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

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.

Keywords: efflux pumps; gonococci; physiology; transcription.

FIG 1

The organization of the gonococcal mtr and gdh loci in N. gonorrhoeae strain FA19, highlighting the position of genes relevant to this study. The length (shown in base pairs) and transcriptional orientation (direction of arrows) of relevant genes are shown. Numbers above the horizontal line refer to the coding regions of the gene, while the sizes of the intergenic regions (shaded boxes) are shown below. The distance between the two loci is 954 bp.

FIG 2

Absence of the GdhR protein confers a fitness advantage in N. gonorrhoeae. Competitive vaginal infections in female BALB/c mice with FA19Str^r and FA19Str^r gdhR::kan (A), FA19Str^r gdhR::kanC3 and FA19Str^r gdhR::kan (B), and FA19Str^r and FA19Str^r gdhR::kanC3 (C). Vaginal swab suspensions were quantitatively cultured on days 1, 3, and 5 post-bacterial inoculation, and the number of CFU of each strain was determined using selective agar as described in Materials and Methods. Each symbol corresponds to the CI from an individual mouse; open circles and open triangles correspond to mice from which only mutant CFU or wild-type CFU were recovered, respectively. Bars represent the geometric mean CI values. Combined data from two experiments are shown, with data points from each experiment indicated in black or grey. Open circles indicate that only the mutant strain was recovered from the vaginal swabs at the indicated time point. Open triangles indicate that only the wild-type strain was recovered from the vaginal swabs at the indicated time point. The differences in the median CI between the FA19Str^r versus FA19Str^r gdhR::kan C3 and the FA19Str^r gdhR::kan versus FA19Str^r gdhR::kan C3 competitions were statistically significant on day 1 (P = 0.02), day 3 (P = 0.07), and day 5 (P = 0.03) postinfection, based on the Kruskal-Wallis test with Dunn’s multiple comparisons test (GraphPad Prism). Comparisons of the FA19Str^r versus FA19Str^r gdhR::kan competition with FA19Str^r versus FA19Str^r gdhR::kan C3 showed that the results approached but did not reach a statistically significant difference; P values for days 3 and 5 in this comparison were 0.055 and 0.056, respectively.

FIG 3

(A) Promoter sequence of the gdhR gene in strain FA19. The −10 and −35 promoter elements, the start of translation (ATG), and the TSSs are represented in blue. Primers are represented by arrows and the putative MtrR-binding site is framed in red. The insertion site of the CE in meningococci is represented in red (TA). The putative GdhR-binding site is underlined in green. (B) Alignment of the MtrR putative binding sites upstream of gdhR and mtrC. Dots between the two sequences indicate identical bases. (C) Quantitative RT-PCR results for gdhR in the wild type (WT) and a strain with mtrR deleted at the late-logarithmic phase of growth. Error bars represent standard deviations of the means of two independent experiments. Normalized expression ratios (NER) were calculated using 16S rRNA expression levels. *, P = 0.0004.

FIG 4

Competitive EMSAs. The MtrR-binding site located on fragment R4/R2 has the highest affinity for the MtrR protein. Lanes: 1, probe R3/R2* alone; 2, probe R3/R2* plus 2 μg of MtrR; 3, probe R3/R2* plus 2 μg of MtrR plus 50× unlabeled R3/R2; 4, probe R3/R2* plus 2 μg of MtrR plus 100× unlabeled R3/R2; 5, probe R3/R2* plus 2 μg of MtrR plus 50× unlabeled R3/R5; 6, probe R3/R2* plus 2 μg of MtrR plus 100× unlabeled R3/R5; 7, probe R3/R2* plus 2 μg of MtrR plus 50× unlabeled R4/R2; 8, probe R3/R2* plus 2 μg of MtrR plus 100× unlabeled R4/R2; 9, probe R3/R2* plus 2 μg of MtrR plus 50× rnpB; 10, probe R3/R2* plus 2 μg of MtrR plus 100× rnpB. An asterisk indicates a radioactive probe. The location of the different gdhR probes are shown in Fig. 3A.

FIG 5

Quantitative RT-PCR results with gdhR (A) or gdhA (B) in wild-type (WT) and gdhR-negative strains at the mid- and late-logarithmic phases of growth. Error bars represent standard deviations from the means of three independent experiments. Normalized expression ratios (NER) were calculated using 16S rRNA expression. *, P = 0.011; **, P = 0.008; NS, not significant.

FIG 6

Alignment of gdhA promoters from gonococcal strain FA19 (top) and meningococcal strain MC58 (bottom). The TSSs determined by primer extension experiments for strain FA19 identified in this study and that of MC58 as reported by Pagliarulo et al. (16) are represented in blue, green, and red, with their respective putative promoter elements. The consensus binding sequence for the GntR protein is underlined. The gdhA translation start site is represented in purple.

FIG 7

The GdhR protein binds to the gdhA promoter in a specific manner. Lanes: 1, probe PgdhA* alone; 2, probe PgdhA* plus 1 μg of GdhR; 3, probe PgdhA* plus 1 μg of GdhR plus 50× unlabeled PgdhA; 4, probe PgdhA* plus 1 μg of GdhR plus 100× unlabeled PgdhA; 5, probe PgdhA* plus 1 μg of GdhR plus 50× unlabeled rnpB; 6, probe PgdhA* plus 1 μg of GdhR plus 100× unlabeled rnpB. Asterisk refers to radioactive probe.