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ABSTRACT Escherichia coli signal peptidase I (LepB) has been shown to inefficiently cleave secreted proteins with aromatic amino acids at the second position after the signal peptidase cleavage site (P2′). The Bacillus subtilis exported protein TasA contains a phenylalanine at P2′, which in B. subtilis is cleaved by a dedicated archaeal-organism-like signal peptidase, SipW. We have previously shown that when the TasA signal peptide is fused to maltose binding protein (MBP) up to the P2′ position, the TasA-MBP fusion protein is cleaved very inefficiently by LepB. However, the precise reason why the TasA signal peptide hinders cleavage by LepB is not known. In this study, a set of 11 peptides were designed to mimic the inefficiently cleaved secreted proteins, wild-type TasA and TasA-MBP fusions, to determine whether the peptides interact with and inhibit the function of LepB. The binding affinity and inhibitory potential of the peptides against LepB were assessed by surface plasmon resonance (SPR) and a LepB enzyme activity assay. Molecular modeling of the interaction between TasA signal peptide and LepB indicated that the tryptophan residue at P2 (two amino acids before the cleavage site) inhibited the active site serine-90 residue on LepB from accessing the cleavage site. Replacing the P2 tryptophan with alanine (W26A) allowed for more efficient processing of the signal peptide when the TasA-MBP fusion was expressed in E. coli. The importance of this residue to inhibit signal peptide cleavage and the potential to design LepB inhibitors based on the TasA signal peptide are discussed. IMPORTANCE Signal peptidase I is an important drug target, and understanding its substrate is critically important to develop new bacterium-specific drugs. To that end, we have a unique signal peptide that we have shown is refractory to processing by LepB, the essential signal peptidase I in E. coli, but previously has been shown to be processed by a more human-like signal peptidase found in some bacteria. In this study, we demonstrate how the signal peptide can bind but is unable to be processed by LepB, using a variety of methods. This can inform the field on how to better design drugs that can target LepB and understand the differences between bacterial and human-like signal peptidases.

ABSTRACT 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.

ABSTRACT MtrA is a member of the AraC family of transcriptional regulators and has been shown to play an important role in enhancing transcription of the mtrCDE operon, which encodes a tripartite multidrug efflux pump, when gonococci are exposed to a sublethal level of antimicrobials. Heretofore, the DNA-binding properties of MtrA were unknown. In order to understand how MtrA activates mtrCDE expression, we successfully purified MtrA and found that it could bind specifically to the mtrCDE promoter region. The affinity of MtrA for the mtrCDE promoter increased 2-fold in the presence of a known effector and substrate of the MtrCDE pump, the nonionic detergent Triton X-100 (TX-100). When placed in competition with MtrR, the transcriptional repressor of mtrCDE, MtrA was found to bind with apparent lower affinity than MtrR to the same region. However, preincubation of MtrA with TX-100 prior to addition of the promoter-containing DNA probe increased MtrA binding and greatly reduced its dissociation from the promoter upon addition of MtrR. Two independent approaches (DNase I footprinting and a screen for bases important in MtrA binding) defined the MtrA-binding site 20–30 bp upstream of the known MtrR-binding site. Collectively, these results suggest that the MtrA and MtrR-binding sites are sterically close and that addition of an effector increases the affinity of MtrA for the mtrCDE promoter such that MtrR binding is negatively impacted. Our results provide a mechanism for transcriptional activation of mtrCDE by MtrA and highlight the complexity of transcriptional control of drug efflux systems possessed by gonococci. IMPORTANCE Antibiotic resistance in Neisseria gonorrhoeae has been increasing in recent years, such that in 2007 the Centers for Disease Control and Prevention listed N. gonorrhoeae as a “superbug.” One of the major contributors to antibiotic resistance in N. gonorrhoeae is the MtrCDE efflux pump. Until now, most work on the regulation of the genes encoding this efflux pump has been done on the transcriptional repressor, MtrR. This study is the first one to purify and define the DNA-binding ability of the transcriptional activator, MtrA. Understanding how levels of the MtrCDE efflux pump are regulated increases our knowledge of gonococcal biology and how the gonococcus can respond to various stresses, including antimicrobials.

ABSTRACT The MtrC-MtrD-MtrE multidrug efflux pump of Neisseria gonorrhoeae confers resistance to a diverse array of antimicrobial agents by transporting these toxic compounds out of the gonococcus. Frequently in gonococcal strains, the expression of the mtrCDE operon is differentially regulated by both a repressor, MtrR, and an activator, MtrA. The mtrR gene lies 250 bp upstream of and is transcribed divergently from the mtrCDE operon. Previous research has shown that mutations in the mtrR coding region and in the mtrR-mtrCDE intergenic region increase levels of gonococcal antibiotic resistance and in vivo fitness. Recently, a C-to-T transition mutation 120 bp upstream of the mtrC start codon, termed mtr120, was identified in strain MS11 and shown to be sufficient to confer high levels of antimicrobial resistance when introduced into strain FA19. Here we report that this mutation results in a consensus −10 element and that its presence generates a novel promoter for mtrCDE transcription. This newly generated promoter was found to be stronger than the wild-type promoter and does not appear to be subject to MtrR repression or MtrA activation. Although rare, the mtr120 mutation was identified in an additional clinical isolate during sequence analysis of antibiotic-resistant strains cultured from patients with gonococcal infections. We propose that cis-acting mutations can develop in gonococci that significantly alter the regulation of the mtrCDE operon and result in increased resistance to antimicrobials. IMPORTANCE Gonorrhea is the second most prevalent sexually transmitted bacterial infection and a worldwide public health concern. As there is currently no vaccine against Neisseria gonorrhoeae, appropriate diagnostics and subsequent antibiotic therapy remain the primary means of infection control. However, the effectiveness of antibiotic treatment is constantly challenged by the emergence of resistant strains, mandating a thorough understanding of resistance mechanisms to aid in the development of new antimicrobial therapies and genetic methods for antimicrobial resistance testing. This study was undertaken to characterize a novel mechanism of antibiotic resistance regulation in N. gonorrhoeae. Here we show that a single base pair mutation generates a second, stronger promoter for mtrCDE transcription that acts independently of the known efflux system regulators and results in high-level antimicrobial resistance.