A laboratory-based predictive pathway for the development of Neisseria gonorrhoeae high-level resistance to corallopyronin A, an inhibitor of bacterial RNA polymerase.

The continued emergence of Neisseria gonorrhoeae strains that express resistance to multiple antibiotics, including the last drug for empiric monotherapy (ceftriaxone), necessitates the development of new treatment options to cure gonorrheal infections. Toward this goal, we recently reported that corallopyronin A (CorA), which targets the switch region of the ß' subunit (RpoC) of bacterial DNA-dependent RNA polymerase (RNAP), has potent anti-gonococcal activity against a panel of multidrug-resistant clinical strains. Moreover, in that study, CorA could eliminate gonococcal infection of primary human epithelial cells and gonococci in a biofilm state. To determine if N. gonorrhoeae could develop high-level resistance to CorA in a single step, we sought to isolate spontaneous mutants expressing any CorA resistance phenotypes. However, no single-step mutants with high-level CorA resistance were isolated. High-level CorA resistance could only be achieved in this study through a multi-step pathway involving over-expression of the MtrCDE drug efflux pump and single amino acid changes in the ß and ß' subunits (RpoB and RpoC, respectively) of RNAP. Molecular modeling of RpoB and RpoC interacting with CorA was used to deduce how the amino acid changes in RpoB and RpoC could influence gonococcal resistance to CorA. Bioinformatic analyses of whole genome sequences of clinical gonococcal isolates indicated that the CorA resistance determining mutations in RpoB/C, identified herein, are very rare (≤ 0.0029%), suggesting that the proposed pathway for resistance is predictive of how this phenotype could potentially evolve if CorA is used therapeutically to treat gonorrhea in the future. IMPORTANCE: The continued emergence of multi-antibiotic-resistant strains of Neisseria gonorrhoeae necessitates the development of new antibiotics that are effective against this human pathogen. We previously described that the RNA polymerase-targeting antibiotic corallopyronin A (CorA) has potent activity against a large collection of clinical strains that express different antibiotic resistance phenotypes including when such gonococci are in a biofilm state. Herein, we tested whether a CorA-sensitive gonococcal strain could develop spontaneous resistance. Our finding that CorA resistance could only be achieved by a multi-step process involving over-expression of the MtrCDE efflux pump and single amino acid changes in RpoB and RpoC suggests that such resistance may be difficult for gonococci to evolve if this antibiotic is used in the future to treat gonorrheal infections that are refractory to cure by other antibiotics.

Neisseria gonorrhoeae MtrCDE Efflux Pump During In Vivo Experimental Genital Tract Infection in Men and Mice Reveals the Presence of Within-Host Colonization Bottleneck.

The MtrCDE efflux pump of Neisseria gonorrhoeae exports a wide range of antimicrobial compounds that the gonococcus encounters at mucosal surfaces during colonization and infection. Here, we evaluate the role of this efflux pump system in strain FA1090 in human male urethral infection with a Controlled Human Infection Model. Using the strategy of competitive multi-strain infection with wild-type FA1090 and an isogenic mutant strain that does not contain a functional MtrCDE pump, we found that the presence of the efflux pump during human experimental infection did not confer a competitive advantage. This finding is in contrast to previous findings in female mice, which demonstrated that gonococci of strain FA19 lacking a functional MtrCDE pump had a significantly reduced fitness compared to the wild type strain in the lower genital tract of female mice. We conducted competitive infections in female mice with FA19 and FA1090 strains, including mutants that do not assemble a functional Mtr efflux pump, demonstrating the fitness advantage provided byt the MtrCDE efflux pump during infection of mice is strain dependent. Our data indicate that new gonorrhea treatment strategies targeting the MtrCDE efflux pump functions may not be universally efficacious in naturally occurring infections. Owing to the equal fitness of FA1090 strains in men, our experiments unexpectedly demonstrated the likely presence of an early colonization bottleneck of N. gonorrhoeae in the human male urethra. TRIAL REGISTRATION: Clinicaltrials.gov NCT03840811 .

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.

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.

A Single Amino Acid Substitution in Elongation Factor G Can Confer Low-Level Gentamicin Resistance in Neisseria gonorrhoeae.

The continued emergence of Neisseria gonorrhoeae isolates which are resistant to first-line antibiotics has reinvigorated interest in alternative therapies such as expanded use of gentamicin (Gen). We hypothesized that expanded use of Gen promotes emergence of gonococci with clinical resistance to this aminoglycoside. To understand how decreased susceptibility of gonococci to Gen might develop, we selected spontaneous low-level Gen-resistant (GenR) mutants (Gen MIC = 32 µg/mL) of the Gen-susceptible strain FA19. Consequently, we identified a novel missense mutation in fusA, which encodes elongation factor G (EF-G), causing an alanine (A) to valine (V) substitution at amino acid position 563 in domain IV of EF-G; the mutant allele was termed fusA2. Transformation analysis showed that fusA2 could increase the Gen MIC by 4-fold. While possession of fusA2 did not impair either in vitro gonococcal growth or protein synthesis, it did result in a fitness defect during experimental infection of the lower genital tract in female mice. Through bioinformatic analysis of whole-genome sequences of 10,634 international gonococcal clinical isolates, other fusA alleles were frequently detected, but genetic studies revealed that they could not decrease Gen susceptibility in a similar manner to fusA2. In contrast to these diverse international fusA alleles, the fusA2-encoded A563V substitution was detected in only a single gonococcal clinical isolate. We hypothesize that the rare occurrence of fusA2 in N. gonorrhoeae clinical isolates is likely due to a fitness cost during infection, but compensatory mutations which alleviate this fitness cost could emerge and promote GenR in global strains.

The serogroup B meningococcal outer membrane vesicle-based vaccine 4CMenB induces cross-species protection against Neisseria gonorrhoeae.

There is a pressing need for a gonorrhea vaccine due to the high disease burden associated with gonococcal infections globally and the rapid evolution of antibiotic resistance in Neisseria gonorrhoeae (Ng). Current gonorrhea vaccine research is in the stages of antigen discovery and the identification of protective immune responses, and no vaccine has been tested in clinical trials in over 30 years. Recently, however, it was reported in a retrospective case-control study that vaccination of humans with a serogroup B Neisseria meningitidis (Nm) outer membrane vesicle (OMV) vaccine (MeNZB) was associated with reduced rates of gonorrhea. Here we directly tested the hypothesis that Nm OMVs induce cross-protection against gonorrhea in a well-characterized female mouse model of Ng genital tract infection. We found that immunization with the licensed Nm OMV-based vaccine 4CMenB (Bexsero) significantly accelerated clearance and reduced the Ng bacterial burden compared to administration of alum or PBS. Serum IgG and vaginal IgA and IgG that cross-reacted with Ng OMVs were induced by 4CMenB vaccination by either the subcutaneous or intraperitoneal routes. Antibodies from vaccinated mice recognized several Ng surface proteins, including PilQ, BamA, MtrE, NHBA (known to be recognized by humans), PorB, and Opa. Immune sera from both mice and humans recognized Ng PilQ and several proteins of similar apparent molecular weight, but MtrE was only recognized by mouse serum. Pooled sera from 4CMenB-immunized mice showed a 4-fold increase in serum bactericidal50 titers against the challenge strain; in contrast, no significant difference in bactericidal activity was detected when sera from 4CMenB-immunized and unimmunized subjects were compared. Our findings directly support epidemiological evidence that Nm OMVs confer cross-species protection against gonorrhea, and implicate several Ng surface antigens as potentially protective targets. Additionally, this study further defines the usefulness of murine infection model as a relevant experimental system for gonorrhea vaccine development.

Mechanistic Basis for Decreased Antimicrobial Susceptibility in a Clinical Isolate of Neisseria gonorrhoeae Possessing a Mosaic-Like mtr Efflux Pump Locus.

Recent reports suggest that mosaic-like sequences within the mtr (multiple transferable resistance) efflux pump locus of Neisseria gonorrhoeae, likely originating from commensal Neisseria sp. by transformation, can increase the ability of gonococci to resist structurally diverse antimicrobials. Thus, acquisition of numerous nucleotide changes within the mtrR gene encoding the transcriptional repressor (MtrR) of the mtrCDE efflux pump-encoding operon or overlapping promoter region for both along with those that cause amino acid changes in the MtrD transporter protein were recently reported to decrease gonococcal susceptibility to numerous antimicrobials, including azithromycin (Azi) (C. B. Wadsworth, B. J. Arnold, M. R. A. Satar, and Y. H. Grad, mBio 9:e01419-18, 2018, https://doi.org/10.1128/mBio.01419-18). We performed detailed genetic and molecular studies to define the mechanistic basis for why such strains can exhibit decreased susceptibility to MtrCDE antimicrobial substrates, including Azi. We report that a strong cis-acting transcriptional impact of a single nucleotide change within the -35 hexamer of the mtrCDE promoter as well gain-of-function amino acid changes at the C-terminal region of MtrD can mechanistically account for the decreased antimicrobial susceptibility of gonococci with a mosaic-like mtr locus.IMPORTANCE Historically, after introduction of an antibiotic for treatment of gonorrhea, strains of N. gonorrhoeae emerge that display clinical resistance due to spontaneous mutation or acquisition of resistance genes. Genetic exchange between members of the Neisseria genus occurring by transformation can cause significant changes in gonococci that impact the structure of an antibiotic target or expression of genes involved in resistance. The results presented here provide a framework for understanding how mosaic-like DNA sequences from commensal Neisseria that recombine within the gonococcal mtr efflux pump locus function to decrease bacterial susceptibility to antimicrobials, including antibiotics used in therapy of gonorrhea.

Control of gdhR Expression in Neisseria gonorrhoeae via Autoregulation and a Master Repressor (MtrR) of a Drug Efflux Pump Operon.

The MtrCDE efflux pump of Neisseria gonorrhoeae contributes to gonococcal resistance to a number of antibiotics used previously or currently in treatment of gonorrhea, as well as to host-derived antimicrobials that participate in innate defense. Overexpression of the MtrCDE efflux pump increases gonococcal survival and fitness during experimental lower genital tract infection of female mice. Transcription of mtrCDE can be repressed by the DNA-binding protein MtrR, which also acts as a global regulator of genes involved in important metabolic, physiologic, or regulatory processes. Here, we investigated whether a gene downstream of mtrCDE, previously annotated gdhR in Neisseria meningitidis, is a target for regulation by MtrR. In meningococci, GdhR serves as a regulator of genes involved in glucose catabolism, amino acid transport, and biosynthesis, including gdhA, which encodes an l-glutamate dehydrogenase and is located next to gdhR but is transcriptionally divergent. We report here that in N. gonorrhoeae, expression of gdhR is subject to autoregulation by GdhR and direct repression by MtrR. Importantly, loss of GdhR significantly increased gonococcal fitness compared to a complemented mutant strain during experimental murine infection. Interestingly, loss of GdhR did not influence expression of gdhA, as reported for meningococci. This variance is most likely due to differences in promoter localization and utilization between gonococci and meningococci. We propose that transcriptional control of gonococcal genes through the action of MtrR and GdhR contributes to fitness of N. gonorrhoeae during infection.IMPORTANCE The pathogenic Neisseria species are strict human pathogens that can cause a sexually transmitted infection (N. gonorrhoeae) or meningitis or fulminant septicemia (N. meningitidis). Although they share considerable genetic information, little attention has been directed to comparing transcriptional regulatory systems that modulate expression of their conserved genes. We hypothesized that transcriptional regulatory differences exist between these two pathogens, and we used the gdh locus as a model to test this idea. For this purpose, we studied two conserved genes (gdhR and gdhA) within the locus. Despite general conservation of the gdh locus in gonococci and meningococci, differences exist in noncoding sequences that correspond to promoter elements or potential sites for interacting with DNA-binding proteins, such as GdhR and MtrR. Our results indicate that implications drawn from studying regulation of conserved genes in one pathogen are not necessarily translatable to a genetically related pathogen.

The MisR Response Regulator Is Necessary for Intrinsic Cationic Antimicrobial Peptide and Aminoglycoside Resistance in Neisseria gonorrhoeae.

During infection, the sexually transmitted pathogen Neisseria gonorrhoeae (the gonococcus) encounters numerous host-derived antimicrobials, including cationic antimicrobial peptides (CAMPs) produced by epithelial and phagocytic cells. CAMPs have both direct and indirect killing mechanisms and help link the innate and adaptive immune responses during infection. Gonococcal CAMP resistance is likely important for avoidance of host nonoxidative killing systems expressed by polymorphonuclear granulocytes (e.g., neutrophils) and intracellular survival. Previously studied gonococcal CAMP resistance mechanisms include modification of lipid A with phosphoethanolamine by LptA and export of CAMPs by the MtrCDE efflux pump. In the related pathogen Neisseria meningitidis, a two-component regulatory system (2CRS) termed MisR-MisS has been shown to contribute to the capacity of the meningococcus to resist CAMP killing. We report that the gonococcal MisR response regulator but not the MisS sensor kinase is involved in constitutive and inducible CAMP resistance and is also required for intrinsic low-level resistance to aminoglycosides. The 4- to 8-fold increased susceptibility of misR-deficient gonococci to CAMPs and aminoglycosides was independent of phosphoethanolamine decoration of lipid A and the levels of the MtrCDE efflux pump and seemed to correlate with a general increase in membrane permeability. Transcriptional profiling and biochemical studies confirmed that expression of lptA and mtrCDE was not impacted by the loss of MisR. However, several genes encoding proteins involved in membrane integrity and redox control gave evidence of being MisR regulated. We propose that MisR modulates the levels of gonococcal susceptibility to antimicrobials by influencing the expression of genes involved in determining membrane integrity.

Phosphoethanolamine Modification of Neisseria gonorrhoeae Lipid A Reduces Autophagy Flux in Macrophages.

Autophagy, an ancient homeostasis mechanism for macromolecule degradation, performs an important role in host defense by facilitating pathogen elimination. To counteract this host defense strategy, bacterial pathogens have evolved a variety of mechanisms to avoid or otherwise dysregulate autophagy by phagocytic cells so as to enhance their survival during infection. Neisseria gonorrhoeae is a strictly human pathogen that causes the sexually transmitted infection, gonorrhea. Phosphoethanolamine (PEA) addition to the 4' position of the lipid A (PEA-lipid A) moiety of the lipooligosaccharide (LOS) produced by gonococci performs a critical role in this pathogen's ability to evade innate defenses by conferring decreased susceptibility to cationic antimicrobial (or host-defense) peptides, complement-mediated killing by human serum and intraleukocytic killing by human neutrophils compared to strains lacking this PEA decoration. Heretofore, however, it was not known if gonococci can evade autophagy and if so, whether PEA-lipid A contributes to this ability. Accordingly, by using murine macrophages and human macrophage-like phagocytic cell lines we investigated if PEA decoration of gonococcal lipid A modulates autophagy formation. We report that infection with PEA-lipid A-producing gonococci significantly reduced autophagy flux in murine and human macrophages and enhanced gonococcal survival during their association with macrophages compared to a PEA-deficient lipid A mutant. Our results provide further evidence that PEA-lipid A produced by gonococci is a critical component in the ability of this human pathogen to evade host defenses.

Phase-variable expression of lptA modulates the resistance of Neisseria gonorrhoeae to cationic antimicrobial peptides.

Phosphoethanolamine (PEA) decoration of lipid A produced by Neisseria gonorrhoeae has been linked to bacterial resistance to cationic antimicrobial peptides/proteins (CAMPs) and in vivo fitness during experimental infection. We now report that the lptA gene, which encodes the PEA transferase responsible for this decoration, is in an operon and that high-frequency mutation in a polynucleotide repeat within lptA can influence gonococcal resistance to CAMPs.

Lipid A's structure mediates Neisseria gonorrhoeae fitness during experimental infection of mice and men.

UNLABELLED: Phosphoethanolamine (PEA) on Neisseria gonorrhoeae lipid A influences gonococcal inflammatory signaling and susceptibility to innate host defenses in in vitro models. Here, we evaluated the role of PEA-decorated gonococcal lipid A in competitive infections in female mice and in male volunteers. We inoculated mice and men with mixtures of wild-type N. gonorrhoeae and an isogenic mutant that lacks the PEA transferase, LptA. LptA production conferred a marked survival advantage for wild-type gonococci in the murine female genital tract and in the human male urethra. Our studies translate results from test tube to animal model and into the human host and demonstrate the utility of the mouse model for studies of virulence factors of the human-specific pathogen N. gonorrhoeae that interact with non-host-restricted elements of innate immunity. These results validate the use of gonococcal LptA as a potential target for development of novel immunoprophylactic strategies or antimicrobial treatments. IMPORTANCE: Gonorrhea is one of the most common bacterial sexually transmitted infections, and increasing antibiotic resistance threatens the use of currently available antimicrobial therapies. In this work, encompassing in vitro studies and in vivo studies of animal and human models of experimental genital tract infection, we document the importance of lipid A's structure, mediated by a single bacterial enzyme, LptA, in enhancing the fitness of Neisseria gonorrhoeae. The results of these studies suggest that novel agents targeting LptA may offer urgently needed prevention or treatment strategies for gonorrhea.

Lipooligosaccharide Structure is an Important Determinant in the Resistance of Neisseria Gonorrhoeae to Antimicrobial Agents of Innate Host Defense.

The strict human pathogen Neisseria gonorrhoeae has caused the sexually transmitted infection termed gonorrhea for thousands of years. Over the millennia, the gonococcus has likely evolved mechanisms to evade host defense systems that operate on the genital mucosal surfaces in both males and females. Past research has shown that the presence or modification of certain cell envelope structures can significantly impact levels of gonococcal susceptibility to host-derived antimicrobial compounds that bathe genital mucosal surfaces and participate in innate host defense against invading pathogens. In order to facilitate the identification of gonococcal genes that are important in determining levels of bacterial susceptibility to mediators of innate host defense, we used the Himar I mariner in vitro mutagenesis system to construct a transposon insertion library in strain F62. As proof of principle that this strategy would be suitable for this purpose, we screened the library for mutants expressing decreased susceptibility to the bacteriolytic action of normal human serum (NHS). We found that a transposon insertion in the lgtD gene, which encodes an N-acetylgalactosamine transferase involved in the extension of the α-chain of lipooligosaccharide (LOS), could confer decreased susceptibility of strain F62 to complement-mediated killing by NHS. By complementation and chemical analyses, we demonstrated both linkage of the transposon insertion to the NHS-resistance phenotype and chemical changes in LOS structure that resulted from loss of LgtD production. Further truncation of the LOS α-chain or loss of phosphoethanolamine (PEA) from the lipid A region of LOS also impacted levels of NHS-resistance. PEA decoration of lipid A also increased gonococcal resistance to the model cationic antimicrobial polymyxin B. Taken together, we conclude that the Himar I mariner in vitro mutagenesis procedure can facilitate studies on structures involved in gonococcal pathogenesis.

Phosphoethanolamine substitution of lipid A and resistance of Neisseria gonorrhoeae to cationic antimicrobial peptides and complement-mediated killing by normal human serum.

The capacity of Neisseria gonorrhoeae to cause disseminated gonococcal infection requires that such strains resist the bactericidal action of normal human serum. The bactericidal action of normal human serum against N. gonorrhoeae is mediated by the classical complement pathway through an antibody-dependent mechanism. The mechanism(s) by which certain strains of gonococci resist normal human serum is not fully understood, but alterations in lipooligosaccharide structure can affect such resistance. During an investigation of the biological significance of phosphoethanolamine extensions from lipooligosaccharide, we found that phosphoethanolamine substitutions from the heptose II group of the lipooligosaccharide beta-chain did not impact levels of gonococcal (strain FA19) resistance to normal human serum or polymyxin B. However, loss of phosphoethanolamine substitution from the lipid A component of lipooligosaccharide, due to insertional inactivation of lptA, resulted in increased gonococcal susceptibility to polymyxin B, as reported previously for Neisseria meningitidis. In contrast to previous reports with N. meningitidis, loss of phosphoethanolamine attached to lipid A rendered strain FA19 susceptible to complement killing. Serum killing of the lptA mutant occurred through the classical complement pathway. Both serum and polymyxin B resistance as well as phosphoethanolamine decoration of lipid A were restored in the lptA-null mutant by complementation with wild-type lptA. Our results support a role for lipid A phosphoethanolamine substitutions in resistance of this strict human pathogen to innate host defenses.

Characterization of the MacA-MacB efflux system in Neisseria gonorrhoeae.

OBJECTIVES: A homologue of the MacA-MacB ABC transporter of Escherichia coli, which recognizes and exports macrolides, was identified in Neisseria gonorrhoeae. This study was undertaken to determine whether gonococci could use the MacA-MacB homologue to express decreased susceptibility to macrolides. METHODS: Techniques of DNA sequencing, gene cloning and expression of recombinant proteins in E. coli, gene mutation construction, transcriptional analysis and antimicrobial susceptibility testing were used in the study. RESULTS: Although the gonococcal MacA-MacB efflux pump enhanced bacterial resistance to macrolides when overexpressed in an E. coli background, its loss in a gonococcal clinical isolate only slightly decreased bacterial resistance to azithromycin and erythromycin. However, a mutation in the -10 sequence of the promoter used in macAB expression enhanced the macrolide resistance of gonococci that produced a defective MtrC-MtrD-MtrE pump, which also recognizes macrolides. CONCLUSIONS: The results from this study indicate that gonococci can employ both the MacA-MacB and MtrC-MtrD-MtrE efflux pumps to develop resistance to macrolides, particularly if mutations develop in the promoter that drives transcription of macAB.

Modulation of the mtrCDE-encoded efflux pump gene complex of Neisseria meningitidis due to a Correia element insertion sequence.

The mtr (multiple transferable resistance) gene complex in Neisseria gonorrhoeae encodes an energy-dependent efflux pump system that is responsible for export of anti-bacterial hydrophobic agents. Expression of the mtrCDE operon in gonococci is negatively regulated by the MtrR protein. Hydrophobic agent resistance mediated by the mtr system is also inducible, which results from an AraC-like protein termed MtrA. In this work, we identified and characterized a pump similar to the gonococcal mtr system in various strains of Neisseria meningitidis. Unlike the situation with gonococci, the mtr system in meningococci is not subject to the MtrR or MtrA regulatory schemes. An analysis of the promoter region of the mtrCDE operon in a panel of meningococcal strains revealed the presence of one or two classes of insertion sequence elements. A 155-159 bp insertion sequence element known as the Correia element, previously identified elsewhere in the gonococcal and meningococcal genomes, was present in the mtrCDE promoter region of all meningococcal strains tested. In addition to the Correia element, a minority of strains had a tandemly linked, intact copy of IS1301. As described previously, a binding site for the integration host factor (IHF) was present at the centre of the Correia element upstream of mtrCDE genes. IHF was found to bind specifically to this site and deletion of the IHF binding site enhanced mtrC transcription. We also identified a post-transcriptional regulation of the mtrCDE transcript by cleavage in the inverted repeat of the Correia element, as previously described by Mazzone et al. [Gene278: 211-222 (2001)] and De Gregorio et al. [Biochim Biophys Acta 1576: 39-44 (2002)]for other Correia element. We conclude that the mtr efflux system in meningococci is subject to transcriptional regulation by IHF and post-transcriptional regulation by cleavage in the inverted repeat of the Correia element.

The major cold shock gene, cspA, is involved in the susceptibility of Staphylococcus aureus to an antimicrobial peptide of human cathepsin G.

A Tn551 insertional library of Staphylococcus aureus strain ISP479 was challenged with an antimicrobial peptide (CG 117-136) derived from human neutrophil cathepsin G (CG). After repeated selection and screening of surviving colonies, a mutant was identified that expressed increased resistance to CG 117-136. Southern hybridization analysis revealed that the Tn551 insert in this mutant (SK1) was carried on a 10.6-kb EcoRI chromosomal DNA fragment. Subsequent physical mapping of this Tn551 insert revealed that it was positioned between a putative promoter sequence and the translational start codon of the cspA gene, which encodes a protein (CspA) highly similar to the major cold shock proteins CspA and CspB of Escherichia coli and Bacillus subtilis, respectively. This Tn551 insertion as well as a separate deletion-insertion mutation in cspA decreased the capacity of S. aureus to respond to the stress of cold shock and increased resistance to CG 117-136. The results indicate for the first time that a physiologic link exists between bacterial susceptibility to an antimicrobial peptide and a stress response system.

The NorM efflux pump of Neisseria gonorrhoeae and Neisseria meningitidis recognizes antimicrobial cationic compounds.

In Neisseria gonorrhoeae and Neisseria meningitidis, we identified a gene that would encode a protein highly similar to NorM of Vibrio parahaemolyticus (Y. Morita et al., Antimicrob. Agents Chemother. 42:1778-1782, 1998). A nonpolar insertional mutation in either the gonococcal or meningococcal norM gene resulted in increased bacterial sensitivity to compounds harboring a quaternary ammonium on an aromatic ring (e.g., ethidium bromide, acriflavine hydrochloride, 2-N-methylellipticinium, and berberine). The presence of point mutations within the -35 region of a putative norM promoter or a likely ribosome binding site resulted in an increased resistance of gonococci and meningococci to the same compounds, as well as to norfloxacin and ciprofloxacin. Structure-activity relationship studies with putative NorM substrates have found that a cationic moiety is essential for NorM recognition.

Phase variable changes in genes lgtA and lgtC within the lgtABCDE operon of Neisseria gonorrhoeae can modulate gonococcal susceptibility to normal human serum.

The alpha-chain of the core oligosaccharide of the lipo-oligosaccharide (LOS) produced by Neisseria gonorrhoeae can undergo reversible and rapid changes in structure due to phase-variable production of certain enzymes employed in the biosynthesis of the lacto-N-neotetraose structure. Five of these enzymes are encoded by the lgtABCDE operon, and polynucleotide tracts within three of these genes (lgtA, lgtC and lgtD) can be substrates for slipped-strand mispairing events that lead to nucleotide insertions or deletion events which result in variable production of their respective gene products. We now report that phase-variable synthesis of the lgtA and lgtC gene products in strain FA19 results in the production of elongated LOS alpha-chains and that the presence of these LOS species can result in gonococci being sensitive to the bacteriolytic action of serum-antibody and complement. Hence, phase variation within the lgtABCDE operon can significantly impact the ability of gonococci to subvert this important host defense system.

Inducible, but not constitutive, resistance of gonococci to hydrophobic agents due to the MtrC-MtrD-MtrE efflux pump requires TonB-ExbB-ExbD proteins.

The MtrC-MtrD-MtrE efflux pump possessed by Neisseria gonorrhoeae is very similar to the MexA-MexB-OprM efflux pump of Pseudomonas aeruginosa. Because the antimicrobial resistance property afforded by the MexA-MexB-OprM efflux pump also requires the TonB protein, we asked whether a similar requirement exists for the gonococcal efflux pump. Unlike earlier studies with P. aeruginosa, we found that constitutive levels of gonococcal resistance to hydrophobic antimicrobial agents (i.e., Triton X-100 [TX-100]) did not require the TonB, ExbB, or ExbD protein. However, inducible levels of TX-100 resistance in gonococci had an absolute requirement for the TonB-ExbB-ExbD system, suggesting that such resistance in gonococci has an energy requirement above and beyond that required for constitutive pump activity.