MtrR control of a transcriptional regulatory pathway in Neisseria meningitidis that influences expression of a gene (nadA) encoding a vaccine candidate

Abstract

The surface-exposed NadA adhesin produced by a subset of capsular serogroup B strains of Neisseria meningitidis is currently being considered as a vaccine candidate to prevent invasive disease caused by a hypervirulent lineage of meningococci. Levels of NadA are known to be controlled by both transcriptional regulatory factors and a component of human saliva, 4-hydroxyphenylacetic acid. Herein, we confirmed the capacity of a DNA-binding protein termed FarR to negatively control nadA expression. We also found that a known transcriptional regulator of farR in N. gonorrhoeae termed MtrR can have a negative regulatory impact on farR and nadA expression, especially when over-expressed. MtrR-mediated repression of nadA was found to be direct, and its binding to a target DNA sequence containing the nadA promoter influenced formation and/or stability of FarR::nadA complexes. The complexity of the multi-layered regulation of nadA uncovered during this investigation suggests that N. meningitidis modulates NadA adhesin protein levels for the purpose of interacting with host cells yet avoiding antibody directed against surface exposed epitopes.

Figure 1. Schematic of nadA, farR, and mtrR promoter regions used for lacZ fusions and DNA probes.

Representations of the nadA (A), farR (B), and mtrR (C) promoter regions showing regulatory protein binding sites, intergenic sequences of interest, and primer annealing locations with oligonucleotide sizes. White arrows represent each open reading frame with the translation start codon noted by “ATG” and a vertical line to indicate if a primer overlaps the start codon. Grey arrows represent the respective primers including oligonucleotide sizes with the following nomenclature: DNA probe for EMSAs - “orf”_prom_F & R; promoter region with start codon fused with lacZ - “orf”_F_Bam and “orf”_R_lacZ. IHF and Fur are binding sites for integration host factor and ferric uptake regulatory protein, respectively.

Figure 2. Growth phase-dependent expression of nadA, farR, and mtrR in N. meningitidis M7.

(A) Growth curve of strain M7 expressing lacZ fused to nadA (solid line; circle timepoints), farR (dotted line; triangle timepoints), and mtrR (dashed line; square timepoints) promoter regions measured by OD600 optical density. Boxed A, B, and C; timepoints for sample harvest. (B) Specific activity of β-galatosidase activity of lacZ fusions as indicated. Samples harvested from liquid culture at various growth phases (A, B, C) were compared with O/N growth on agar plates. Inset; magnified view of mtrR-lacZ expression. NS, not significant; **, P<0.01.

Figure 3. MtrR-regulation of farR.

Specific activity of β-galactosidase activity of farR-lacZ in the strains M7 wild-type, ΔmtrR, and ΔmtrR complemented with the native and inducible-promoter alleles (superscript asterisk), respectively. NS, not significant; *, P<0.05; **, P<0.01.

Figure 4. Expression of nadA by FarR and MtrR.

(A–C) Specific activity of β-galactosidase activity of nadA-lacZ in various M7 backgrounds as indicated. Strains over-expressing MtrR marked with a superscript asterisk. NS, not significant; *, P<0.05; **, P<0.01. (D) Western immunoblot analysis of NadA, FarR, and MtrR levels. Protein samples grown overnight on GC agar plates, collected, and analyzed by electropheoresis through 6% (NadA) or 12% (FarR and MtrR) SDS-PAGE gels followed by immunoblot with the respective antisera. Molecular weight standards are listed to the left. Coomassie-stained gel provided for protein level comparison. Arrows represent minor immunoreactive bands used to determine NadA steady-state differences across strains.

Figure 5. DNA binding properties of FarR-MBP.

(A) Successive increases of FarR-MBP incubated with 10 ng nadA promoter region to assess binding by gel-shift analysis. Arrows; various complexes of DNA and FarR-MBP. (B) Competition assays. ³²P-labeled nadA promoter (384 bp) was incubated with 0.5 µg FarR-MBP and competed with unlabeled nadA, farR (333 bp), farAB (435 bp), and rnpB (354 bp) at 25, 50, and 100 times molar excess of labeled probe (lanes 3 through 14). The competing probe used is listed below each panel. Arrow; ³²P-labeled probe competed away from FarR-MBP by unlabeled probe. Lane 1, labeled probe alone; lane 2, labeled probe and 0.5 µg FarR-MBP without competitor.

Figure 6. DNA binding properties of MtrR-MBP.

Successive increases of MtrR-MBP incubated with 10 ng mtrCDE (A) or nadA (B) promoter regions to assess binding by gel-shift analysis. Arrow; primary complex of DNA and MtrR-MBP. (C) Competition assays. ³²P-labeled nadA promoter (384 bp) was incubated with 1.0 µg MtrR-MBP and competed with unlabeled nadA, mtrCDE (552 bp), and rnpB (354 bp) at 25, 50, and 100 times molar excess of labeled probe (lanes 3 through 14). The competing probe used is listed below each panel. Arrow; ³²P-labeled probe competed away from MtrR-MBP by unlabeled probe. Lane 1, labeled probe alone; lane 2, labeled probe and 0.5 µg MtrR-MBP without competitor.

Figure 7. MtrR influences FarR::DNA complexes.

Shown is an EMSA evaluating the binding of MtrR–MBP and FarR-MBP to ³²P-labeled nadA promoter based on order of protein introduction. Lane assignments from left to right: probe alone; probe plus 1.0 mg MtrR-MBP and increasing amounts of FarR-MBP (0, 0.13, 0.25, 0.50, 1.0, 2.5, and 5.0 mg); probe alone; probe plus 0.5 mg MtrR-MBP and increasing amounts of FarR-MBP (0, 0.13, 0.25, 0.50, 1.0, 2.5, and 5.0 mg). The arrowhead shows the position of MtrR:DNA complex lacking FarR while the arrows show positions of FarR:DNA complexes lacking MtrR.

Figure 8. Identification of a DNase I hypersensitive site at the nadA promoter in the presence of MtrR.

(A) Increasing amounts of MtrR-MBP (0, 1, 5, 10 mg) were incubated with the nadA promoter prior to DNase I incubation. Site of DNase I hypersensitivity is denoted with an asterisk. The nucleotide sequence (G, A, T, C) is listed adjacent to the lanes. (B) Nucleotide sequence of the nadA promoter. Colored boxes, FarR-MBP binding sites 1, 2 & 3; Asterisk, DNase I hypersensitive site. The transcription start site (+1) and translational start site (ATG, bold and underlined) are indicated.