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BMC Microbiology

Open Access

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

BMC Microbiology20077:100

https://doi.org/10.1186/1471-2180-7-100

Received: 07 June 2007

Accepted: 07 November 2007

Published: 07 November 2007

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.

Keywords

PertussisGreen Fluorescent Protein ExpressionPrimer Extension AnalysisFootprint ExperimentPrimer Extension Experiment

Background

The genus Bordetella currently comprises nine species, most of which were found to be associated with host organisms [1, 2]. Medically the most important species is B. pertussis, the etiological agent of whooping cough for which humans are the only known host. B. parapertussis causes milder forms of whooping cough-like disease in humans. B. bronchiseptica is known to cause respiratory disease in various mammalian species, but only rarely in humans [2]. These "classical" Bordetella species are closely related and the recent determination of their genome sequences confirmed previous suggestions that B. pertussis and B. parapertussis are independent descendents of B. bronchiseptica-like ancestors which during specialization to a single host have sustained a significant erosion of their genetic material [3]. In agreement with their close relationship these organisms produce highly related virulence factors such as several toxins and colonization factors [2].

Among these virulence factors the filamentous hemagglutinin FHA is of particular relevance for pathogenesis. It is an important adhesin and it is required for tracheal colonization in animal models [4]. FHA is a huge protein synthesized as a precursor of 367 kDa which is processed to a mature protein of about 220 kDa by extensive N-terminal and C-terminal modifications involving possibly the Lep signal peptidase and the subtilisin-like autotransporter protein SphB1 [5, 6]. FHA is exported to the cell surface and/or secreted via a two-partner export mechanism requiring the FhaC protein located in the outer membrane of the bacteria [7, 8]. It has several distinct binding domains involved in adhesion to different substrates. The carbohydrate recognition domain (CRD) probably enables the bacteria to attach to ciliated respiratory epithelial cells and to macrophages [9]. FHA exhibits lectin-like activity for heparin and dextran sulphate possibly involved in the interaction with nonciliated epithelial cells which also contributes to FHA-mediated hemagglutination [10]. Furthermore, there is an Arg-Gly-Asp (RGD) motif which enables FHA to adhere to human monocytes/macrophages via the leukocyte integrin complement receptor 3 (CR3, alpha M beta 2, CD11b/CD18) [11]. The FHA proteins of the "classical" species are highly related, but not entirely functionally exchangeable. Recently, by use of a B. bronchiseptica strain expressing the B. pertussis FHA protein it was found that the heterologous protein could mediate adherence to various epithelial and macrophage cell lines in vitro. In contrast, in rat infection models significant differences in the host-pathogen interaction were noted for the mutant B. bronchiseptica strain suggesting significantly different activities of the closely related FHA proteins of the classical species and a role of FHA for host adaptation [12]. Two other proteins, FhaL and FhaS, with significant sequence similarity to FHA are present in the members of the B. bronchiseptica cluster, but their functional relevance in virulence is not yet clear [3]. As in case of most other virulence factors, the expression of the fhaB locus is controlled by the BvgAS two-component system which is responsive to environmental stimuli such as temperature or compounds such as MgSO4 or nicotinic acid [13, 14]. The architecture of the fhaB promoter and its activation mechanism by the BvgAS system has been investigated in much detail [15, 16]. Finally, FHA is a protective antigen and it is included in acellular pertussis vaccines [2].

More recently, additional species were included in the genus Bordetella. In 1984, B avium, a respiratory pathogen of birds, was first described. The genome sequence of B. avium was recently established and revealed the presence of several factors orthologous to those of the "classical" Bordetellae including FHA and the BvgAS two-component system [17, 18]. Among other "novel" Bordetella species occasionally isolated from patients with underlying disease such as B. hinzii and B. trematum, B. holmesii has gained most attention in the past years, since its 16S rDNA sequence suggested this organism to be most closely related to B. pertussis [2, 19]. Moreover, B. holmesii is known to contain several copies of the insertion elements IS481 and IS1001, otherwise found only in B. pertussis and B. parapertussis, respectively [20, 21]. In addition to its isolation from immunocompromised patients [2224], B. holmesii was found to be able to cause whooping-cough like symptoms in otherwise healthy persons [25, 26]. Very little was known about the virulence properties of this bacterium and attempts to identify virulence factors related to those of B. pertussis failed. Recently, we succeeded to identify the BvgAS two-component system of B. holmesii by PCR amplification with degenerate oligonucleotides [27]. Interestingly, in contrast to the close relationship of the 16S rDNA sequences of B. holmesii and B. pertussis, the B. holmesii BvgAS system was found to be more closely related to the orthologous BvgAS system of B. avium than to that of B. pertussis [27].

Based again on a PCR approach with degenerate oligonucleotides we attempted to identify additional putative virulence factors of B. holmesii related to those of B. pertussis. Here we describe the identification and initial characterization of an FHA orthologue of B. holmesii. We show that the FHA protein of B. holmesii is more closely related to that of B. avium than to the FHA proteins of the "classical" Bordetellae. Furthermore, we show that similar to FHA of all other species it is transcriptionally regulated by the BvgAS two-component system suggesting similar virulence strategies in the different Bordetella species.

Results and Discussion

Identification and sequence analysis of the fhaB gene of B. holmesii

Based on short regions of highly conserved nucleotide sequences of the 5'- and 3'-ends of the fhaB genes of B. avium and B. pertussis oligonucleotide pairs Fha1F/Fha1R and Fha2F/Fha2R were designed and used for PCR amplification with chromosomal DNA of B. holmesii. For both primer combinations PCR products could be amplified. The sequence analysis of the amplified DNA fragments revealed significant similarity in particular in the deduced amino acid sequence to the respective fhaB sequences of the other Bordetellae. By PCR amplification using the primer pair Fha1F/Fha1R, we found that all B. holmesii strains tested (B. holmesii G7702, B. holmesii G8341, B. holmesii ATCC51541 and B. holmesii No1) harbour orthologues of this fhaB gene (data not shown). Using a genome walking strategy the DNA sequence of the fhaB gene of strain B. holmesii G7702 was completed. The fhaB coding region of B. holmesii starts with a GTG codon and comprises 8,793 bp with a coding capacity for a 304 kDa protein, which is smaller than FHA of B. pertussis (367 kDa) but larger than the respective protein of B. avium (273 kDa). The genome organisation of the fhaB locus of B. holmesii differs from that of the other Bordetella species. In B. holmesii, upstream of the fhaB gene an open reading frame (orfMP) is located with significant similarity to a conserved integral membrane protein of B. bronchiseptica (BB3004) and B. parapertussis (BPP3041) which is transcribed divergently. Directly downstream of the fhaB gene a copy of the insertion element IS1001 is located which is known to occur in B. holmesii and in B. parapertussis. In contrast, in the species belonging to the B. bronchiseptica cluster the fhaB gene is arranged between the divergently transcribed bvgAS locus and the fimbrial operon fimABCD which precedes the fhaC gene whose gene product is involved in the export of FHA. In B. avium an ORF encoding an ABC transporter is present upstream of fhaB, while the downstream gene organization is similar to the B. bronchiseptica cluster (Fig. 1) [3, 17, 18].
Figure 1

Schematic view of the gene loci encoding fhaB in 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.

The overall similarity of the nucleotide sequence of the fhaB gene of B. holmesii with those of B. avium and B. pertussis is low (between 50% and 52%) explaining the failure of previous attempts to detect an fhaB orthologue in this species by Southern hybridization experiments (data not shown). Even in those regions of the B. holmesii fhaB gene (e.g. within the first 600 bp) in which the predicted amino acid sequences of the FHA proteins are quite conserved (see below), the nucleotide sequence similarity is quite limited (59%). On the level of the deduced amino acid sequences, the B. holmesii FHA protein shows an overall similarity of 55% to the FHA protein of B. avium (Fig. 1). The similarity to the FHA, FhaL and FhaS proteins of the members of the B. bronchiseptica complex is less pronounced, e.g. 39.2% to FHA of B. pertussis as calculated using the EMBOSS pairwise alignment algorithms http://www.ebi.ac.uk/emboss/align/ (Table 1). With a sequence similarity of 37.3% the most similar protein outside of the genus Bordetella is an adhesin-like protein from Yersinia frederiksenii, an environmental bacterium that can also be associated with a wide variety of host organisms [28]. This analysis shows that FHA of B. holmesii is more similar to the B. avium protein than to the related factors of the mammalian pathogens, in line with previous observations that the BvgA, BvgS and RecA proteins of B. holmesii and B. avium are more closely related to each other than they are to those of the B. bronchiseptica complex [29, 30]. This contrasts previous data obtained by comparison of the 16S rDNA sequences, which placed B. holmesii as a new species closer to B. pertussis than to B. avium. On the other hand, recent evidence indicated that the gene encoding the 16S RNA of B. holmesii may have been acquired horizontally from B. pertussis [30].
Table 1

Amino acid sequence similarity of the B. holmesii FHA protein and other FHA-like proteins of Bordetella species

 

FHA BP1)

FhaL BP

FhaS BP

FHA BB

FHA BA

FHA BH

39.2/28.12)

29.4/19.9

37.3/26.1

38.0/26.9

55.5/41.5

FHA BP

 

31.5/22.7

49.2/38.8

91.2/89.0

40.0/27.7

FhaL BP

  

30.7/23.1

29.6/21.1

29.2/19.4

FhaS BP

   

43.3/37.3

41.3/27.5

FHA BB

    

39.2/26.8

1) BP: B. pertussis; BB: B. bronchiseptica; BA: B. avium; BH: B. holmesii

2) Sequence similarity in %/Sequence identity in %

For the Sec-dependent secretion across the cytoplasmic membrane, the B. holmesii FHA protein has an extended N-terminal signal sequence, which is very similar to that of the B. pertussis protein. In B. pertussis, the signal sequence is cleaved at an alanine at sequence position 71 probably by the Lep signal peptidase [6]. The B. holmesii FHA protein has also an alanine residue at this position suggesting its processing at this site after transport through the cytoplasmic membrane. In addition, the N-terminus of B. pertussis FHA harbours a domain about 250 amino acid residues in length which is essential for secretion through the outer membrane according to the two-partner secretion model, the so-called TPS domain [31]. The exact nature of the transport signal is not known so far, but two sequence motifs (NPNL and NPNGI) are well conserved among two-partner secreted proteins and at least the NPNL motif plays a role in the secretion of B. pertussis FHA [32]. Both sequence motifs are also present in the FHA protein of B. holmesii suggesting that it is also exported via a two-partner secretion mechanism.

Similarities of the B. holmesii protein with domains of the B. pertussis FHA protein possibly involved in adhesion including the heparin binding (aa 442–862) and carbohydrate recognition domains (CRD) (aa 1141–1279) are very limited and the elucidation of relevant binding activities must await the biochemical characterization of the B. holmesii FHA protein. An intriguing feature of the FHA protein of B. pertussis is the presence of an RGD motif (aa 1097–1099) enabling the protein to interact with integrin receptors [2]. The B. holmesii protein does not contain an RGD motif, instead at sequence position 742–744 it harbours a KGD motif (Fig. 2). In some cases, KGD motifs may be involved in integrin binding [33, 34], however, ascribing such a function to the KGD motif of the B. holmesii protein must await future experimental analysis.
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.

Regulation of expression of the B. holmesii fhaB gene

Upstream of the fhaB gene a divergently transcribed gene is present suggesting that the fhaB gene has a promoter of its own. To analyse the regulatory region of the fhaB gene (P fhaB ) it was cloned upstream of a promoterless gfp gene in the broad host range vector pMMB208 and the resulting plasmid was introduced into B. holmesii. By primer extension analysis with a gfp-specific primer three transcripts (P1 – P3) were identified initiating at sequence positions -58 (P1), -71 (P2) and -88 (P3) with respect to the translational start codon of the fhaB gene (Fig. 3). No obvious -10 and -35 regions could be observed upstream of any of these putative transcriptional start sites. To investigate whether, similar to the other Bordetellae, the BvgAS system is involved in the control of fhaB expression, primer extension analysis was performed on RNA extracted from a B. holmesii bvgAS mutant [27] harbouring the plasmid-borne P fhaB -gfp fusion. Interestingly, the longest of the three transcripts (P3) was not detected in the bvgAS mutant indicating that the synthesis of this transcript is controlled by the BvgAS system, while the other transcripts are constitutively produced under our experimental conditions (Fig. 3). Similarly, the fhaB gene of B. pertussis is controlled by a BvgAS regulated promoter, but some constitutive expression was noted [35].
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.

Although the fhaB gene is transcribed in B. holmesii grown at standard culture conditions, various attempts to detect the B. holmesii FHA protein were not successful so far. The B. holmesii strains used in this study grow very poorly in liquid culture reaching a maximal OD600 of about 0,5. We were unable to detect a protein with a molecular weight corresponding to FHA in the culture supernatants neither by SDS PAGE nor by immune blotting using a polyclonal antiserum against the B. pertussis FHA protein. Similarly, also in whole cell lysates of bacteria grown on BG agar plates no FHA protein could be detected (data not shown). It is therefore possible, that the translation efficiency of the fhaB gene is low, which may be in line with the fact that the open reading frame starts with a GTG codon, or that the protein is processed to smaller fragments than the related proteins of the other Bordetellae.

To further investigate the transcriptional regulation of the fhaB gene by the BvgAS system we performed DNA binding experiments in vitro with purified recombinant BvgABH of B. holmesii [27]. In fact, in band shift experiments binding of the phosphorylated but not of the unphosphorylated BvgABH protein to the fhaB upstream region could be detected (Fig. 4). Binding was specific since addition of unspecific competitor DNA did not interfere with binding of BvgABH-P even in the presence of a 1,000 fold excess of competitor DNA (data not shown). To further characterize BvgA binding to the promoter region, DNase I footprint analysis with BvgABH of B. holmesii was performed. Footprint experiments were carried out on a 312 bp DNA segment ranging from nucleotide position +29 to -283 as numbered with respect to the translational start site of the fhaB gene. The addition of BvgABH-P to the reaction mixture resulted in a large region protected from DNase I digestion ranging from position -40 to -243 with respect to the start codon of fhaB. Within the protected region the appearance of a regular pattern of hypersensitive sites every 10 to 11 nucleotides could be observed (Fig. 5), a phenomenon which was noted previously in the case of the promoter of the bvgAS operon of B. holmesii [27]. Surprisingly, the protected area covers all three transcriptional start sites mapped by primer extension analysis and, accordingly, includes the corresponding core promoter elements. It is not clear whether this observation has in vivo relevance.
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) BvgABH, 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 BvgABH 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 BvgABH (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.

A search for putative BvgA binding sites within the fhaB promoter region revealed the presence of four sequence motifs termed BS1 to BS4, respectively, with similarities to the well defined BvgA binding sites in B. pertussis (Fig. 6) which are located within or close to the region protected by BvgABH-P in the footprint experiments. BS2 and BS3 show high similarity to each other and consist of inverted repeat heptanucleotide sequences centered at position -117 and -65.5 relative to the start site of transcript P3. The left and right half-site motifs of BS2 and BS3 match the consensus half-site motif for binding of B. pertussis BvgA (BvgABP) [15] in 4 and 5 (BS2) and 5 (BS3) out of seven positions. BS1 is arranged as a direct heptanucleotide repeat whose half-sites match the consensus in 5 and 6 positions, respectively, and is centered at position -161. BS4 which consists of an inverted repeat centered at position -47.5 shows the lowest similarity to the consensus heptanucleotide BvgABP binding motif (4 and 3 matches per half-site). The fhaB promoter of B. pertussis comprises a heptanucleotide inverted repeat sequence with high affinity for BvgABP binding centered at position -88.5 relative to the transcriptional start, as well as two additional low affinity binding sites centered at position -67.5 and directly adjacent to the -35 region [14, 15]. Cooperative binding of BvgABP to the secondary binding sites which show only limited similarity to the high-affinity inverted repeat motif is required for full transcriptional activation of the fhaB promoter of B. pertussis [15, 35]. Remarkably, the positions of the low-affinity BvgABP binding sites in the fhaB promoter of B. pertussis and of BS3 and BS4 in the upstream region of fhaB from B. holmesii are almost identical. However, while the high-affinity binding site in the fhaB promoter of B. pertussis is located immediately 5' adjacent to the low-affinity sites, the centers of the inverted repeat sequences BS2 and BS3 are located in a distance of 51 bp. The most prominent sites showing hypersensitivity to DNase I cleavage in footprint experiments map to the region flanked by BS2 and BS3 (Fig. 5).
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 BvgABH-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.

To investigate a functional role of these putative BvgA binding site(s) we performed a deletion analysis of the fhaB promoter region and carried out band shift assays with progressively 5'- truncated DNA fragments lacking BS1 to BS4. As shown in Fig. 7, BvgABH-P bound equally well to DNA fragments comprising the four putative BvgA binding sites BS1 to BS4 and to a DNA fragment lacking BS1 suggesting a negligible role of BS1 for the activation of the BvgA-P dependent promoter of fhaB. In agreement with this assumption, BS1 was only partially protected by BvgABH-P binding in DNase I footprint experiments (Fig. 6). When BvgABH-P was incubated with a DNA fragment lacking both BS1 and BS2 still a distinct DNA-protein complex was formed, however, binding was significantly weaker since a much higher amount of BvgABH-P was required to achieve a band shift. No significant binding of BvgABH-P was detectable to DNA fragments containing only BS4 or no BS box at all. These data suggest a functional role of the highly similar BS2 and BS3 sites for binding of BvgABH-P to the fhaB upstream region.
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 BvgABH protein. Numbers indicate the distance from the Bvg-dependent fhaB transcriptional start site (P3) taken as position +1. (b) Binding of the B. holmesii BvgABH 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 BvgABH. 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.

To test whether these in vitro data have also relevance in vivo, DNA fragments containing various pieces of the fhaB upstream region were cloned in a promoterless gfp expression vector and transferred to the B. holmesii wild type and the bvgAS mutant strain. GFP expression directed from the different constructs was used as a measure for promoter activity. GFP expression was strong in the B. holmesii wild type strain harbouring construct pMMB208-fhaP-gfp0 containing the entire fhaB upstream region, while very low amounts of GFP were detected in the bvgAS mutant harbouring the same plasmid (Fig. 8, compare lanes 1 and 2). These data confirm the BvgAS mediated regulation of fhaB expression which was already observed on the transcriptional level (Fig. 3). Moreover, corroborating the in vitro data, GFP expression was virtually absent in strains B. holmesii G7702 (pMMB208-fhaP-gfp4) and B. holmesii G7702 (pMMB208-fhaP-gfp6) whose gfp expression plasmids contained only site BS4 or did not contain a BS site at all (Fig. 8, lanes 5 and 6). In agreement with the results of the DNA binding experiments a dramatic increase in GFP expression could be noted when the fhaB upstream region in the gfp expression plasmids comprised BS3 or BS2 and BS3 in addition to BS4 (Fig. 8, lanes 4 and 3). The virtually identical GFP expression directed from the pMMB208-fhaP-gfp3 (containing BS3 and BS4) and pMMB208-fhaP-gfp2 (containing BS2 to BS4) plasmids was surprising since the EMSA studies reported above revealed a relatively weak binding of BvgABH-P to a promoter fragment containing BS3 and BS4, while binding to a fragment comprising BS2 to BS4 was very efficient suggesting a prominent role of BS2 for transcriptional activation. Since BS3 is fairly similar to the consensus BvgABP binding motif and is located at the appropriate position to allow the interaction between BvgABH and the C-terminal domain of the α subunit of RNA polymerase, BvgABH binding to BS3 facilitated by DNA topology effects might be sufficient to fully activate the plasmid-borne fhaB promoter in pMMB208-fhaP-gfp3. In the presence of the full length fhaB promoter cooperative protein interactions are likely be involved in the binding of BvgABH to the BS2/BS3 region.
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.

To investigate whether the fhaB promoter of B. holmesii is also recognized by the BvgA protein of B. pertussis, the pMMB208-fhaP-gfp0 plasmid containing the entire promoter region of the B. holmesii fhaB gene fused to GFP was introduced into the B. pertussis strains Tohama I (TI) and BP359. Strong GFP expression was observed by immunoblot analysis in the wildtype strain TI (pMMB208-fhaP-gfp0), while expression of the reporter gene was hardly detectable in the bvgAS mutant BP359 (pMMB208-fhaP-gfp0) (data not shown). Interestingly, primer extension analysis performed with RNA extracted from TI (pMMB208-fhaP-gfp0) and BP359 (pMMB208-fhaP-gfp0) using a gfp-specific oligonucleotide demonstrated that in the wild type strain transcription of gfp starts at two sites, which, however, are identical to the start sites of transcripts P1 and P3 synthesized from constitutive (P1) and bvg-dependent (P3) dependent promoters in B. holmesii. Moreover, as observed in B. holmesii, in B. pertussis the promoter directing the synthesis of transcript P3 is not transcribed anymore when the BvgAS system is inactivated (data not shown). These data suggest that the activation mechanism of the fhaB promoter of B. holmesii by the BvgA proteins of B. holmesii and B. pertussis is remarkably similar. This is surprising since it was recently shown that the BvgA protein of B. holmesii does not bind to and cannot activate the fhaB promoter of B. pertussis, although in particular in its C-terminal output domain it is highly related to the BvgA protein of B. pertussis [27, 36].

Conclusion

Little was known so far about the virulence mechanisms of B. holmesii which can cause pertussis-like disease in humans and within the genus Bordetella was thought to be most closely related to B. pertussis. Previous attempts to identify possible virulence factors related to those of the etiological agent of whooping cough and of the other well-characterized Bordetellae were not successful. Here we describe the identification of a B. holmesii factor related to the major adhesin of the other pathogenic Bordetellae, the filamentous hemagglutinin FHA. This adds to our previous report on the identification of a two-component system in B. holmesii orthologous to the BvgAS two-component system of B. pertussis which in the other pathogenic Bordetellae is the master regulator of virulence gene expression and directly controls the expression of FHA. We show that also in B. holmesii the expression of FHA is regulated by the BvgAS system and that the activation mechanism of the fhaB promoter in B. holmesii resembles that in B. pertussis. These data strongly suggest that basic virulence mechanisms of B. holmesii and of the other pathogenic Bordetellae are related. Furthermore the present study provides further evidence that B. holmesii may be more closely related to the bird pathogen B. avium than to B. pertussis indicating that in the genus Bordetella in different phylogenetic lineages independent strains repeatedly evolved towards being human pathogens.

Methods

Bacterial strains and growth conditions

Bacterial strains used in this study are listed in Table 2. B. holmesii strains, B. pertussis strains and B. bronchiseptica strains were grown on Bordet-Gengou (BG) agar plates supplemented with 20% horse blood [27]. When required, antibiotics were added to the following final concentrations: streptomycin, 100 μg ml-1; spectinomycin, 100 μg ml-1; kanamycin, 50 μg ml-1; gentamycin, 15 μg ml-1; ampicillin, 100 μg ml-1 and chloramphenicol, 20 μg ml-1. Bacterial conjugations were performed as described previously [37], using Escherichia coli SM10 as the donor strain [38]. Protein lysates were prepared from bacteria grown on BG agar plates for 48 h at 37°C which were suspended in saline at a cell density of 1.4 × 108 c.f.u. ml-1.
Table 2

Bacterial strains and plasmids

Bacterial strain or plasmid

Relevant feature(s)

Reference or source

Strains

  

B. holmesii

  

ATCC 515 41

clinical isolate

[19]

No1

clinical isolate

[23]

G8341

clinical isolate

[19]

G7702

clinical isolate

[19]

G7702 bvgA

G7702 with a kanamycin-resistance cassette disrupting the bvgA gene

[27]

B. pertussis

  

Tohama I (TI)

wildtype, but rpsL

[43]

BP359

derivative of TI, bvgA::Tn5

[43]

E. coli

  

DH5α

strain used for high-efficiency transformation

Gibco

SM10

mobilizing strain

[38]

Plasmids

  

PbluescriptSK

high copy number cloning vector

Stratagene

pMMB208

Broad-host-range expression vector

[44]

pKEN2

plasmid containing the promoterless gfp-mut2 gene

[40]

pSK-FP

pSK carrying a 312 bp PCR fragment of B. holmesii G7702 derived from the upstream region of fhaB

This study

pSK-fhaP-gfp0

pSK carrying a fusion between 277 bp of the fhaB promoter region of B. holmesii G7702 harbouring the putative bindings sites BS1–BS4 and the gfp-mut2 gene

This study

pMMB208-fhaP-gfp0

pMMB208 carrying a fusion between 277 bp of the fhaB promoter region of B. holmesii G7702 (BS1–BS4) and the gfp-mut2 gene

This study

pMMB208-fhaP-gfp2

pMMB208 carrying a fusion between 224 bp of the fhaB promoter region of B. holmesii G7702 (BS2–BS4) and the gfp-mut2 gene

This study

pMMB208-fhaP-gfp3

pMMB208 carrying a fusion between 168 bp of the fhaB promoter region of B. holmesii G7702 (BS3–BS4) and the gfp-mut2 gene

This study

pMMB208-fhaP-gfp4

pMMB208 carrying a fusion between 146 bp of the fhaB promoter region of B. holmesii G7702 (BS4) and the gfp-mut2 gene

This study

pMMB208-fhaP-gfp6

pMMB208 carrying a fusion between 111 bp of the fhaB promoter region of B. holmesii G7702 and the gfp-mut2 gene

This study

General techniques

DNA manipulation, cloning procedures and acrylamide gel electrophoresis were carried out according to standard procedures. PCR amplifications were performed with a model T3 thermocycler (Biometra) using Pfu polymerase (Promega) or Taq polymerase (Qbiogene Inc.). Oligonucleotides used in this study are listed in Table 3. All cloned PCR products were subjected to automated sequencing to ensure proper amplification. Immunoblot analysis was performed using a semidry blotting procedure as described previously [39]. Green fluorescent protein (GFP) was detected using rabbit GFP antiserum (Invitrogen).
Table 3

Oligonucleotides used in this study

Oligonucleotide

Sequence (5'-3')*

Restriction sites

Fha1F

5'-CTCATCATCGCCAACCCCAACGG -3'

-

Fha1R

5'-AGCTGGCGCACGCCCAGGCCTG -3'

-

Fha2F

5'-CCCAAGCCCAAGCCCAAGCCCAAGGCC -3'

-

Fha2R

5'-ATAGAAGACCCGGTAGTTCT -3'

-

FhaBamHI

5'-CCTCGGAGGATCC CCTCCATCGA -3'

BamHI

FhaHindIII

5'-TACTTTGCTGAAGCTT AAACGATAG -3'

HindIII

Fhagfp1

5'-CCTCGGAGGATCC CCTCCATCGA -3'

BamHI

Fhagfp2

5'-ACAACGAGAGGATCC GCAGCAA -3'

BamHI

Fhagfp3

5'-CAAAAGGGGATCC ACGGGGCAA -3'

BamHI

Fhagfp4

5'-AGGGTGCGAGGATCC TGACACA -3'

BamHI

Fhagfp6

5'-AAGTGTTGGGATCC GTAGTGTCT -3'

BamHI

Fhagfp7

5'-AACGATCTAGA TCCGCGCTGCCC -3'

XbaI

FhaGR1

5'-GACTATCCTGACACATTGAGGAG -3'

-

FhaGR2

5'-CTACATGTAAGTAGGGCCCTGTG -3'

-

Gfp1

5'-CAAGAATTGGGACAACTCCAGT -3'

-

* Restriction sites introduced for cloning purposes are underlined.

Characterization of the fhaB locus of B. holmesii

Chromosomal DNA of B. holmesii G7702 was used as template for PCR reactions. Primers for PCR reactions were deduced from conserved DNA regions of the fhaB gene of B. pertussis and B. avium. Primer pair Fha1F/Fha1R was deduced from the 5'-end of the fhaB gene (Fha1F: base pair 514 to 536 in fhaB of B. pertussis; base pair 493 to 515 in fhaB of B. avium; Fha1R: base pair 923 to 944 in fhaB of B. pertussis; base pair 902 to 923 in fhaB of B. avium). Primer pair Fha2F/Fha2R was deduced from the 3'-end of the fhaB gene (Fha2F: base pair 10420 to 10445 in fhaB of B. pertussis; base pair 7573 to 7598 in fhaB of B. avium; Fha2R: base pair 10738 to 10757 in fhaB of B. pertussis; base pair 7877 to 7896 in fhaB of B. avium). Using primer pairs Fha1F/Fha1R and Fha2F/Fha2R, two fragments of the expected length (340 bp and 440 bp) could be amplified from chromosomal DNA of B. holmesii G7702. The PCR products were sequenced and the sequence analysis demonstrated that the DNA fragments encoded part of the fhaB homologue of B. holmesii. The entire fhaB gene of B. holmesii was sequenced by a genome walking approach using the Universal Genome Walker Kit (Clontech Inc.).

Construction of B. holmesii and B. pertussis strains containing a plasmid with a fusion of the fhaB promoter region of B. holmesii to a gfp reporter gene

A 277 bp DNA fragment containing the entire promoter region of the fhaB gene was PCR amplified from genomic DNA of B. holmesii G7702 using the primer pair Fhagfp1/Fhagfp7, thereby introducing BamHI and XbaI restriction sites at the 5'- and 3'-terminus, respectively. A DNA fragment containing the promoterless gfp-mut2 gene was excised with XbaI and HindIII from plasmid pKEN [40]. The 277 bp DNA fragment harbouring the fhaB promoter (termed fhaP0) and the gfp fragment were cloned together in plasmid pSK, resulting in plasmid pSK-fhaP-gfp0. The fhaP-gfp0 fragment was then excised by BamHI- and HindIII-digestion and was subsequently ligated into plasmid pMMB208. In the resulting plasmid pMMB208-fhaP-gfp0, the fusion of the promoter fragment and the gfp gene is located in the opposite orientation to the plasmid-borne tac promoter. pMMB208-fhaP-gfp0 was subsequently transformed into E. coli SM10 and transferred by conjugation into various B. holmesii and B. pertussis strains. The same protocol was applied to generate the following constructs, which contain fusions of different fhaB promoter fragments of B. holmesii G7702 with the gfp reporter gene: pMMB208-fhaP-gfp2 (fhaP2, 224 bp, amplified with Fhagfp2/Fhagfp7), pMMB208-fhaP-gfp3 (fhaP3, 168 bp, amplified with Fhagfp3/Fhagfp7), pMMB208-fhaP-gfp4 (fhaP4, 146 bp, amplified with Fhagfp4/Fhagfp7), and pMMB208-fhaP-gfp6 (fhaP6, 111 bp, amplified with Fhagfp6/Fhagfp7).

Primer extension experiments

Total RNA was prepared from bacteria grown on BG agar plates for 48 h at 37°C. Primer extension experiments were carried out essentially as described previously [27] with the primer oligonucleotide Gfp1 (Table 2). Sequencing reaction mixtures, with plasmid pSK-fhaP-gfp0 as template DNA and the appropriate oligonucleotide primer, were analysed on 6% urea-polyacrylamide gels and used as standards for determination of the transcription initiation sites.

Gel retardation experiments

A 277 bp DNA fragment (probe I) containing part of the fhaB upstream region was PCR amplified from genomic DNA of B. holmesii G7702 using primer pair Fhagfp1/Fhagfp7. The PCR fragment was 5'-end labelled with [γ-32P]-ATP using T4 polynucleotide kinase (MBI) and purified using the QIAquick Nucleotide Removal Kit (Qiagen Inc.). The His6-BvgABH protein described previously [27] was diluted in 1 × dilution buffer (2 mM MgCl2, 50 mM KCl, 0.1% Igepal CA 630, 10 mM DTT) and was phosphorylated by incubation with 50 mM acetyl phosphate (Sigma Inc.) for 20 min at room temperature. Increasing amounts of the protein were added to approximately 15,000 cpm of the labelled DNA probe in 20 μl of 1 × binding buffer (10 mM Tris/HCl, pH 8, 10 mM KCl, 5 mM EDTA, 1 mM DTT, 10% glycerol, v/v). The samples were incubated for 20 min at room temperature and were then loaded onto a non-denaturing 4% polyacrylamide gel. Gels were run for 2.5 h at 150 V and subsequently the dried gels were autoradiographed. The same procedure was applied using the following DNA probes, which were amplified from the fhaB upstream region of B. holmesii G7702: probe II (224 bp, amplified by Fhagfp2/Fhagfp7), probe III (163 bp, amplified by FhaGR1/Fhagfp7), probe IV (135 bp, amplified by FhaGR2/Fhagfp7) and probe V (111 bp, amplified by Fhagfp6/Fhagfp7).

DNase I footprinting

DNase I footprint experiments were performed essentially as described previously [41]. A 312 bp DNA fragment containing part of the upstream region of the fhaB gene was PCR amplified from chromosomal DNA of B. holmesii G7702 using primer pair FhaBamHI/FhaHindIII, thereby introducing BamHI and HindIII restriction sites at the 5'- and 3'-terminus, respectively. The purified fhaB upstream fragment was cloned into plasmid pSK. The resulting plasmid pSK-FP was digested with BamHI and 5'-end labelled with [γ-32P]-ATP using T4 polynucleotide kinase. The labelled promoter fragment was excised from the plasmid by HindIII digestion, purified by gel electrophoresis and eluted in 4 ml elution buffer (10 mM Tris/HCl, pH 8, 1 mM EDTA, 300 mM sodium acetate, 0.2% SDS). The eluted probe was then extracted with phenol/chloroform (1:1, v/v) and ethanol precipitated. Binding reaction mixtures contained various concentrations of BvgABH protein and approximately 100,000 cpm of labelled DNA probe in 50 μl of 1 × binding buffer (10 mM Tris/HCl, pH 8, 2 mM MgCl2, 0.1 mM CaCl2, 1 mM DTT, 10% glycerol, v/v). The samples were incubated 20 min at room temperature and then the nucleolytic reactions were initiated by the addition of 1 U DNase I in 1 × binding buffer. After 1 min digestions were terminated by the addition of 140 μl stop buffer (192 mM sodium acetate, 0.14% SDS, 62 μg ml-1 yeast tRNA). The samples were extracted with phenol/chloroform (1:1, v/v), ethanol precipitated and run on a 6% polyacrylamide-urea sequencing gel. A G+A sequencing reaction was also conducted in parallel with the labelled DNA probe and subjected to electrophoresis on the same gel [42].

Accession Number

The DNA sequence reported in this manuscript can be retrieved by the accession number [EMBL:AM491633].

Declarations

Acknowledgements

We thank Susanne Bauer for technical assistance. This work was supported by the priority program SFB479-A2 by the Deutsche Forschungsgemeinschaft.

Authors’ Affiliations

(1)
Lehrstuhl für Mikrobiologi, Biozentrum der Universität Würzburg, Am Hubland, Germany

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