Identification and regulation of expression of a gene encoding a filamentous hemagglutinin-related protein in Bordetella holmesii

Abstract

Background: Bordetella holmesii is a human pathogen closely related to B. pertussis, the etiological agent of whooping cough. It is able to cause disease in immunocompromised patients, but also whooping cough-like symptoms in otherwise healthy individuals. However, virtually nothing was known so far about the underlying virulence mechanisms and previous attempts to identify virulence factors related to those of B. pertussis were not successful.

Results: By use of a PCR approach we were able to identify a B. holmesii gene encoding a protein with significant sequence similarities to the filamentous hemagglutinin (FHA) of B. avium and to a lesser extent to the FHA proteins of B. pertussis, B. parapertussis, and B. bronchiseptica. For these human and animal pathogens FHA is a crucial virulence factor required for successful colonization of the host. Interestingly, the B. holmesii protein shows a relatively high overall sequence similarity with the B. avium protein, while sequence conservation with the FHA proteins of the human and mammalian pathogens is quite limited and is most prominent in signal sequences required for their export to the cell surface. In the other Bordetellae expression of the fhaB gene encoding FHA was shown to be regulated by the master regulator of virulence, the BvgAS two-component system. Recently, we identified orthologs of BvgAS in B. holmesii, and here we show that this system also contributes to regulation of fhaB expression in B. holmesii. Accordingly, the purified BvgA response regulator of B. holmesii was shown to bind specifically in the upstream region of the fhaB promoter in vitro in a manner similar to that previously described for the BvgA protein of B. pertussis. Moreover, by deletion analysis of the fhaB promoter region we show that the BvgA binding sites are relevant for in vivo transcription from this promoter in B. holmesii.

Conclusion: The data reported here show that B. holmesii is endowed with a factor highly related to filamentous hemagglutinin (FHA), a prominent virulence factor of the well characterized pathogenic Bordetellae. We show that like in the other Bordetellae the virulence regulatory BvgAS system is also involved in the regulation of fhaB expression in B. holmesii. Taken together these data indicate that in contrast to previous notions B. holmesii may in fact make use of virulence mechanisms related to those described for the other Bordetellae.

Figure 1

Schematic view of the gene loci encoding fhaBin B. pertussis, B. avium and B. holmesii. Relevant genes are represented as boxes. The arrows below the boxes show the transcriptional polarity of each gene. The open reading frame orfMP of B. holmesii encoding a putative membrane is not yet completely sequenced. The shaded regions (marked A to D) within the fhaB gene of B. holmesii indicate the degree of similarity of the deduced protein sequence with that of the FHA protein of B. avium. Region A comprises approximately amino acids 1–850, region B amino acids 950 to 1,640, region C amino acids 1,900 to 2,600, and region D amino acids 2,830 to 2,930 of the B. holmesii FHA protein; these regions correspond to amino acids 1–820, 840–1,540, 1,600–2310, and 2,520–2621 of the B. avium protein, respectively.

Figure 2

Schematic representation of the overall structureof FHA proteins of B. pertussis and B. holmesii. Protein regions with known or presumable functional importance are represented by dark grey boxes. The arrow indicates the maturation site for proteolytic cleavage of the B. pertussis FHA by the SphB1 protease. Abbreviations: N, N terminus; C, C terminus; SP, signal peptide; TPS, two-partner secretion domain; HBD, heparin binding domain; RGD, arginine-glycine-aspartic acid motif; CRD, carbohydrate binding motif; KGD, lysine-glycine-aspartic acid motif. Numbers indicate amino acid positions within the FHA proteins.

Figure 3

Determination of transcriptional start sites of the gfp reporter gene fused to the upstream region of fhaB of B. holmesii by primer extension analysis. Equal amounts of total RNA extracted from B. holmesii G7702 (pMMB208-fhaP-gfp0) (lane 1) and B. holmesii G7702 bvgA (pMMB208-fhaP-gfp0) (lane 2) were hybridized with radiolabelled oligonucleotide gfp.PE. The cDNAs corresponding to the transcripts P1 to P3 are indicated by arrows. A part of the fhaB promoter sequence is shown on the left. Transcriptional start points are indicated by arrows. The sequencing reaction (lanes A, C, G and T) was performed using oligonucleotide gfp.PE and plasmid pSK-fhaP-gfp0 as a template.

Figure 4

Binding of BvgA_BH to the fhaB upstream region of B. holmesii. A radiolabelled 277 bp PCR fragment containing the fhaB upstream region of B. holmesii was incubated with 150, 350, 500 and 650 ng of unphosphorylated (lanes 1–4) and in vitro phosphorylated (lanes 5–8) BvgA_BH, respectively. Lane 9 contains the radiolabelled DNA probe. The reaction mixtures were run on a non-denaturating 4% polyacrylamide gel. F, free DNA; C, DNA-protein complex.

Figure 5

Binding of the B. holmesii BvgA_BH protein to the fhaB upstream region of B. holmesii. The footprint shows the entire region protected by BvgA_BH as indicated by the bar on the right side of the figure. DNase I footprint experiments were performed on a 312 bp BamHI-HindIII DNA fragment from plasmid pSK-FP labelled at its BamHI site containing the entire fhaB upstream sequence including all four putative binding sites (BS1–BS4). The 5'-labelled probe was incubated with 0.5, 1.0, 2.0, 3.0, 4.0, 6.0 and 8.0 μg of in vitro phosphorylated BvgA_BH (lanes 4–10, respectively). No protein was added to the reaction mixture loaded in lane 3. Lane 1 and 2 are G+A sequencing reactions on the DNA probe used as a size marker [42]. Numbers on the left indicate the distance from the translational start codon of the fhaB gene. The positions of the start sites of transcripts P1–P3 are indicated.

Figure 6

Representation of the intergenic region between orfMP and fhaB of B. holmesii. Partial amino acid sequences are shown below the respective coding DNA sequences. The GTG start codon of fhaB and the ATG start codon of the neighbouring orfMP coding for a putative membrane protein are given in bold letters. The putative Shine/Dalgarno sequence of fhaB is shown in italics and underlined. Transcriptional start sites of the constitutively synthesized transcripts P1 and P2 and of the bvg-dependent transcript P3 of fhaB are marked by arrows. The region protected from DNaseI digestion in footprint experiments with BvgA_BH-P is underlined. The sequence motifs BS1 to BS4 showing similarity to the BvgA consensus binding site in B. pertussis promoters are indicated by horizontal arrows above and below the DNA sequence. Nucleotides which match the consensus sequence are marked in bold letters.

Figure 7

Characterization of putative BvgA binding sitesinside the fhaB upstream region of B. holmesii. (a) Schematic representation of the fhaB upstream region containing the four putative BvgA binding sites BS1–BS4 and representation of the PCR amplified DNA probes I-V used for band shift experiments with the purified B. holmesii BvgA_BH protein. Numbers indicate the distance from the Bvg-dependent fhaB transcriptional start site (P3) taken as position +1. (b) Binding of the B. holmesii BvgA_BH protein to DNA probes I-V, containing different amounts of putative BvgA binding sites (a); the radiolabelled PCR fragments were incubated with 150 ng (lane 2), 300 ng (lanes 3 and 8), 400 ng (lanes 4 and 9), 500 ng (lanes 5, 10, 13, 17 and 21), 600 ng (lane 22), 700 ng (lanes 6, 11, 14, 18 and 23) and 800 ng (lanes 15, 19 and 24) of in vitro phosphorylated BvgA_BH. No protein was added in lane 1 (DNA probe I), lane 7 (DNA probe II), lane 12 (DNA probe III), lane 16 (DNA probe IV) and lane 20 (DNA probe V). The reaction mixtures were run on a non-denaturating 4% polyacrylamide gel. F, free DNA probes; C, DNA-protein complexes.

Figure 8

Immunoblot analysis of protein lysates of B. holmesii strains with a polyclonal anti-GFP antiserum. B. holmesii G7702 (pMMB208-fhaP-gfp0; BS1 to BS4) (lane 1), B. holmesii G7702 bvgA (pMMB208-fhaP-gfp0; BS1 to BS4) (lane 2), B. holmesii G7702 (pMMB208-fhaP-gfp2; BS2 to BS4) (lane 3), B. holmesii G7702 (pMMB208-fhaP-gfp3; BS3 and BS4) (lane 4), B. holmesii G7702 (pMMB208-fhaP-gfp4; BS4) (lane 5), B. holmesii G7702 (pMMB208-fhaP-gfp6; no BS) (lane 6). Lysates of E. coli (pSK-fhaP-gfp0) expressing GFP under control of the pSK lac promoter (lane 7) and of E. coli (pMMB208-fhaP-gfp0) (lane 9) were analysed as positive and negative control, respectively. BH-WT, B. holmesii wild type; BH-bvgA, B. holmesii bvgA mutant.