Transcriptional regulation of a gonococcal gene encoding a virulence factor (L-lactate permease).
ABSTRACT: 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.
Project description:A cytochrome c peroxidase (CCP) produced by Neisseria gonorrhoeae has been shown to have novel characteristics by investigating its location, expression and role in Neisseria gonorrhoeae and by expression in Escherichia coli. Analysis of the N-terminus of CCP indicated that it is a lipoprotein with a signal peptide for cleavage by signal peptidase II. Expression of the gonococcal CCP in E. coli revealed that it is first synthesized as a pro-apo-cytochrome that is translocated across the cytoplasmic membrane. The signal peptide is cleaved and haem is attached in the periplasm. The gonococcal CCP was associated with the membrane of both E. coli and N. gonorrhoeae. The expression of a MalE-CCP fusion protein has allowed characterization of CCP in vitro. Evidence is presented that CCP protects gonococci from hydrogen peroxide, presumably in the periplasmic compartment of the cell. The expression of CCP is dependent on the transcription factor FNR, but is repressed by nitrite, indicating that it could be most important in the stationary-phase response. These data support the hypothesis that the gonococcal lipoprotein CCP is anchored to the membrane in the periplasm, where it might be responsible for the reduction of hydrogen peroxide. Other putative CCP lipoproteins have been identified, representing a new subclass of bacterial CCP proteins.
Project description:The MtrR transcriptional-regulatory protein is known to repress transcription of the mtrCDE operon, which encodes a multidrug efflux pump possessed by Neisseria gonorrhoeae that is important in the ability of gonococci to resist certain hydrophobic antibiotics, detergents, dyes, and host-derived antimicrobials. In order to determine whether MtrR can exert regulatory action on other gonococcal genes, we performed a whole-genome microarray analysis using total RNA extracted from actively growing broth cultures of isogenic MtrR-positive and MtrR-negative gonococci. We determined that, at a minimum, 69 genes are directly or indirectly subject to MtrR control, with 47 being MtrR repressed and 22 being MtrR activated. rpoH, which encodes the general stress response sigma factor RpoH (sigma 32), was found by DNA-binding studies to be directly repressed by MtrR, as it was found to bind to a DNA sequence upstream of rpoH that included sites within the rpoH promoter. MtrR also repressed the expression of certain RpoH-regulated genes, but this regulation was likely indirect and a reflection of MtrR control of rpoH expression. Inducible expression of MtrR was found to repress rpoH expression and to increase gonococcal susceptibility to hydrogen peroxide (H(2)O(2)) and an antibiotic (erythromycin) recognized by the MtrC-MtrD-MtrE efflux pump system. We propose that, apart from its ability to control the expression of the mtrCDE-encoded efflux pump operon and, as a consequence, levels of gonococcal resistance to host antimicrobials (e.g., antimicrobial peptides) recognized by the efflux pump, the ability of MtrR to regulate the expression levels of rpoH and RpoH-regulated genes also modulates levels of gonococcal susceptibility to H(2)O(2).
Project description:Symptomatic gonococcal infection, caused exclusively by the human-specific pathogen Neisseria gonorrhoeae (the gonococcus), is characterized by the influx of polymorphonuclear leukocytes (PMNs) to the site of infection. Although PMNs possess a potent antimicrobial arsenal comprising both oxidative and non-oxidative killing mechanisms, gonococci survive this interaction, suggesting that the gonococcus has evolved many defenses against PMN killing. We previously identified the NG1686 protein as a gonococcal virulence factor that protects against both non-oxidative PMN-mediated killing and oxidative killing by hydrogen peroxide. In this work, we show that deletion of ng1686 affects gonococcal colony morphology but not cell morphology and that overexpression of ng1686 does not confer enhanced survival to hydrogen peroxide on gonococci. NG1686 contains M23B endopeptidase active sites found in proteins that cleave bacterial cell wall peptidoglycan. Strains of N. gonorrhoeae expressing mutant NG1686 proteins with substitutions in many, but not all, conserved metallopeptidase active sites recapitulated the hydrogen peroxide sensitivity and altered colony morphology of the ?ng1686 mutant strain. We showed that purified NG1686 protein degrades peptidoglycan in vitro and that mutations in many conserved active site residues abolished its degradative activity. Finally, we demonstrated that NG1686 possesses both dd-carboxypeptidase and endopeptidase activities. We conclude that the NG1686 protein is a M23B peptidase with dual activities that targets the cell wall to affect colony morphology and resistance to hydrogen peroxide and PMN-mediated killing.
Project description: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.
Project description:The ferric uptake regulatory protein, Fur, functions as a global regulatory protein of gene transcription in the mucosal pathogen Neisseria gonorrhoeae. We have shown previously that several N. gonorrhoeae Fur-repressed genes are expressed in vivo during mucosal gonococcal infection in men, which suggests that this organism infects in an iron-limited environment and that Fur is expressed under these conditions. In this study we have demonstrated expression of the gonococcal fur gene in vitro, in human cervical epithelial cells, and in specimens from female subjects with uncomplicated gonococcal infection. In vitro studies confirmed that the expression of the gonococcal fur gene was repressed during growth under iron-replete growth conditions but that a basal level of the protein was maintained. Using GFP transcriptional fusions constructed from specific Fur binding sequences within the fur promoter/operator region, we determined that this operator region was functional during N. gonorrhoeae infection of cervical epithelial cells. Furthermore, reverse transcription-PCR analysis, as well as microarray analysis, using a custom Neisseria Fur and iron regulon microarray revealed that several Fur- and iron-regulated genes were expressed during N. gonorrhoeae infection of cervical epithelial cells. Microarray analysis of specimens obtained from female subjects with uncomplicated gonococcal infection corroborated our in vitro findings and point toward a key role of gonococcal Fur- and iron-regulated genes in gonococcal disease.
Project description:In this work we have determined by RNA-Seq the whole set of genes regulated by the N. gonorrhoeae GdhR transcriptional regulator (NGO1360 gene in reference strain FA1090). The results showed that GdhR regulates the expression of 2.3% of all the genes in the gonococcal (strain FA19) genome, of which 39 were activated and 11 were repressed. Most of the GdhR-regulated genes lie in the category of fimbrial proteins and membrane antigens or transporters. GdhR was found not be a cryptic regulator since there is an overlap in the set of GdhR-regulated genes between the wild type and a strain expressing gdhR ectopically from the lac promoter. Among the GdhR-regulated genes linked to gonococcal pathogenesis we found that lctP, encoding a unique L-lactate transporter, was the highest of GdhR-repressed genes. Overall design: Total RNA samples were isolated from N. gonorrhoeae cultures grown to late exponential phase (OD600 1.0), depleted from ribosomal RNA and subjected to paired-end high throughput sequencing on the Illumina NextSeq 500 platform (Illumina Inc., San Diego, CA). Each strain (condition) was sequenced in duplicates. Transcript differential expression analyses were conducted between the transcriptomes of a wild type FA19 strain and its isogenic gdhR mutant, and between the gdhR mutant and its complemented strain expressing gdhR ectopically from the lac promoter.
Project description:CR3-mediated endocytosis is a primary mechanism by which Neisseria gonorrhoeae elicits membrane ruffling and cellular invasion of the cervical epithelia. Our data indicate that, upon infection of cervical epithelia, N. gonorrhoeae specifically releases proteins, including a phospholipase D (PLD) homolog, which facilitate membrane ruffling. To elucidate the function of gonococcal PLD in infection of the cervical epithelia, we constructed an N. gonorrhoeae PLD mutant. By comparative association and/or invasion assays, we demonstrated that PLD mutant gonococci are impaired in their ability to adhere to and to invade primary cervical cells. This defect can be rescued by the addition of supernatants obtained from wild-type-infected cell monolayers but not by exogenously added Streptomyces PLD. The decreased level of total cell association (i.e., adherence and invasion) observed for mutant gonococci is, in part, attributed to the inability of these bacteria to recruit CR3 to the cervical cell surface with extended infection. Using electron microscopy, we demonstrate that gonococcal PLD may be necessary to potentiate membrane ruffling and clustering of gonococci on the cervical cell surface. These data may be indicative of the inability of PLD mutant gonococci to recruit CR3 to the cervical cell surface. Alternatively, in the absence of gonococcal PLD, signal transduction events required for CR3 clustering may not be activated. Collectively, our data indicate that PLD augments CR3-mediated gonococcus invasion of and survival within cervical epithelia.
Project description:BACKGROUND:Gonorrhea, caused by the bacterium Neisseria gonorrhoeae, is a globally prevalent sexually transmitted infection. The dynamics of gonococcal population biology have been poorly defined due to a lack of resolution in strain typing methods. METHODS:In this study, we assess how the core genome can be used to improve our understanding of gonococcal population structure compared with current typing schemes. RESULTS:A total of 1668 loci were identified as core to the gonococcal genome. These were organized into a core genome multilocus sequence typing scheme (N gonorrhoeae cgMLST v1.0). A clustering algorithm using a threshold of 400 allelic differences between isolates resolved gonococci into discrete and stable core genome groups, some of which persisted for multiple decades. These groups were associated with antimicrobial genotypes and non-overlapping NG-STAR and NG-MAST sequence types. The MLST-STs were more widely distributed among core genome groups. CONCLUSIONS:Clustering with cgMLST identified globally distributed, persistent, gonococcal lineages improving understanding of the population biology of gonococci and revealing its population structure. These findings have implications for the emergence of antimicrobial resistance in gonococci and how this is associated with lineages, some of which are more predisposed to developing antimicrobial resistance than others.
Project description:Antibodies against reduction modifiable protein (anti-Rmp Abs) can block complement-dependent killing of Neisseria gonorrhoeae by otherwise bactericidal Abs. An anti-lipooligosaccharide bactericidal monoclonal Ab (mAb) 2C7, a gonococcal vaccine candidate Ab, attenuates vaginal colonization by gonococci in BALB/c mice. Here we show that anti-Rmp Abs block the efficacy of mAb 2C7 in mice in a dose-dependent manner. Anti-Rmp Abs also counteract 2C7-mediated enhancement of C3 deposition on gonococci in vivo. The mouse model will prove useful to study how blocking Abs influence the efficacy of gonococcal vaccines. Preexisting anti-Rmp Abs will be an important consideration in evaluating the efficacy of gonococcal vaccine candidates.
Project description:Neisseria gonorrhoeae is a human-specific organism that is not usually exposed to UV light or chemicals but is likely to encounter reactive oxygen species during infection. Exposure of N. gonorrhoeae to sublethal hydrogen peroxide revealed that the ng1427 gene was upregulated sixfold. N. gonorrhoeae was thought to lack an SOS system, although NG1427 shows amino acid sequence similarity to the SOS response regulator LexA from Escherichia coli. Similar to LexA and other S24 peptidases, NG1427 undergoes autoproteolysis in vitro, which is facilitated by either the gonococcal or E. coli RecA proteins or high pH, and autoproteolysis requires the active and cleavage site residues conserved between LexA and NG1427. NG1427 controls a three gene regulon: itself; ng1428, a Neisseria-specific, putative integral membrane protein; and recN, a DNA repair gene known to be required for oxidative damage survival. Full NG1427 regulon de-repression requires RecA following methyl methanesulphonate or mitomycin C treatment, but is largely RecA-independent following hydrogen peroxide treatment. NG1427 binds specifically to the operator regions of the genes it controls, and DNA binding is abolished by oxidation of the single cysteine residue encoded in NG1427. We propose that NG1427 is inactivated independently of RecA by oxidation.