Jacqueline T. Balthazar

Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents is modulated by the mtrRCDE efflux system

The mtr (multiple transferable resistance) system of Neisseria gonorrhoeae determines levels of gonococcal resistance to hydrophobic agents (HAs), including detergent-like fatty acids and bile salts that bathe certain mucosal surfaces. The genetic organization of the mtr system was determined and found to consist of the mtrR gene, which encodes a transcriptional regulator (MtrR), and three tandemly linked genes termed mtrCDE. The mtrCDE genes were organized in the same apparent transcriptional unit, upstream and divergent from the mtrR gene. The mtrCDE-encoded proteins of N. gonorrhoeae were analogous to a family of bacterial efflux/transport proteins, notably the MexABOprK proteins of Pseudomonas aeruginosa and the AcrAE and EnvCD proteins of Escherichia coli, that mediate resistance to drugs, dyes, and detergents. Inactivation of the mtrC gene resulted in loss of the MtrC lipoprotein and rendered gonococci hypersusceptible to structurally diverse HAs; this revealed the importance of the mtr system in determining HAR in gonococci. Further support for a role of the mtrCDE gene complex in determining levels of HAR in gonococci was evident when transformants bearing mutations in the mtrR gene were analysed. In this respect, missense and null mutations in the mtrR gene were found to result in increased levels of MtrC and HAR. However, high levels of MtrC and HAR, similar to those observed for clinical isolates, were associated with a single bp deletion in a 13 bp inverted repeat sequence that intervened the divergent mtrR and mtrC genes. We propose that the 13 bp inverted-repeat sequence represents a transcriptional control element that regulates expression of the mtrRCDE gene complex, thereby modulating levels of gonococcal susceptibility to HAs.

Missense mutations that alter the DNA-binding domain of the MtrR protein occur frequently in rectal isolates of Neisseria gonorrhoeae that are resistant to faecal lipids.

Resistance of Neisseria gonorrhoeae to structurally diverse hydrophobic agents (HAs) has been associated with missense or deletion mutations in the mtrR (multiple transferable resistance Regulator) gene of laboratory-derived strains but their prevalence in clinical isolates was heretofore unknown. Since faecal lipids provide strong selective pressure for the emergence of variants resistant to HAs (HAR), the nucleotide sequence of the mtrR gene from rectal isolates of N. gonorrhoeae, which displayed different levels of HAR, was determined. Compared to the mtrR gene possessed by the HA-sensitive strain FA19, each clinical isolate contained mutations in the coding and/or promoter regions of their mtrR gene. A missense mutation in codon 45 (Gly-45 to Asp) was the most common mutation found in the strains studied and impacted the structure of the helix-turn-helix domain of the MtrR protein thought to be important in DNA-binding activity. Two clinical isolates bearing a missense mutation in codon 45 also contained a single basepair deletion in a 13 bp inverted sequence positioned within the mtrR promoter region. Introduction of mtrR sequences amplified from the clinical isolates into strain FA19 revealed that acquisition of the single basepair deletion was correlated with high level HAR while mutations in the mtrR-coding region provided for an intermediate level of HAR.

The MtrD protein of Neisseria gonorrhoeae is a member of the resistance/nodulation/division protein family constituting part of an efflux system

The mtr (multiple transferable resistance) system of Neisseria gonorrhoeae mediates resistance of gonococci to structurally diverse hydrophobic agents (HAs) through an energy-dependent efflux process. Recently, complete or partial ORFs that encode membrane proteins (MtrC, MtrD, MtrE) forming an efflux pump responsible for removal of HAs from gonococci were identified and appeared to constitute a single transcriptional unit. In this study, the complete nucleotide sequence of the mtrD gene was determined, permitting the characterization of the MtrD protein. The full-length MtrD protein has a predicted molecular mass of nearly 114 kDa, putatively containing a 56 amino acid signal peptide. MtrD displays significant amino acid sequence similarity to a family of cytoplasmic membrane proteins, termed resistance/nodulation/division (RND) proteins, which function as energy-dependent transporters of antibacterial agents and secrete bacterial products to the extracellular fluid. The predicted topology of the MtrD transporter protein revealed 12 potential membrane-spanning domains, which were clustered within the central and C-terminal regions of the primary sequence. Loss of MtrD due to insertional inactivation of the mtrD gene rendered gonococci hypersusceptible to several structurally diverse HAs, including two fatty acids (capric acid and palmitic acid) and a bile salt (cholic acid), but not hydrophilic antibiotics such as ciprofloxacin and streptomycin. Since gonococci often infect mucosal sites rich in toxic fatty acids and bile salts, the expression of the mtr efflux system may promote growth of gonococci under hostile conditions encountered in vivo.

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.

Phosphoethanolamine Substitution of Lipid A and Resistance of Neisseria gonorrhoeae to Cationic Antimicrobial Peptides and Complement-Mediated Killing by Normal Human Serum

ABSTRACT 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 β-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.

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

ABSTRACT 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.

Lipid A’s Structure Mediates Neisseria gonorrhoeae Fitness during Experimental Infection of Mice and Men

ABSTRACT 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. 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.

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

The α-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 α-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.

Loss-of-function mutations in the mtr efflux system of Neisseria gonorrhoeae.

Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents (HAs) has been ascribed to the mtr (multiple transferable resistance) operon. This operon is composed of the mtrR gene, which encodes a transcriptional repressor (MtrR), and a three-gene complex (mtrCDE), which encodes cell envelope proteins (MtrC-MtrD-MtrE) that form an energy-dependent efflux pump. HA-hypersusceptible strains are often isolated from patients, but the genetic basis for such hypersusceptibility was heretofore unknown. The genetic basis of HA hypersusceptibility in laboratory-derived strains BR54 and BR87 was studied to learn if this trait could be linked to mutations in the mtr operon. Mutations in the mtrR gene of these strains that could be phenotypically suppressed by mutations in their mtrC or mtrD genes were identified. Thus, small deletions (4-10 bp) in the mtrC or mtrD genes of strains BR87 and BR54 that would result in the production of truncated efflux pump proteins that serve as a membrane fusion protein (MtrC) or transporter of HAs (MtrD) were found to be responsible for their HA-hypersusceptible property.