined that GdhR directly inhibits transcription by binding to an inverted repeat sequence located 9 bases downstream of the lctP TSS. Epistasis analysis revealed that, while loss of lctP increased susceptibility of gonococci to hydrogen peroxide (H2O2) the loss of gdhR enhanced resistance; however, this GdhR-endowed property was reversed in a double gdhR lctP null mutant. We assessed the effect of different carbon sources on lctP expression and found that D-glucose, but not L-lactate or pyruvate, repressed lctP expression within a physiological concentration range but in a GdhR-independent manner. Moreover, we found that adding glucose to the medium enhanced susceptibility of gonococci to hydrogen peroxide. We propose a model for the role of lctP regulation via GdhR and glucose in the pathogenesis of N. gonorrhoeae.

Molecular basis for the differential expression of the global regulator VieA in Vibrio cholerae biotypes directed by H-NS, LeuO and quorum sensing.

VieA is a cyclic diguanylate phosphodiesterase that modulates biofilm development and motility in Vibrio cholerae O1 of the classical biotype. vieA is part of an operon encoding the VieSAB signal transduction pathway that is nearly silent in V. cholerae of the El Tor biotype. A DNA pull-down assay for proteins interacting with the vieSAB promoter identified the LysR-type regulator LeuO. We show that in classical biotype V. cholerae, LeuO cooperates with the nucleoid-associated protein H-NS to repress vieSAB transcription. LeuO and H-NS interacted with the vieSAB promoter of both biotypes with similar affinities and protected overlapping DNA sequences. H-NS was expressed at similar levels in both cholera biotypes. In contrast, El Tor biotype strains expressed negligible LeuO under identical conditions. In El Tor biotype vibrios, transcription of vieSAB is repressed by the quorum sensing regulator HapR, which is absent in classical biotype strains. Restoring HapR expression in classical biotype V. cholerae repressed vieSAB transcription by binding to its promoter. We propose that double locking of the vieSAB promoter by H-NS and HapR in the El Tor biotype prior to the cessation of exponential growth results in a more pronounced decline in VieA specific activity compared to the classical biotype.

Repression by H-NS of genes required for the biosynthesis of the Vibrio cholerae biofilm matrix is modulated by the second messenger cyclic diguanylic acid.

Expression of Vibrio cholerae genes required for the biosynthesis of exopolysacchide (vps) and protein (rbm) components of the biofilm matrix is enhanced by cyclic diguanylate (c-di-GMP). In a previous study, we reported that the histone-like nucleoid structuring (H-NS) protein represses the transcription of vpsA, vpsL and vpsT. Here we demonstrate that the regulator VpsT can disrupt repressive H-NS nucleoprotein complexes at the vpsA and vpsL promoters in the presence of c-di-GMP, while H-NS could disrupt the VpsT-promoter complexes in the absence of c-di-GMP. Chromatin immunoprecipitation-Seq showed a remarkable trend for H-NS to cluster at loci involved in biofilm development such as the rbmABCDEF genes. We show that the antagonistic relationship between VpsT and H-NS regulates the expression of the rbmABCDEF cluster. Epistasis analysis demonstrated that VpsT functions as an antirepressor at the rbmA/F, vpsU and vpsA/L promoters. Deletion of vpsT increased H-NS occupancy at these promoters while increasing the c-di-GMP pool had the opposite effect and included the vpsT promoter. The negative effect of c-di-GMP on H-NS occupancy at the vpsT promoter required the regulator VpsR. These results demonstrate that c-di-GMP activates the transcription of genes required for the biosynthesis of the biofilm matrix by triggering a coordinated VpsR- and VpsT-dependent H-NS antirepression cascade.

Role of methylthioadenosine/S-adenosylhomocysteine nucleosidase in Vibrio cholerae cellular communication and biofilm development.

In Vibrio cholerae, the genes required for biofilm development are repressed by quorum sensing at high cell density due to the accumulation in the medium of two signaling molecules, cholera autoinducer 1 (CAI-1) and autoinducer 2 (AI-2). A significant fraction of toxigenic V. cholerae isolates, however, exhibit dysfunctional quorum sensing pathways. It was reported that transition state analogs of the enzyme methylthioadenosine/S-adenosylhomocysteine nucleosidase (MtnN) required to make AI-2 inhibited biofilm formation in the prototype quorum sensing-deficient strain N16961. This finding prompted us to examine the role of both autoinducers and MtnN in biofilm development and virulence gene expression in a quorum sensing-deficient genetic background. Here we show that deletion of mtnN encoding methylthioadenosine/S-adenosylhomocysteine nucleosidase, cqsA (CAI-1), and/or luxS (AI-2) do not prevent biofilm development. However, two independent mtnN mutants exhibited diminished growth rate and motility in swarm agar plates suggesting that, under certain conditions, MtnN could influence biofilm formation indirectly. Nevertheless, MtnN is not required for the development of a mature biofilm.

The LuxR-type regulator VpsT negatively controls the transcription of rpoS, encoding the general stress response regulator, in Vibrio cholerae biofilms.

Cholera is a waterborne diarrheal disease caused by Vibrio cholerae strains of serogroups O1 and O139. Expression of the general stress response regulator RpoS and formation of biofilm communities enhance the capacity of V. cholerae to persist in aquatic environments. The transition of V. cholerae between free-swimming (planktonic) and biofilm life-styles is regulated by the second messenger cyclic di-GMP (c-di-GMP). We previously reported that increasing the c-di-GMP pool by overexpression of a diguanylate cyclase diminished RpoS expression. Here we show that c-di-GMP repression of RpoS expression is eliminated by deletion of the genes vpsR and vpsT, encoding positive regulators of biofilm development. To determine the mechanism of this regulation, we constructed a strain expressing a vpsT-FLAG allele from native transcription and translation signals. Increasing the c-di-GMP pool induced vpsT-FLAG expression. The interaction between VpsT-FLAG and the rpoS promoter was demonstrated by chromatin immunoprecipitation. Furthermore, purified VpsT interacted with the rpoS promoter in a c-di-GMP-dependent manner. Primer extension analysis identified two rpoS transcription initiation sites located 43 bp (P1) and 63 bp (P2) upstream of the rpoS start codon. DNase I footprinting showed that the VpsT binding site at the rpoS promoter overlaps the primary P1 transcriptional start site. Deletion of vpsT significantly enhanced rpoS expression in V. cholerae biofilms that do not make HapR. This result suggests that VpsT and c-di-GMP contribute to the transcriptional silencing of rpoS in biofilms prior to cells entering the quorum-sensing mode.

Transcriptional responses of Neisseria gonorrhoeae to glucose and lactate: implications for resistance to oxidative damage and biofilm formation.

Understanding how bacteria adapt to different environmental conditions is crucial for advancing knowledge regarding pathogenic mechanisms that operate during infection as well as efforts to develop new therapeutic strategies to cure or prevent infections. Here, we investigated the transcriptional response of Neisseria gonorrhoeae, the causative agent of gonorrhea, to L-lactate and glucose, two important carbon sources found in the host environment. Our study revealed extensive transcriptional changes that gonococci make in response to L-lactate, with 37% of the gonococcal transcriptome being regulated, compared to only 9% by glucose. We found that L-lactate induces a transcriptional program that would negatively impact iron transport, potentially limiting the availability of labile iron, which would be important in the face of the multiple hydrogen peroxide attacks encountered by gonococci during its lifecycle. Furthermore, we found that L-lactate-mediated transcriptional response promoted aerobic respiration and dispersal of biofilms, contrasting with an anaerobic condition previously reported to favor biofilm formation. Our findings suggest an intricate interplay between carbon metabolism, iron homeostasis, biofilm formation, and stress response in N. gonorrhoeae, providing insights into its pathogenesis and identifying potential therapeutic targets.IMPORTANCEGonorrhea is a prevalent sexually transmitted infection caused by the human pathogen Neisseria gonorrhoeae, with ca. 82 million cases reported worldwide annually. The rise of antibiotic resistance in N. gonorrhoeae poses a significant public health threat, highlighting the urgent need for alternative treatment strategies. By elucidating how N. gonorrhoeae responds to host-derived carbon sources such as L-lactate and glucose, this study offers insights into the metabolic adaptations crucial for bacterial survival and virulence during infection. Understanding these adaptations provides a foundation for developing novel therapeutic approaches targeting bacterial metabolism, iron homeostasis, and virulence gene expression. Moreover, the findings reported herein regarding biofilm formation and L-lactate transport and metabolism contribute to our understanding of N. gonorrhoeae pathogenesis, offering potential avenues for preventing and treating gonorrhea infections.

Hormonal steroids induce multidrug resistance and stress response genes in Neisseria gonorrhoeae by binding to MtrR.

Transcriptional regulator MtrR inhibits the expression of the multidrug efflux pump operon mtrCDE in the pathogenic bacterium Neisseria gonorrhoeae. Here, we show that MtrR binds the hormonal steroids progesterone, ß-estradiol, and testosterone, which are present at urogenital infection sites, as well as ethinyl estrogen, a component of some hormonal contraceptives. Steroid binding leads to the decreased affinity of MtrR for cognate DNA, increased mtrCDE expression, and enhanced antimicrobial resistance. Furthermore, we solve crystal structures of MtrR bound to each steroid, thus revealing their binding mechanisms and the conformational changes that induce MtrR.

Hormonal steroids bind the Neisseria gonorrhoeae multidrug resistance regulator, MtrR, to induce a multidrug binding efflux pump and stress-response sigma factor.

Overexpression of the multidrug efflux pump MtrCDE, a critical factor of multidrug-resistance in Neisseria gonorrhoeae , the causative agent of gonorrheae, is repressed by the transcriptional regulator, MtrR (multiple transferable resistance repressor). Here, we report the results from a series of in vitro experiments to identify innate, human inducers of MtrR and to understand the biochemical and structural mechanisms of the gene regulatory function of MtrR. Isothermal titration calorimetry experiments reveal that MtrR binds the hormonal steroids progesterone, ß-estradiol, and testosterone, all of which are present at significant concentrations at urogenital infection sites as well as ethinyl estrogen, a component of some birth control pills. Binding of these steroids results in decreased affinity of MtrR for cognate DNA, as demonstrated by fluorescence polarization-based assays. The crystal structures of MtrR bound to each steroid provided insight into the flexibility of the binding pocket, elucidated specific residue-ligand interactions, and revealed the conformational consequences of the induction mechanism of MtrR. Three residues, D171, W136 and R176 are key to the specific binding of these gonadal steroids. These studies provide a molecular understanding of the transcriptional regulation by MtrR that promotes N. gonorrhoeae survival in its human host.

Interaction of the histone-like nucleoid structuring protein and the general stress response regulator RpoS at Vibrio cholerae promoters that regulate motility and hemagglutinin/protease expression.

The bacterium Vibrio cholerae colonizes the human small intestine and secretes cholera toxin (CT) to cause the rice-watery diarrhea characteristic of this illness. The ability of this pathogen to colonize the small bowel, express CT, and return to the aquatic environment is controlled by a complex network of regulatory proteins. Two global regulators that participate in this process are the histone-like nucleoid structuring protein (H-NS) and the general stress response regulator RpoS. In this study, we address the role of RpoS and H-NS in the coordinate regulation of motility and hemagglutinin (HA)/protease expression. In addition to initiating transcription of hapA encoding HA/protease, RpoS enhanced flrA and rpoN transcription to increase motility. In contrast, H-NS was found to bind to the flrA, rpoN, and hapA promoters and represses their expression. The strength of H-NS repression at the above-mentioned promoters was weaker for hapA, which exhibited the strongest RpoS dependency, suggesting that transcription initiation by RNA polymerase containing σ(S) could be more resistant to H-NS repression. Occupancy of the flrA and hapA promoters by H-NS was demonstrated by chromatin immunoprecipitation (ChIP). We show that the expression of RpoS in the stationary phase significantly diminished H-NS promoter occupancy. Furthermore, RpoS enhanced the transcription of integration host factor (IHF), which positively affected the expression of flrA and rpoN by diminishing the occupancy of H-NS at these promoters. Altogether, we propose a model for RpoS regulation of motility gene expression that involves (i) attenuation of H-NS repression by IHF and (ii) RpoS-dependent transcription initiation resistant to H-NS.

The histone-like nucleoid structuring protein (H-NS) is a repressor of Vibrio cholerae exopolysaccharide biosynthesis (vps) genes.

The capacity of Vibrio cholerae to form biofilms has been shown to enhance its survival in the aquatic environment and play important roles in pathogenesis and disease transmission. In this study, we demonstrated that the histone-like nucleoid structuring protein is a repressor of exopolysaccharide (vps) biosynthesis genes and biofilm formation.

Structural Basis of Peptide-Based Antimicrobial Inhibition of a Resistance-Nodulation-Cell Division Multidrug Efflux Pump.

Bacterial efflux pumps in the resistance-nodulation-cell division (RND) family of Gram-negative bacteria contribute significantly to the development of antimicrobial resistance by many pathogens. In this study, we selected the MtrD transporter protein of Neisseria gonorrhoeae as it is the sole RND pump possessed by this strictly human pathogen and can export multiple antimicrobials, including antibiotics, bile salts, detergents, dyes, and antimicrobial peptides. Using knowledge from our previously published structures of MtrD in the presence or absence of bound antibiotics as a model and the known ability of MtrCDE to export cationic antimicrobial peptides, we hypothesized that cationic peptides could be accommodated within MtrD binding sites. Furthermore, we thought that MtrD-bound peptides lacking antibacterial action could sensitize bacteria to an antibiotic normally exported by the MtrCDE efflux pump or other similar RND-type pumps possessed by different Gram-negative bacteria. We now report the identification of a novel nonantimicrobial cyclic cationic antimicrobial peptide, which we termed CASP (cationic antibiotic-sensitizing peptide). By single-particle cryo-electron microscopy, we found that CASP binds within the periplasmic cleft region of MtrD using overlapping and distinct amino acid contact sites that interact with another cyclic peptide (colistin) or a linear human cationic antimicrobial peptide derived from human LL-37. While CASP could not sensitize Neisseria gonorrhoeae to an antibiotic (novobiocin) that is a substrate for RND pumps, it could do so against multiple Gram-negative, rod-shaped bacteria. We propose that CASP (or future derivatives) could serve as an adjuvant for the antibiotic treatment of certain Gram-negative infections previously thwarted by RND transporters. IMPORTANCE RND efflux pumps can export numerous antimicrobials that enter Gram-negative bacteria, and their action can reduce the efficacy of antibiotics and provide decreased susceptibility to various host antimicrobials. Here, we identified a cationic antibiotic-sensitizing peptide (CASP) that binds within the periplasmic cleft of an RND transporter protein (MtrD) produced by Neisseria gonorrhoeae. Surprisingly, CASP was able to render rod-shaped Gram-negative bacteria, but not gonococci, susceptible to an antibiotic that is a substrate for the gonococcal MtrCDE efflux pump. CASP (or its future derivatives) could be used as an adjuvant to treat infections for which RND efflux contributes to multidrug resistance.

Gonococcal Clinical Strains Bearing a Common gdhR Single Nucleotide Polymorphism That Results in Enhanced Expression of the Virulence Gene lctP Frequently Possess a mtrR Promoter Mutation That Decreases Antibiotic Susceptibility.

GdhR is a transcriptional repressor of the virulence factor gene lctP, which encodes a unique l-lactate permease that has been linked to pathogenesis of Neisseria gonorrhoeae, and loss of gdhR can confer increased fitness of gonococci in a female mouse model of lower genital tract infection. In this work, we identified a single nucleotide polymorphism (SNP) in gdhR, which is often present in both recent and historical gonococcal clinical strains and results in a proline (P)-to-serine (S) change at amino acid position 6 (P6S) of GdhR. This mutation (gdhR6) was found to reduce GdhR transcriptional repression at lctP in gonococcal strains containing the mutant protein compared to wild-type GdhR. By using purified recombinant proteins and in vitro DNA-binding and cross-linking experiments, we found that gdhR6 impairs the DNA-binding activity of GdhR at lctP without an apparent effect on protein oligomerization. By analyzing a panel of U.S. (from 2017 to 2018) and Danish (1928 to 2013) clinical isolates, we observed a statistical association between gdhR6 and the previously described adenine deletion in the promoter of mtrR (mtrR-P A-del), encoding the repressor (MtrR) of the mtrCDE operon that encodes the MtrCDE multidrug efflux pump that can export antibiotics, host antimicrobials, and biocides. The frequent association of gdhR6 with the mtrR promoter mutation in these clinical isolates suggests that it has persisted in this genetic background to enhance lctP expression, thereby promoting virulence. IMPORTANCE We report the frequent appearance of a novel SNP in the gdhR gene (gdhR6) possessed by Neisseria gonorrhoeae. The resulting amino acid change in the GdhR protein resulted in enhanced expression of a virulence gene (lctP) that has been suggested to promote gonococcal survival during infection. The mutant GdhR protein expressed by gdhR6 had a reduced ability to bind to its target DNA sequence upstream of lctP. Interestingly, gdhR6 was found in clinical gonococcal strains isolated in the United States and Denmark at a high frequency and was frequently associated with a mutation in the promoter of the gene encoding a repressor (MtrR) of both the mtrCDE antimicrobial efflux pump operon and gdhR. Given this frequent association and the known impact of these regulatory mutations, we propose that virulence and antibiotic resistance properties are often phenotypically linked in contemporary gonococcal strains.

Potent In Vitro and Ex Vivo Anti-Gonococcal Activity of the RpoB Inhibitor Corallopyronin A.

Gonorrhea remains a major global public health problem because of the high incidence of infection (estimated 82 million cases in 2020) and the emergence and spread of Neisseria gonorrhoeae strains resistant to previous and current antibiotics used to treat infections. Given the dearth of new antibiotics that are likely to enter clinical practice in the near future, there is concern that cases of untreatable gonorrhea might emerge. In response to this crisis, the World Health Organization (WHO), in partnership with the Global Antibiotic Research and Development Partnership (GARDP), has made the search for and development of new antibiotics against N. gonorrhoeae a priority. Ideally, these antibiotics should also be active against other sexually transmitted organisms, such as Chlamydia trachomatis and/or Mycoplasma genitalium, which are often found with N. gonorrhoeae as co-infections. Corallopyronin A is a potent antimicrobial that exhibits activity against Chlamydia spp. and inhibits transcription by binding to the RpoB switch region. Accordingly, we tested the effectiveness of corallopyronin A against N. gonorrhoeae. We also examined the mutation frequency and modes of potential resistance against corallopyronin A. We report that corallopyronin A has potent antimicrobial action against antibiotic-susceptible and antibiotic-resistant N. gonorrhoeae strains and could eradicate gonococcal infection of cultured, primary human cervical epithelial cells. Critically, we found that spontaneous corallopyronin A-resistant mutants of N. gonorrhoeae are exceedingly rare (≤10-10) when selected at 4× the MIC. Our results support pre-clinical studies aimed at developing corallopyronin A for gonorrheal treatment regimens. IMPORTANCE The high global incidence of gonorrhea, the lack of a protective vaccine, and the emergence of N. gonorrhoeae strains expressing resistance to currently used antibiotics demand that new treatment options be developed. Accordingly, we investigated whether corallopyronin A, an antibiotic which is effective against other pathogens, including C. trachomatis, which together with gonococci frequently cause co-infections in humans, could exert anti-gonococcal action in vitro and ex vivo, and potential resistance emergence. We propose that corallopyronin A be considered a potential future treatment option for gonorrhea because of its potent activity, low resistance development, and recent advances in scalable production.

Interplay among cyclic diguanylate, HapR, and the general stress response regulator (RpoS) in the regulation of Vibrio cholerae hemagglutinin/protease.

Vibrio cholerae secretes the Zn-dependent metalloprotease hemagglutinin (HA)/protease (mucinase), which is encoded by hapA and displays a broad range of potential pathogenic activities. Expression of HA/protease has a stringent requirement for the quorum-sensing regulator HapR and the general stress response regulator RpoS. Here we report that the second messenger cyclic diguanylic acid (c-di-GMP) regulates the production of HA/protease in a negative manner. Overexpression of a diguanylate cyclase to increase the cellular c-di-GMP pool resulted in diminished expression of HA/protease and its positive regulator, HapR. The effect of c-di-GMP on HapR was independent of LuxO but was abolished by deletion of the c-di-GMP binding protein VpsT, the LuxR-type regulator VqmA, or a single-base mutation in the hapR promoter that prevents autorepression. Though expression of HapR had a positive effect on RpoS biosynthesis, direct manipulation of the c-di-GMP pool at a high cell density did not significantly impact RpoS expression in the wild-type genetic background. In contrast, increasing the c-di-GMP pool severely inhibited RpoS expression in a ΔhapR mutant that is locked in a regulatory state mimicking low cell density. Based on the above findings, we propose a model for the interplay between HapR, RpoS, and c-di-GMP in the regulation of HA/protease expression.

A high-throughput screening assay for inhibitors of bacterial motility identifies a novel inhibitor of the Na+-driven flagellar motor and virulence gene expression in Vibrio cholerae.

Numerous bacterial pathogens, particularly those that colonize fast-flow areas in the bladder and gastrointestinal tract, require motility to establish infection and spread beyond the initially colonized tissue. Vibrio cholerae strains of serogroups O1 and O139, the causative agents of the diarrheal illness cholera, express a single polar flagellum powered by sodium motive force and require motility to colonize and spread along the small intestine. Therefore, motility may be an attractive target for small molecules that can prevent and/or block the infective process. In this study, we describe a high-throughput screening (HTS) assay to identify small molecules that selectively inhibit bacterial motility. The HTS assay was used to screen an ∼8,000-compound structurally diverse chemical library for inhibitors of V. cholerae motility. The screen identified a group of quinazoline-2,4-diamino analogs that completely suppressed motility without affecting the growth rate in broth. A further study on the effects of one analog, designated Q24DA, showed that it induces a flagellated but nonmotile (Mot(-)) phenotype and is specific for the Na(+)-driven flagellar motor of pathogenic Vibrio species. A mutation conferring phenamil-resistant motility did not eliminate inhibition of motility by Q24DA. Q24DA diminished the expression of cholera toxin and toxin-coregulated pilus as well as biofilm formation and fluid secretion in the rabbit ileal loop model. Furthermore, treatment of V. cholerae with Q24DA impacted additional phenotypes linked to Na(+) bioenergetics, such as the function of the primary Na(+) pump, Nqr, and susceptibility to fluoroquinolones. The above results clearly show that the described HTS assay is capable of identifying small molecules that specifically block bacterial motility. New inhibitors such as Q24DA may be instrumental in probing the molecular architecture of the Na(+)-driven polar flagellar motor and in studying the role of motility in the expression of other virulence factors.

Transcriptional control of the gonococcal ompA gene by the MisR/MisS two-component regulatory system.

Neisseria gonorrhoeae, the causative agent of gonorrhea, is an exclusive human pathogen whose growing antibiotic resistance is causing worldwide concern. The increasing rise of antibiotic resistance expressed by gonococci highlights the need to find alternative approaches to current gonorrhea treatment such as vaccine development or novel therapeutics. The gonococcal OmpA protein was previously identified as a potential vaccine candidate due to its conservation and stable expression amongst strains of Neisseria gonorrhoeae. However, factors that might modulate levels of OmpA and therefore potential vaccine efficacy are unknown. Earlier work indicated that ompA is part of the MisR/MisS regulon and suggested that it was a MisR-activated gene. Herein, we confirmed MisR/MisS regulation of ompA and report that the MisR response regulator can bind upstream of the ompA translational start codon. Further, we describe the contribution of a DNA sequence upstream of the ompA promoter that is critical for MisR activation of ompA transcription. Our results provide a framework for understanding the transcription of gonococcal ompA through a regulatory system known to be important for survival of gonococci during experimental infection.

A proteome-wide screen to identify transcription factors interacting with the Vibrio cholerae rpoS promoter.

We describe a proteomic approach to identify transcription factors binding to a target promoter. The method's usefulness was tested by identifying proteins binding to the Vibrio cholerae rpoS promoter in response to cell density. Proteins identified in this screen included the nucleoid-associated protein Fis and the quorum sensing regulator HapR.

Chromatin Immunoprecipitation.

Chromatin immunoprecipitation (ChIP) measures the physical association between a protein and DNA in the cell. In combination with next-generation sequencing, the technique enables the identification of DNA targets for the corresponding protein across an entire genome. Here we describe the immunoprecipitation of Vibrio cholerae DNA bound to the histone-like nucleoid structuring protein (H-NS) tagged with the Flag epitope. The quality of the DNA obtained in this protocol is suitable for next-generation sequencing. The procedure described herein can be readily adapted to other bacteria and DNA-binding proteins.