Identification and regulation of expression of a gene encoding a filamentous hemagglutinin-related protein in Bordetella holmesii
© Link et al; licensee BioMed Central Ltd. 2007
Received: 07 June 2007
Accepted: 07 November 2007
Published: 07 November 2007
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.
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.
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.
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 . 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 . In agreement with their close relationship these organisms produce highly related virulence factors such as several toxins and colonization factors .
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 . 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 . 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 . 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) . 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 . 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 . 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 .
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 [22–24], 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 . 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 .
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
Amino acid sequence similarity of the B. holmesii FHA protein and other FHA-like proteins of Bordetella species
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 . 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 . 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 . 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.
Regulation of expression of the B. holmesii fhaB gene
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 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].
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.
Bacterial strains and growth conditions
Bacterial strains and plasmids
Bacterial strain or plasmid
Reference or source
ATCC 515 41
G7702 with a kanamycin-resistance cassette disrupting the bvgA gene
Tohama I (TI)
wildtype, but rpsL
derivative of TI, bvgA::Tn5
strain used for high-efficiency transformation
high copy number cloning vector
Broad-host-range expression vector
plasmid containing the promoterless gfp-mut2 gene
pSK carrying a 312 bp PCR fragment of B. holmesii G7702 derived from the upstream region of fhaB
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
pMMB208 carrying a fusion between 277 bp of the fhaB promoter region of B. holmesii G7702 (BS1–BS4) and the gfp-mut2 gene
pMMB208 carrying a fusion between 224 bp of the fhaB promoter region of B. holmesii G7702 (BS2–BS4) and the gfp-mut2 gene
pMMB208 carrying a fusion between 168 bp of the fhaB promoter region of B. holmesii G7702 (BS3–BS4) and the gfp-mut2 gene
pMMB208 carrying a fusion between 146 bp of the fhaB promoter region of B. holmesii G7702 (BS4) and the gfp-mut2 gene
pMMB208 carrying a fusion between 111 bp of the fhaB promoter region of B. holmesii G7702 and the gfp-mut2 gene
Oligonucleotides used in this study
5'-CCTCGGAGGATCC CCTCCATCGA -3'
5'-TACTTTGCTGAAGCTT AAACGATAG -3'
5'-CCTCGGAGGATCC CCTCCATCGA -3'
5'-ACAACGAGAGGATCC GCAGCAA -3'
5'-CAAAAGGGGATCC ACGGGGCAA -3'
5'-AGGGTGCGAGGATCC TGACACA -3'
5'-AAGTGTTGGGATCC GTAGTGTCT -3'
5'-AACGATCTAGA TCCGCGCTGCCC -3'
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 . 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  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  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 . 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 .
The DNA sequence reported in this manuscript can be retrieved by the accession number [EMBL:AM491633].
We thank Susanne Bauer for technical assistance. This work was supported by the priority program SFB479-A2 by the Deutsche Forschungsgemeinschaft.
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