INSERM U447, Institut de Biologie de Lille1 and Département de Microbiologie des Ecosystèmes, Institut Pasteur de Lille2, 1 rue Calmette, F-59019 Lille Cedex, France
Departments of Medicine and Microbiology & Immunology, Stanford University, Stanford, CA 94305, USA3
VA Palo Alto Health Care System 154T, 3801 Miranda Avenue, Palo Alto, CA 94304, USA4
Author for correspondence: David A. Relman. Tel: +1 650 852 3308. Fax: +1 650 852 3291. e-mail: relman{at}cmgm.stanford.edu
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ABSTRACT |
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Keywords: Bordetella bronchiseptica, filamentous haemagglutinin, FHA, secretion, adherence
Abbreviations: FHA, filamentous haemagglutinin; GFP, green fluorescent protein
The GenBank accession numbers for the sequences reported in this paper are AF111794, AF111796, AF111797 and AF111798.
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INTRODUCTION |
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These three closely related organisms produce similar arrays of virulence factors (Rappuoli, 1994 ). Among these, filamentous haemagglutinin (FHA) is regarded as the dominant attachment factor (Arico et al., 1993
; Leininger et al., 1993
; Relman et al., 1989
; Urisu et al., 1986
). B. pertussis FHA (FHA Bp) is also highly immunogenic in humans and is a protective antigen in animal models (Amsbaugh et al., 1993
; Cahill et al., 1993
; Shahin et al., 1992
). The multiple binding activities of the mature 220 kDa protein have been the focus of many studies. FHA is recognized by lactose-containing glycolipids on ciliated respiratory epithelial cells (Tuomanen et al., 1988
). It binds to sulphated carbohydrates of sulphatides and proteoglycans on the surface of epithelial cells or in the extracellular matrix (Brennan et al., 1991
; Menozzi et al., 1991
, 1994a
). In addition, it possesses an RGD motif, which is a canonical recognition sequence for members of the integrin family. This FHA RGD site within the mature protein is recognized by the beta-3 integrin, leucocyte response integrin, in concert with integrin-associated protein (Ishibashi et al., 1994
; Relman et al., 1990
). As the dominant adhesin, differences in FHA-mediated function might contribute to differences in Bordetella host species tropism.
B. bronchiseptica FHA (FHA Bb) has a molecular mass and haemagglutination properties that are similar to those of FHA Bp (Sakurai et al., 1993 ). Electron microscopy studies of FHA Bb preparations suggest similar structure and dimensions to those of FHA Bp (Ohgitani et al., 1991
). Most monoclonal antibodies generated against FHA Bp cross-react with FHA Bb, indicating shared epitopes (Menozzi et al., 1994a
, b
). FHA Bb is required for B. bronchiseptica colonization of the rat trachea (Cotter et al., 1998
).
A noteworthy feature of FHA is its high level of secretion by B. pertussis (Locht et al., 1993 ). FHA represents the most abundant polypeptide in the culture supernatant of B. pertussis grown in vitro. It is also associated with the bacterial outer membrane. Coating of the bacterial outer surface with FHA is thought to be responsible for autoagglutination of the bacteria by FHAFHA homotypic interactions (Menozzi et al., 1994b
).
In B. pertussis, the mature, 220 kDa, form of FHA derives from a 370 kDa FhaB precursor by an as-yet-uncharacterized proteolytic removal of the large C-terminal portion (Domenighini et al., 1990 ; Renauld-Mongenie et al., 1996
). FHA is exported by a signal-peptide-dependent pathway across the cytoplasmic membrane and it requires a single specific accessory protein, FhaC, for translocation across the outer membrane (Jacob-Dubuisson et al., 1996
; Lambert-Buisine et al., 1998
; Willems et al., 1994
). An N-proximal 115-residue-long region of FHA, called the secretion domain, is essential for FHA secretion (Jacob-Dubuisson et al., 1997
). This region probably interacts with FhaC in a specific manner to drive translocation of FHA through the outer membrane. The molecular details of this step are still under investigation. FHA appears to cross both membranes in a coupled fashion and acquires its native conformation upon extrusion from the outer membrane (Guédin et al., 1998
).
FHA appears to be produced and/or secreted at lower levels by B. bronchiseptica than by B. pertussis (Leininger et al., 1993 ; Menozzi et al., 1994a
). Furthermore, as a dominant adherence factor, FHA may play a role in differential host species tropism and receptor recognition. In this study, we set out to characterize the genetic basis for B. bronchiseptica FHA expression and secretion, compare these findings with the corresponding features in B. pertussis, and explore some of the possible factors responsible for differences in FHA expression by these two species.
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METHODS |
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Bordetella culture conditions were as described by Locht et al. (1992) . Phenotypic modulation of GP1 was achieved by the addition of 20 mM MgSO4 to modified StainerScholte (SS) media, or by incubation at room temperature of BordetGengou agar plates.
DNA cloning and other manipulations.
A chromosomal library from B. bronchiseptica GP1 was created by partial Sau3AI restriction of genomic DNA, followed by ligation with BamHI-restricted pHC79 (Hohn & Collins, 1980 ), and transformation of Escherichia coli DH5
. Cosmid gp1#4 was selected after recognition by colony hybridization with a bvgAS gene probe. An EcoRI restriction map suggested that this cosmid also included the fhaB gene. EcoRI restriction fragments of cosmid gp1#4 were subcloned into pBSKSII (Stratagene). pBK117 consists of the pBSKII+ vector plus a 10 kb EcoRI insert comprising the 5' end of B. bronchiseptica fhaB subcloned from cosmid gp1#4. pBK116 consists of the same vector with an additional, naturally occurring 1·2 kb EcoRI fragment from gp1#4 containing the 3' end of B. bronchiseptica fhaB. pBK123 contains a 5 kb BamHI insert corresponding to the 3' half of the 10 kb EcoRI insert of pBK117. These plasmid inserts were further subcloned, and both strands were sequenced using a variety of internal primers. Independent amplification and subcloning of specific regions were performed to confirm the sequence at ambiguous positions. DNA sequence analysis of pBK123 indicated the absence of two Gs at positions 5561 and 9764 relative to the B. pertussis fhaB sequence; these deletions result in frameshifts in the B. bronchiseptica fhaB sequence, leading to premature termination of the predicted protein. Direct PCR amplification of these regions from the chromosome of GP1 and several other B. bronchiseptica isolates showed that both missing nucleotides were present in all the strains. Therefore, we concluded that their absence in pBK123 was due to either cloning artefacts or sequencing errors.
The fhaBgfp fusions were generated as follows. The parent vector, pBK152, was constructed by insertion of a promoterless 729 bp gfpmut3 fragment from an EcoRI/HindIII digest of pGFPmut3 into pBBR1MCS-5 (Cormack et al., 1996 ; Kovach et al., 1994
). Segments (320 bp) of DNA encompassing the promoter region of fhaB were amplified by PCR using chromosomal DNA from the indicated strains and the primers FHA-192FR (5'-GGAAAATTCTGAATTCCCGCGC-3') and FHA328RR (5'-CGGTGgAATtCTCGCTCACGG-3'). The EcoRI sites (underlined) were used for the cloning of the promoter regions upstream of the green fluorescent protein (GFP) coding sequence in pBK152; the lower case letters in FHA328RR reflect nucleotide changes for incorporation of an EcoRI site. The PCR was performed using 30 cycles of 94 °C for 30 s, 45 °C for 30 s and 72 °C for 30 s. The reaction mixture included a final concentration of 5% (v/v) glycerol. Proper orientation of the inserts was confirmed by restriction enzyme digestion and sequence analysis. At least two independent clones were completely sequenced for each construct to detect and avoid errors due to the PCR. Plasmids were transformed into Escherichia coli SM10 and introduced by conjugation into B. bronchiseptica GP1 and B. pertussis BPGR4. These strains were grown in modified SS media supplemented with 100 µg streptomycin ml-1 and 20 µg gentamicin ml-1 at room temperature, at 37 °C or at 37 °C in the presence of 20 mM MgSO4.
pBG4 was described earlier (Renauld-Mongenie et al., 1996 ). It contains a 2·8 kb EcoRIBamHI fragment comprising the first third of B. pertussis fhaB encoding Fha44. pEC40 encodes the B. bronchiseptica Fha44. It was generated using the same procedure as described for pBG4 (Renauld-Mongenie et al., 1996
). Briefly, the 10 kb EcoRI fragment of pBK117 was cloned into pBBR122, a vector that is able to replicate in Bordetella spp. (Antoine & Locht, 1992
). The internal 7·1 kb BamHI fragment was then deleted by digesting the resulting plasmid with BamHI and re-ligating it, thereby yielding pEC40.
pFJD16 was obtained as follows. The unique PvuI site of pBBR1MCS was removed using a T4 polymerase treatment of the PvuI-restricted plasmid, followed by re-ligation. The 2·2 kb SalIXbaI fragment from pFJD16 (Jacob-Dubuisson et al., 1996
), encoding B. pertussis fhaC, was then introduced into the corresponding sites of the modified pBBR1MCS, giving rise to pFJD16
. For the expression of B. bronchiseptica fhaC, the 1·7 kb PvuISacI fhaC fragment of pFJD16
was replaced by the corresponding fragment of B. bronchiseptica fhaC, giving rise to pEC46 (SacI site was located within vector polylinker). This construct was verified by the absence of two restriction sites in B. bronchiseptica fhaC that are present in B. pertussis fhaC, and was partly sequenced to confirm the gene replacement. It should be noted that although this cloning procedure resulted in the replacement of the first 44 residues of FhaC Bb by those of FhaC Bp, sequence data indicate that only one substitution occurs in that region (Willems et al., 1993
and results presented in this paper).
The B. bronchiseptica FhaC coding sequence was PCR amplified from B. bronchiseptica GP1 chromosomal DNA prepared with the Qiagen Genomic DNA kit, using the oligonucleotides 5'-ATGACTGACGCAACGAACCGTTTCC-3' and 5'-GCGTTCTCGCCGGGCTCAGAAACTG-3' as primers. The amplicon was cloned into EcoRV-restricted pZERO (InVitrogen) and sequenced on both strands using the universal and reverse vector-based primers, as well as several internal primers. Two independent clones were sequenced entirely and a third clone was sequenced partially as the sequences of the first two differed at two positions.
Sequencing strategy.
pBK117, pBK123 and pBK116 were used as templates for the sequencing of B. bronchiseptica fhaB. The fhaB gene was sequenced by using the universal and reverse M13 vector-based primers, as well as a series of internal primers that were designed based on the sequences generated. Sequencing was performed using ABI Prism 377 and ABI 373 DNA Sequencers and the kits supplied by the manufacturer (Perkin Elmer). Alignments of sequences and generation of contigs were performed using the DNASTAR program and ABI Sequence Navigator and AutoAssembler (Perkin Elmer). The database accession numbers are as follows: B. bronchiseptica GP1 fhaB, AF111796; B. bronchiseptica GP1 fhaC, AF111794; B. bronchiseptica RB50 upstream region of fhaB, AF111797; B. parapertussis 8234 region upstream of fhaB, AF111798.
Measurement of GFP activity.
GP1 or BPGR4 cells containing fhagfp fusion plasmids were swabbed from plates into modified SS media or taken directly from liquid cultures and diluted to an OD600 of 0·05. Cells were analysed during log and stationary phases and under modulating (20 mM MgSO4 or room temperature) and non-modulating conditions. Cells were fixed in 1% paraformaldehyde in PBS. Median fluorescence at 488 nm was measured from 10000 cells that were gated from 10 to 10000 units (Becton Dickinson FACScan). Four independent exconjugants for each plasmid construct were analysed separately on each of three occasions. The mean value of median fluorescence from each of these occasions was calculated.
Protein analyses.
Proteins from supernatants or cell extracts were analysed by SDS-PAGE on 8 or 10% polyacrylamide gels and stained with Coomassie brilliant blue, or transferred to membranes and probed with antibody. For the comparison of FHA production/secretion between the different strains, haemagglutination assays on fresh culture supernatants or intact cells were performed as described previously (Jacob-Dubuisson et al., 1996 ). ELISA was performed as described previously (Jacob-Dubuisson et al., 1996
). The titres of the supernatants corresponded to the dilution yielding an A420 value threefold higher than the background value. Polyclonal chicken anti-FHA IgY and polyclonal anti-FhaC rat IgG were made by Eurogentec. FHA and Fha44 were purified from BPGR4 culture supernatants by heparin-Sepharose chromatography as described by Menozzi et al. (1991)
. Membrane extracts for the detection of FhaC were prepared as described by Jacob-Dubuisson et al. (1996)
except that the Sarkosyl extraction step was omitted. N-terminal sequencing was performed at the CNRS URA1309, Institut Pasteur de Lille. Sample preparation was as described previously (Jacob-Dubuisson et al., 1996
).
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RESULTS |
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The DNA sequences downstream of the B. pertussis and B. bronchiseptica fhaB genes diverge significantly 69 nt after the stop codon (not shown). The conserved 3' untranslated segment includes a 9 bp perfect inverted repeat both in B. bronchiseptica and in B. pertussis, potentially acting as a rho-independent terminator for fhaB transcription (Domenighini et al., 1990 ; Willems et al., 1992
). All five B. bronchiseptica isolates sequenced in that region are identical, except for two positions. In addition, we sequenced a 44 bp region encompassing the 3' end of the fhaB gene and the region immediately downstream of the termination codon from the B. parapertussis PEP clinical isolate. This sequence was also identical to the corresponding region of the B. bronchiseptica GP1 genome (not shown). The sequence divergence observed 69 nt after the fhaB stop codon between B. bronchiseptica and B. parapertussis on the one hand, and B. pertussis on the other, is consistent with the recently reported 419 bp insertion corresponding to the beginning of a complete fimA gene present in B. bronchiseptica and B. parapertussis, which is truncated in B. pertussis (Boschwitz et al., 1997
).
Analysis of the predicted FHA protein sequence
FHA Bb is predicted to comprise 3634 residues, with an amino acid composition and general sequence features very similar to those of FHA Bp (Domenighini et al., 1990 ). The two proteins are 93·5% identical, with a total of 44 additional residues in the FHA Bb protein (Fig. 2
). This high level of amino acid identity occurs throughout the B. bronchiseptica FHA sequence and is not restricted to the portion encoding the mature protein (approx. 2199 amino acids beginning at the N terminus). The most striking difference between the two proteins lies in the R1 repeat region, between residues 343 and 900 (Fig. 2
), which has been proposed to form one side of the ß hairpin structure of FHA (Makhov et al., 1994
). In the R1 region, FHA Bb contains 40 tandem copies of a 19-residue repeat, two more than in FHA Bp (Fig. 2
). The additional two copies are inserted between the seventh and the eighth repeats as defined by Makhov et al. (1994)
. Both proteins contain 13 tandem copies of a 19-residue repeat in the R2 region, which is located between residues 1475 and 1727 in FHA Bb (Fig. 2
). As in FHA Bp, sequences of the B. bronchiseptica repeats all fit the R1 and R2 consensus sequences defined by Makhov et al. (1994)
, although no residue is strictly conserved within each group.
All the features of FHA Bp with demonstrated or putative biological function are conserved in the predicted FHA Bb. Both RGD sequences, one in the mature FHA sequence (residues 10971099) and the other in the C-terminal processed region of the precursor, are predicted to be present at identical positions in the FHA Bb sequence. Other FHA domains with putative adhesin activity, the heparin-binding domain (FHA Bp residues 442862) (Hannah et al., 1994 ) and the putative carbohydrate-binding domain (positions 11411279) (Liu et al., 1997
; Prasad et al., 1993
) are more than 90 % identical between the two proteins. The ETKEVDG sequence (positions 20962102) with a possible role in integrin recognition of FHA (Rozdzinski et al., 1995
), is identical between the two proteins. An additional feature common to both proteins is an arginine-rich region (RRARR), which serves as a proteolytic cleavage site in mature FHA (Delisse-Gathoye et al., 1990
; Domenighini et al., 1990
; Relman et al., 1989
). The region between FHA Bp residues 1929 and 2019 and, in particular, residues 20012015, contains linear epitopes that are dominant in eliciting an antibody response to FHA (Leininger et al., 1993
; Wilson et al., 1998
); 7 of 91 and 2 of 15 residues differ within these segments, respectively, between the two FHA homologues.
The sequence regions that are important for the secretion of FHA are strictly conserved between the two protein homologues. The NPNL and NPNG motifs shown to be critical for FHA Bp secretion (Jacob-Dubuisson et al., 1997 ) are also present in FHA Bb. The 71-residue signal peptides are identical with the exception of two residues. The N-terminal residue of mature FHA Bp has recently been determined to be a modified glutamine, encoded by the 72nd codon after the initiation codon (Lambert-Buisine et al., 1998
). To determine the first residue of FHA Bb, we overproduced a truncated, N-terminal portion of the GP1 FHA in B. pertussis. This protein, which is the B. bronchiseptica equivalent of Fha44 (Renauld-Mongenie et al., 1996
), was efficiently secreted in B. pertussis, suggesting that the secretion signals of FHA Bb are fully functional in the heterologous host. In a fashion similar to that of B. pertussis Fha44, most of B. bronchiseptica Fha44 was not amenable to N-terminal Edman degradation, suggesting that the N-terminal residue of the B. bronchiseptica FHA is also blocked. In the first few cycles, minute chromatographic peaks corresponding to the QGLVP sequence were detectable (data not shown), indicating that the first residue of FHA Bb is also a modified glutamine, and that the FHA Bb precursor also contains a 71-residue-long signal peptide.
fhaB promoter analysis
Sequence comparison of the ~180 bp promoter/regulatory regions located 5' to the B. pertussis and B. bronchiseptica fhaB genes revealed a number of differences, most of which are located upstream of the transcriptional start site defined for B. pertussis fhaB 70 nt upstream of the ATG (Roy & Falkow, 1991 ; Scarlato et al., 1991
) (Fig. 3
). This region was also amplified by PCR from B. bronchiseptica RB50 and B. parapertussis 8234, and sequenced. A single nucleotide difference between the B. bronchiseptica GP1 (low FHA producer) and the B. bronchiseptica RB50 (intermediate FHA producer) promoters was found 11 bases upstream of the transcription initiation site (Fig. 3
). The GP1 sequence is unique, with a G at this position; all other fhaB promoters analysed contain an A at this position instead. Other substitutions shown in Fig. 3
(e.g. 31 bp differences between GP1 and BP536) distinguish the B. bronchiseptica (low or intermediate FHA producers), B. parapertussis (intermediate FHA producer) and B. pertussis (high FHA producer) fhaB promoter/regulatory regions.
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To investigate whether any of these differences might have an effect on promoter activity, the four promoter/regulatory regions shown in Fig. 3 were cloned upstream of a promoterless GFP-encoding sequence. These constructs were introduced into B. bronchiseptica GP1 as episomal elements and fluorescence was measured as a marker of fhaB promoter activity. B. pertussis, B. parapertussis and B. bronchiseptica RB50 promoters induced similar levels of fluorescence in a Bvg-dependent manner (Fig. 4
). In contrast, the fluorescence of the B. bronchiseptica GP1 GFP fusion was much lower. These observations indicate that the A at position -11 is important for FHA expression, as the RB50 sequence is otherwise identical to that of GP1 in that region. They also indicate that the nucleotide differences upstream of the transcriptional start site in the three species do not have a net effect on FHA promoter activity; however, their possible effect on transcript stability and processing was not addressed. It is interesting to note that the nucleotide found at the -11 position in nine other Bvg-regulated promoters is also an A (Scarlato et al., 1991
). This nucleotide lies at the fourth position of a sequence in the -10 region, 5'-CAG(A/G)CT-3', which bears some resemblance to the prokaryotic
70 -10 consensus motif, 5'-TATAAT-3'. The expression of the fhaB gene is under the control of the two-component system BvgAS, which is down-modulated at low temperatures as well as in the presence of 20 mM MgSO4. Growth at low temperature (Fig. 4
) and in the presence of MgSO4 (not shown) resulted in the complete absence of fluorescence, indicating that none of the nucleotide substitutions affects BvgA regulation of FHA expression. The same GFP reporter constructs were also introduced into B. pertussis BPGR4 under non-modulating conditions and the results obtained were similar to those measured in GP1 (data not shown). This suggests that the differences in genetic backgrounds of the two species do not grossly affect fhaB expression.
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DISCUSSION |
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The dependence of B. pertussis and B. bronchiseptica on FHA during early stages of pathogenesis emphasizes the importance of characterizing the relevant genetic loci and their expressed products. As a first step toward the molecular characterization of FHA Bb secretion, we analysed the B. bronchiseptica fhaB promoter, FHA Bb primary structure and the FhaC Bb accessory protein. Both the structural fhaB gene and the fhaC gene were found to be highly similar to the B. pertussis homologues. Interspecies complementation experiments indicated that the FhaC proteins are interchangeable. In addition, FHA secretion determinants defined in B. pertussis were found to be well conserved in B. bronchiseptica and they were fully functional.
Downstream of the secretion domain, FHA contains two repeat regions, previously defined as R1 and R2 (Makhov et al., 1994 ). FHA Bb contains two additional 19-residue repeats in the R1 region. However, this addition did not appear to interfere with the secretion competence or the stability of the protein, as shown by the efficient secretion of B. bronchiseptica Fha44, which contains the first 27 of the 40 R1 repeats, in B. pertussis. This difference in the number of R1 repeats is therefore not critical for secretion and protein stability, as was also suggested by the isolation of a spontaneous B. pertussis Fha44 mutant with a 39-residue deletion corresponding to two repeats in the R1 region. This mutant Fha44 protein remained highly proficient for secretion (our unpublished observation). The R1 repeat region thus tolerates a certain level of variation, possibly indicating some structural plasticity. It should also be noted that both the B. bronchiseptica Fha44 homologue and the B. pertussis mutant Fha44 were able to bind to heparinSepharose as efficiently as B. pertussis Fha44, and all three proteins eluted under similar conditions (our unpublished observations). This indicates that the binding site for sulphated sugars, previously mapped in the R1 repeat region (Hannah et al., 1994
), is not grossly altered by these structural variations. Other domains with putative or proven roles in FHA-associated adherence functions are predicted to be highly conserved at the level of primary structure in these two species.
The C-terminal domains of the FhaB precursors are highly conserved as well. The function of this domain is not fully understood. A complete deletion of the gene segment corresponding to the 150 kDa C-terminal domain severely hampers FHA secretion in B. pertussis (Renauld-Mongenie et al., 1996 ). However, this region is widely tolerant to smaller deletions (our unpublished observation) with respect to the secretion of FHA. Nevertheless, without creating unmarked fhaB chromosomal allelic exchange strains (which presents substantial technical challenges), we cannot fully assess the role of the C-terminal FHA domain in FHA secretion.
An interesting difference between the B. bronchiseptica GP1 and the B. pertussis BP536 fhaB genes lies in their promoter regions. In the GP1 promoter, a conserved A in the putative -10 motif was replaced by a G. This A to G change was not found in the other B. bronchiseptica fhaB promoter sequences analysed in the course of this work. Remarkably, an A is invariably found at this position in Bvg-regulated promoters (Scarlato et al., 1991 ). The A to G substitution in the GP1 promoter resulted in a significant decrease in fhaB expression, as assessed by gfp reporter gene fusion experiments, and the decrease in promoter strength is likely to be responsible for the significantly lower FHA production in GP1 compared to other B. bronchiseptica strains. It is not known whether this low level of FHA expression reflects an adaptation of the GP1 strain to a particular host or niche and confers some advantage upon this isolate. We cannot rule out the possibility that other interspecies fhaB upstream sequence polymorphisms (Fig. 3
) might result in altered fhaB transcript processing or stability, and explain some of the differences in levels of expressed FHA protein.
The cloning and sequencing of the B. bronchiseptica fhaC gene revealed that the accessory protein is also very similar to its B. pertussis homologue. Complementation of the B. pertussis fhaC gene by the B. bronchiseptica fhaC gene resulted in high levels of FHA production and secretion in B. pertussis, indicating that the accessory proteins are also functionally interchangeable. However, given our fhaC-cloning strategy (see Methods), we can not assess the possible role of the GP1 FhaC G14V substitution in modifying FhaC function. We have also shown that FhaC is produced at levels similar to those in B. pertussis in two B. bronchiseptica strains which produce and secrete FHA at low and intermediate levels, respectively. Therefore, the primary structure or the amount of the FhaC accessory protein in B. bronchiseptica probably does not account for the lower level of FHA secretion by that species.
The function of bacterial adhesins is dictated and regulated at the levels of primary sequence, promoter activity, transcript and protein stability, secretion, and by the context in which adhesins are presented at the bacterial surface. FHA is an important Bordetella adherence factor and may play a role in determining host range. We and others have found that FHA is expressed and secreted at different levels by B. bronchiseptica and B. pertussis. Our data suggest that this variation in FHA secretion/expression may be due to differences in cell-envelope composition, or to differences in fhaB-transcript or FHA-protein stability (protein degradation) between the two organisms. These data provide a starting point for further analysis of FHA function and Bordetella host species tropism.
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ACKNOWLEDGEMENTS |
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Received 1 November 1999;
revised 13 January 2000;
accepted 26 January 2000.