Expression of factor H binding protein of meningococcus responds to oxygen limitation through a dedicated FNR-regulated promoter

Abstract

Factor H binding protein (fHBP) is a surface-exposed lipoprotein in Neisseria meningitidis, which is a component of several investigational vaccines against serogroup B meningococcus (MenB) currently in development. fHBP enables the bacterium to evade complement-mediated killing by binding factor H, a key downregulator of the complement alternative pathway, and, in addition, fHBP is important for meningococcal survival in the presence of the antimicrobial peptide LL-37. In this study, we investigate the molecular mechanisms involved in transcription and regulation of the fHBP-encoding gene, fhbp. We show that the fHBP protein is expressed from two independent transcripts: one bicistronic transcript that includes the upstream gene and a second shorter monocistronic transcript from its own dedicated promoter, P(fhbp). Transcription from the promoter P(fhbp) responds to oxygen limitation in an FNR-dependent manner, and, accordingly, the FNR protein binds to a P(fhbp) probe in vitro. Furthermore, expression in meningococci of a constitutively active FNR mutant results in the overexpression of the fHBP protein. Finally, the analysis of fHBP regulation was extended to a panel of strains expressing different fHBP allelic variants at different levels, and we demonstrate that FNR is involved in the regulation of this antigen in all but one of the strains tested. Our data suggest that oxygen limitation may play an important role in inducing the expression of fHBP from a dedicated FNR-regulated promoter. This implies a role for this protein in microenvironments lacking oxygen, for instance in the submucosa or intracellularly, in addition to its demonstrated role in serum resistance in the blood.

FIG. 1.

(A) Schematic representation of the fhbp locus within the genome of wild-type N. meningitidis strain MC58 and mutant strains Δnmb1869 and Δfhbp. In the null mutants, an erythromycin cassette used to replace the respective genes by allelic exchange is orientated in the same direction (Δfhbp) or in the opposite direction (Δnmb1869). Hairpin structures indicate the position of predicted rho-independent terminators. The stem-loop downstream of the fhbp gene is comprised of a 12-nt perfect palindromic sequence including the GCCGTCTGAA DNA uptake sequence (DUS) separated by 13 nt of loop and followed by a stretch of four Ts. DUS are often found in the base-paired stem of transcription terminators (48). The putative rho-independent terminator in the nmb1869-fhbp intergenic region is comprised of a similar 12-nt imperfect palindromic sequence in which the second DUS sequence has two mismatches, separated by 4 nt and followed by a stretch of three Ts. The relative positions of the radioactively labeled probes used in the Northern blot analysis are indicated under each gene. The figure provides a diagrammatic representation of the long bicistronic transcriptional unit linking the nmb1869 and fhbp genes and of the two monocistronic transcripts independently transcribed from their own dedicated promoters. (B and C) Northern blot analysis. Total RNA prepared from MC58, Δfhbp, and Δnmb1869 cultures grown to mid-log phase was run on an 0.8% denaturing agarose gel, transferred to a nylon membrane, and probed with radioactively labeled PCR products equivalent to 187 bp of the fhbp gene (B) and 142 bp of the nmb1869 gene (C). The relative positions of the molecular weight RNA ladder are shown.

FIG. 2.

Mapping of the 5′ end of the nmb1869-specific transcript (A) and the fhbp-specific transcript (B) by primer extension. Total RNA (20 μg) prepared from cultures of the wild-type strain (MC58) grown to mid-logarithmic phase was hybridized with gene-specific primers (741PE1 and 1869PE1) (Table 1) and elongated with reverse transcriptase. Sequence reactions (G, A, T, and C) were performed with the same primers on the respective cloned promoter regions and run in parallel. The elongated primer bands mapping the major 5′ end of the corresponding gene transcripts are indicated. The corresponding +1 nt of transcriptional initiation and the upstream −10 and −35 promoter elements are underlined and in bold face in the nucleotide sequences of the respective intergenic regions shown underneath. An FNR box located at −40.5 bp is highlighted in gray. The pentanucleotide ATATT (in the box) is the target of insertion for a 181-bp AT-rich nucleotide sequence (ATR) in the genome of MenA strain Z2491 (37).

FIG. 3.

Regulation of fHBP by FNR. (A) Northern blot analysis showing the regulation of mRNA transcripts. The generation of the null mutants and Δfnr complemented strain Δfnr_C (in the MC58 and H44/76 background) and their growth under aerobic (+) and oxygen-limiting (−) conditions are described in Materials and Methods. Aliquots (5 μg) of total RNA were subjected to electrophoresis, transferred onto nylon filters, and hybridized with the fhbp-specific probe. In each lane, the amount of RNA loaded was normalized to the amount of rRNA. (B) Western blot analysis showing the FNR-regulated protein expression. Equal amounts of total protein from overnight plates cultured under microaerobic conditions were fractionated by SDS-PAGE, blotted onto nitrocellulose filters, and stained with mouse polyclonal antiserum raised against the NMB1869 protein, the fHBP protein, or the FNR protein. Where IPTG was necessary to induce the expression of FNR, it was added to plates at a final concentration of 1 mM.

FIG. 4.

Footprinting analysis of purified FNR(D148A) on the aniA (A) and fhbp (B) intergenic probes. The probes were labeled at one end and prepared as described in Materials and Methods. Lane 1 is a G+A sequence reaction obtained with each probe and used as a molecular weight marker. Footprinting reactions containing purified FNR(D148A) protein at final concentrations of 0 nM, 13 nM, and 40 nM in panel A (lanes 2 to 4, respectively) and at 0 nM, 8 nM, 40 nM, 200 nM, and 1 μM in panel B (lanes 2 to 6, respectively). The vertical bar on the right side of each panel indicates the area of DNase I protection in the promoter region; small arrows refer to DNase I hypersensitive sites. The numbers indicate the positions of the relevant nucleotides with respect to the +1 transcriptional start site. The nucleotides corresponding to the FNR binding site are indicated, with mismatches to the FNR box of E. coli displayed on the right.

FIG. 5.

(A) Western blot analysis of NMB1869 and fHBP protein expression in a broad panel of 40 N. meningitidis strains. Whole bacterial lysates were prepared from overnight plate cultures and subjected to immunoblot analysis with mouse polyclonal antibody against each protein. The asterisk marks a nonspecific cross-reactive band that is shown as a loading control. The fHBP protein is expressed at different levels in different strains. In this representative Western blot, it is not possible to visualize fHBP expression in some of the isolates (M2671, 5/99, OX99.30304, M1239) due to their low level of expression that was evident only with long exposure of the film (data not shown). (B) Western blot analysis of protein expression on a subset of wild-type strains and their respective FNR mutant derivative expressing the FNR(D148A) protein (+). The site-directed mutant allele of the fnr gene, fnr(D148A), was inserted under the control of the P_tac-inducible promoter in the genomes of these strains, and FNR expression is induced by growth on 1 mM IPTG. Immunoblot analysis was performed using antisera against NMB1869, fHBP, and the FNR protein. N, Norway; UK, United Kingdom; USA, United States; TCH, Chad; F, Finland; D, Denmark; NL, the Netherlands; RUS, Russia.