JB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Donato, G. M.
Right arrow Articles by Hewlett, E. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Donato, G. M.
Right arrow Articles by Hewlett, E. L.
Journal of Bacteriology, November 2005, p. 7579-7588, Vol. 187, No. 22
0021-9193/05/$08.00+0     doi:10.1128/JB.187.22.7579-7588.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Adenylate Cyclase Toxin (ACT) from Bordetella hinzii: Characterization and Differences from ACT of Bordetella pertussis

Gina M. Donato, Hung-Lun J. Hsia, Candace S. Green, and Erik L. Hewlett*

Division of Infectious Diseases and International Health, Departments of Medicine and Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908

Received 6 May 2005/ Accepted 6 September 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bordetella hinzii is a commensal respiratory microorganism in poultry but is increasingly being recognized as an opportunistic pathogen in immunocompromised humans. Although associated with a variety of disease states, practically nothing is known about the mechanisms employed by this bacterium. In this study, we show by DNA sequencing and reverse transcription-PCR that both commensal and clinical strains of B. hinzii possess and transcriptionally express cyaA, the gene encoding adenylate cyclase toxin (ACT) in other pathogenic Bordetella species. By Western blotting, we also found that B. hinzii produces full-length ACT protein in quantities that are comparable to those made by B. pertussis. In contrast to B. pertussis ACT, however, ACT from B. hinzii is less extractable from whole bacteria, nonhemolytic, has a 50-fold reduction in adenylate cyclase activity, and is unable to elevate cyclic AMP levels in host macrophages (nontoxic). The decrease in enzymatic activity is attributable, at least in part, to a decreased binding affinity of B. hinzii ACT for calmodulin, the eukaryotic activator of B. pertussis ACT. In addition, we demonstrate that the lack of intoxication by B. hinzii ACT may be due to the absence of expression of cyaC, the gene encoding the accessory protein required for the acylation of B. pertussis ACT. These results demonstrate the expression of ACT by B. hinzii and represent the first characterization of a potential virulence factor of this organism.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There are currently a total of eight members of the genus Bordetella. The four "classical" species, B. pertussis, B. parapertussis, B. bronchiseptica, and B. avium, all cause similar upper respiratory tract infections in a variety of hosts. The strictly human pathogen, B. pertussis, is the causative agent of the disease whooping cough (pertussis) (35). B. parapertussis causes a milder form of pertussis in humans and sheep. B. bronchiseptica is primarily a veterinary pathogen infecting a wide range of animals, including dogs, rabbits, and horses (29), whereas B. avium is the etiological agent of bordetellosis in turkeys and other birds (43, 74).

The remaining four Bordetella species have been discovered in the past decade. B. holmesii has most often been associated with septicemia in patients with underlying disorders (61, 73, 79, 91) but has also been isolated from immunocompetent hosts (60, 68) and nasopharyngeal specimens of patients with pertussis-like symptoms (56, 94). B. trematum has been isolated from human wounds and ear infections (83). B. petrii is the only one of the Bordetella species with an environmental reservoir, having been found in an anaerobic bioreactor culture enriched from river sediment (86). Finally, B. hinzii represents an interesting member of the Bordetella genus, since it has been isolated from both animals and multiple sites within humans. Originally identified as a commensal organism in poultry (84), B. hinzii has subsequently been isolated from humans in sputum and bronchoalveolar lavage fluid (22, 84), respiratory secretions (from cystic fibrosis patients) (13, 21), blood cultures (15, 42), and bile secretions (2). A previously misidentified bacterial strain from a rabbit was also confirmed to be the first nonhuman mammalian B. hinzii isolate (65). Despite being increasingly recognized as an important opportunistic pathogen (22) associated with human disease, virtually nothing is known about the virulence factors employed by this species or the pathogenesis of disease it causes.

The three species (B. pertussis, B. parapertussis, and B. bronchiseptica) whose genomes are completely sequenced and annotated (63) are closely related, maintaining a common set of virulence determinants, including adhesins (fimbriae, filamentous hemagglutinin, and pertactin) and toxins (adenylate cyclase toxin [ACT], dermonecrotic toxin, and tracheal cytotoxin). B. pertussis also possesses the species-specific pertussis toxin. The bacteria coordinate the differential regulation of virulence gene expression through the two-component regulatory system BvgAS (1, 78). The inner membrane BvgS sensor recognizes a stimulatory signal, initiating a phosphorelay cascade which culminates in the phosphorylation of the cytoplasmic response regulator, BvgA (81, 82). Activated BvgA~P, in turn, binds to DNA sequences within the promoter region and transcriptionally activates the expression of specific virulence genes (7-9, 40, 41, 48, 71).

Among the genes whose expression is turned on by BvgAS is cyaA, the locus encoding ACT. ACT is a large, modular, bifunctional protein with both adenylate cyclase and hemolytic activities (27, 28). The C-terminal hemolysin domain is homologous to the RTX (repeats-in-toxin) family of bacterial toxins, while the N-terminal 400 amino acids contain the catalytic domain responsible for enzymatic activity (52). Once the catalytic adenylate cyclase domain of the toxin enters a susceptible target cell, it binds to and is activated by eukaryotic calmodulin, converting ATP to cyclic AMP (cAMP) (30, 34, 92). This ACT-mediated production of supraphysiological levels of intracellular cAMP intoxicates host immune cells, leading to the inhibition of phagocytosis, chemotaxis, and the oxidative burst (14, 20, 64, 87). In addition to suppressing normal inflammatory cell function, ACT is also capable of promoting apoptotic cell death in macrophages (44, 46). Presumably by disarming host immune effectors, ACT allows the bacteria to escape host defenses.

In this study, we begin to characterize factors potentially involved in Bordetella hinzii infection, in the hope of augmenting the minimal knowledge of this intriguing organism that can act as both a commensal and a pathogen. We have focused on ACT as a likely candidate in B. hinzii pathogenesis because in other Bordetella species it affects different cell types, possesses multiple important functions, and is essential to virulence (32, 45, 88, 90). Contrary to a previous report by Gerlach et al. (25), we have found that B. hinzii expresses cyaA and produces a protein that retains adenylate cyclase enzymatic activity but differs in some other properties relative to the ACT of B. pertussis.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and growth conditions. B. hinzii type strain LMG 13501 (84) was purchased from the American Type Culture Collection (no. 51783). BC-306 (42) is a clinical isolate from a septicemia patient and was kindly provided by Brad Cookson (University of Washington). BP338 is a wild-type B. pertussis laboratory strain (89). B. pertussis was grown on Bordet-Gengou (BG) agar (Difco) containing 10% defibrinated sheep blood (Cocalico) for 3 days at 37°C and modified Stainer-Scholte liquid medium (SSM) (77) at 35.5°C. B. hinzii strains were grown overnight in media identical to those for B. pertussis or on trypticase soy agar (TSA) and broth (TSB) (Difco), as suggested by the source of each strain (American Type Culture Collection) (83, 84). For growth curves, overnight cultures were diluted into the appropriate media to a starting optical density at 650 nm (OD650) of <0.1, and aliquots were removed over the time course, serially diluted, and plated on TSA. Plates were incubated overnight at 37°C, and CFU/ml were determined.

General techniques. Genomic DNA was prepared according to the manufacturer's instructions using the DNeasy tissue kit (QIAGEN). All the PCR primers utilized for the presence of cyaA and cyaC were derived from the B. pertussis genomic sequence (GenBank accession numbers Y00545 and M57286, respectively), and reactions were performed with a GeneAmp 2400 (Perkin-Elmer) under empirically determined conditions. Due to the high GC content of Bordetella species, all DNA was amplified with BIO-X-ACT short DNA polymerase (Bioline). PCR products were visualized on 1% ethidium bromide-stained agarose gels with a FOTO/Analyst Investigator Eclipse imaging system (Fotodyne) and purified using the QIAquick kit (QIAGEN) before being sequenced at the University of Virginia Biomolecular Research Facility.

Reverse transcription-PCR (RT-PCR). Bacterial strains grown in SSM to equivalent cell densities (~3 x 109 to 6 x 109 bacteria/ml) were centrifuged, and RNA was isolated from the pellets with the Aurum total RNA kit following the manufacturer's protocol (Bio-Rad). DNase-treated RNA (DNA free; Ambion) was run on an agarose gel to verify rRNA band integrity. Equal amounts of RNA (480 ng), as determined by using RiboGreen (Molecular Probes), were added to the iScript cDNA synthesis (Bio-Rad) reaction mixture, and the reaction was carried out in the presence and absence of reverse transcriptase. An equal volume of each cDNA was used as a template for amplification with the following gene-specific primers: cyaA forward, 5'-CAACCCCCATTCCACCAG-3', and reverse, 5'-GCGAGCGATTTTCCACAAC-3' (primers were generated from GenBank accession no. Y00545); ptx forward, 5'-ACCGCAAGAACAGGCTG-3', and reverse, 5'-GTCGATCGGCATGCTGTTC-3' (primer sequences were taken from Kinnear et al. [47] and GenBank accession no. M14378); recA forward, 5'-GTCGAACACGACATCCAG-3', and reverse, 5'-CCGAGTCGATGACGATCAG-3' (primers from GenBank accession no. X53457); and cyaC forward, 5'-CGACGACTTCGCGGCACTG-3', reverse-A, 5'-CATAGGAGAGTTCGGTGTCG-3', and reverse-B, 5'-TCAGGCGGTGCCCCGGCC-3' (primers were from GenBank accession no. M57286). PCR products were electrophoresed on 1% or 2% agarose gels, stained with ethidium bromide, detected with a Fotodyne imaging system, and quantitated with ImageQuant 5.2 software (Molecular Dynamics). Band intensities were normalized to recA, a non-bvg-regulated housekeeping gene (49, 57). Similar results were also obtained using sodB (48) as the normalizing standard. Mock reactions in the absence of reverse transcriptase produced no PCR product, indicating the absence of contaminating genomic DNA in the RNA preparations. We ensured that the number of rounds of PCR chosen was suitable by removing aliquots at different cycles, quantitating product band intensities, and determining the linear portion of the PCR amplification curve.

Protein preparation and Western blotting. Strains were grown to logarithmic phase in the appropriate medium (SSM for BP338 and TSB for B. hinzii). An aliquot (1 ml) of each was centrifuged, and the bacterial pellet was resuspended in 1 ml of fresh medium. Equal lysate volumes were solubilized, electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide Criterion gels (Bio-Rad), transferred to polyvinylidene difluoride (PVDF; Osmonics, Inc.), probed with polyclonal ACT antiserum (1:1,000 dilution) and goat anti-rabbit horseradish peroxidase-conjugated immunoglobulin G (Jackson ImmunoResearch), and detected chromogenically with 4-chloro-1-naphthol substrate.

ACT purification. Each strain was grown in 1 liter of SSM, 0.2 mg/ml thimerosol was added, and bacteria were centrifuged at 13,000 x g (9,000 rpm) for 50 min. Pellets were resuspended in 20 ml medium, sonicated on ice in seven 1-min bursts, mixed with 4 M urea, and rotated overnight at 4°C. Suspensions were centrifuged at 27,000 x g (15,000 rpm) for 1 h, and the urea-extracted supernatants were diluted 1:4 in 50 mM Tris, pH 7.5, to reduce the final urea concentration to 1 M. To this material, 1 mM CaCl2 and 1 ml of prewashed calmodulin-Sepharose 4B (Amersham) was added and rotated overnight at 4°C. Columns were packed, nonspecific proteins were washed off with 50 mM Tris, pH 7.5, 0.5 M NaCl, 1 mM CaCl2, and ACT was eluted in a TEE/8 M urea buffer (TEE = 10 mM tricine, pH 8.0, 0.5 mM EGTA, 0.5 mM EDTA). Purification fractions were solubilized, and ACT was detected in Western blot assays as described above. Full-length ACT protein bands were quantitated with ImageQuant 5.2 software. Protein concentrations were determined with a BCA protein assay kit (Pierce). The Blue-Sepharose purification scheme was essentially the same except reconstituted and prewashed Blue-Sepharose CL-6B (Sigma) replaced calmodulin (CaM)-Sepharose as the column matrix.

Adenylate cyclase enzymatic activity. Adenylate cyclase enzymatic activity was measured by the conversion of [32P]ATP to [32P]cAMP in a cell-free system as previously described (38). Briefly, each sample contained purified ACT diluted into a buffer containing 60 mM tricine, 10 mM MgCl2, 2 mM ATP, 1 µM calmodulin, and approximately 2 x 105 cpm of [{alpha}-32P]ATP at pH 8.0. Reactions were carried out at 30°C for 10 min and terminated by adding a stop solution (1% SDS, 20 mM ATP, and 6.24 mM cAMP with 2 x 104 cpm of [3H]cAMP). Radiolabeled cAMP was separated and measured by the double column method of Salomon et al. (70). Final urea concentration in the buffer control was approximately 1 M and ranged from 1 mM to 100 mM in the experimental samples.

Intoxication. J774A.1 cells (murine macrophage cell line) were grown to ~80% confluence in 24-well tissue culture plates in Dulbecco's modified Eagle's medium with high glucose (Gibco) plus 10% heat-inactivated fetal bovine serum (Gibco) at 37°C in 5% CO2. Cells were washed with Hank's balanced salt solution (HBSS; Gibco), purified ACT was added at various concentrations, and the total final volume was adjusted to 1 ml/well with HBSS. Cells were incubated for 1 h at 37°C in 5% CO2 and then washed three times with cold HBSS and lysed by incubation with 0.1 N HCl at room temperature for at least 30 min. Cells and cellular debris were pelleted by centrifugation, and intracellular cAMP from the supernatant was determined by radioimmunoassay (10).

Blue-Sepharose binding. The binding of ACT to Blue-Sepharose was performed basically as described by Ladant (50). Briefly, purified ACT was diluted 10-fold in a buffer consisting of 50 mM Tris-HCl, pH 8.0, 0.1 mM CaCl2, 0.1% Nonidet P-40, and 20% glycerol. An aliquot (200 µl) was mixed with 10 µl prewashed Blue-Sepharose CL-6B in the absence or presence of 100 nM calmodulin at 4°C for 5 h. Tubes were centrifuged, and ACT enzyme activity in the supernatants was measured. Total activity was determined by incubating equivalent toxin samples with CaM in the absence of Blue-Sepharose, under the same conditions.

Nucleotide sequence accession numbers. B. hinzii type (accession no. DQ007078) and BC-306 (accession no. DQ102773) cyaA and type (accession no. DQ141547) and BC-306 (accession no. DQ141548) cyaC DNA sequences were deposited in GenBank.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
B. hinzii growth characteristics. For decades before B. hinzii was classified as its own species, isolates were often identified as B. avium, B. bronchiseptica, or Alcaligenes species (13, 65), due to their similar biochemical profiles (2, 42). More recently, however, it has been shown by whole-cell protein patterns and rRNA ribotyping that genetically, B. hinzii is most closely related to B. bronchiseptica and B. parapertussis (21, 42, 84). Using the B. hinzii type strain isolated from a chicken trachea (LMG 13501, herein referred to as B. hinzii type) (84) and a clinical isolate, BC-306 (42), we have observed that both strains grow well in liquid and semisolid media. Colonies were visible after overnight incubation (16 to 20 h) at 37°C on TSA. On BG agar supplemented with blood, colonies were raised, gray/white, and nonhemolytic. This differs from the hemolytic colony phenotype attributed to ACT production in B. pertussis and B. bronchiseptica. Both strains were indistinguishable from other Bordetella species in a Gram stain, appearing as small, lightly stained gram-negative coccobacilli.

In order to determine which laboratory media would sustain B. hinzii growth, we cultured each strain in peptone-rich TSB and chemically defined SSM designed for B. pertussis (77). As shown in the growth curves in Fig. 1, the B. hinzii strains grew equivalently to each other and consistently reached higher OD values in TSB than SSM (Fig. 1A). There was no discernible difference in the number of live bacteria between the two liquid media at early time points, but after 11 h there were two- to fourfold fewer viable bacteria grown in SSM relative to TSB (Fig. 1B). This difference in bacterial density during stationary phase may indicate that nutrients necessary for B. hinzii growth are scarce or depleted more readily in SSM than in the nutrient-rich TSB.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1. B. hinzii growth in SSM and TSB. Strains were grown in the indicated media at 35.5°C, aliquots were removed at specific time points, and the OD650 was measured with a spectrophotometer (A) and serial dilutions were plated on TSA and viable colonies were counted (B). Graphs are representative of typical results, and data are plotted on a logarithmic scale.

 
cyaA expression. Although the existence of B. hinzii isolates has been known for many years, there remains a paucity of data on the components expressed by this microorganism. We investigated whether B. hinzii possesses cyaA, since this gene encodes a protein (ACT) that is both conserved among most pathogenic bordetellae (16, 75), with the exception of B. avium, and required for virulence (32, 45, 88, 90). We amplified overlapping cyaA PCR fragments from BP338, B. hinzii type, and BC-306 chromosomal DNA and sequenced both strands of the products. The sequences of cyaA in all three strains were identical to each other (GenBank accession no. DQ007078 and DQ102773), as well as to the whole genome-derived sequence from B. pertussis strain Tohama I (63). Sequence identity encompassed the entire cyaA coding region plus additional DNA upstream to bp –305, relative to the B. pertussis cyaA transcriptional start site (53). Thus, not only are there no mutations within the cyaA structural gene, but the putative promoter carrying all regulatory elements (ribosome binding site, –10 Pribnow box, and –35 region) appears intact.

To ensure that cyaA is not a cryptic or pseudogene in B. hinzii, we performed RT-PCR assays. Gene-specific PCR primers were used to amplify cDNA synthesized from RNA isolated from B. hinzii type, BC-306, and BP338 grown to equivalent bacterial concentrations. As depicted in Fig. 2 and Table 1, both B. hinzii strains expressed cyaA, although there appeared to be a considerable difference in transcript levels between the two strains. While the amount of the internal control recA mRNA was comparable between B. hinzii type and BC-306, cyaA transcription was greater than twofold higher in BC-306 than B. hinzii type. Due to the difference in visible recA product between B. hinzii and B. pertussis, when cyaA expression was normalized to this housekeeping gene (49, 57), there was slightly more, but not statistically significantly more (P > 0.3), relative cyaA production in the B. hinzii strains (Table 1). Whether this represents a true differential expression of cyaA between the species or is due solely to the decreased basal level of recA in B. hinzii is unclear. As a control for RNA cross-contamination between strains, we also assessed ptx expression, a B. pertussis-specific gene. As expected, ptx was only expressed from BP338 (Fig. 2). We concluded from these data that commensal and clinical isolates of B. hinzii contain a chromosomally located cyaA gene that is both intact and transcriptionally active.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 2. RT-PCR analysis of cyaA mRNA expression. An equal volume of each RT-PCR mixture was electrophoresed on the same 1% agarose gel and visualized by ethidium bromide staining. Gene-specific panels were separated for visual purposes only. Product sizes: cyaA, 610 bp; recA, 337 bp; ptx, 834 bp. M, hyperladder I DNA standards (Bioline) with sizes indicated in kb to the left. Type, B. hinzii type; 306, BC-306; Bp, B. pertussis BP338.

 

View this table:
[in this window]
[in a new window]
  		TABLE 1. Quantitation of band intensities from RT-PCRa

 
ACT protein production, purification, and activity. To test whether B. hinzii cyaA transcription correlates to protein production, we probed for ACT from solubilized bacterial pellets. As shown in the Western blot in Fig. 3, the B. hinzii strains produced full-length ACT that was equivalent to B. pertussis in both size and total quantity. As we would surmise from the cyaA gene sequence, there seems to be no gross alteration in the corresponding ACT protein product from B. hinzii.



View larger version (83K):
[in this window]
[in a new window]
  		FIG. 3. Western blot of ACT expression. Equal volumes of whole-cell bacterial lysates were electrophoresed on a 15% SDS-polyacrylamide gel, transferred to PVDF, and probed with polyclonal rabbit anti-ACT serum. Proteins were detected chromogenically. Std, Precision Plus dual color protein standards (Bio-Rad) with sizes in kDa to the left; arrow, full-length ACT; Bp, B. pertussis BP338; type, B. hinzii type; 306, BC-306.

 
The purification of endogenous ACT from B. pertussis (39) and B. pertussis ACT synthesized in Escherichia coli (72) makes use of the toxin's specificity for binding CaM. By virtue of its identical DNA sequence to B. pertussis, the B. hinzii cyaA gene is expected to encode a functional calmodulin binding domain, so we employed techniques developed for B. pertussis ACT isolation to purify ACT from B. hinzii. By utilizing sonication, urea extraction, and CaM affinity chromatography, we found that B. hinzii type and BC-306 both made ACT protein that was capable of binding calmodulin beads (Fig. 4). However, when compared to ACT purified from B. pertussis (BP338) under the same conditions, there were some noticeable differences. First, although the starting culture for each strain contained roughly an equal number of bacteria, there was far less full-length ACT protein extracted from B. hinzii than BP338. Secondly, the B. hinzii ACT appeared to bind CaM less well than its BP338 counterpart. When quantitated, the amount of full-length ACT in the unbound fraction accounted for less than 10% of the BP338 ACT total, whereas an equal proportion of B. hinzii type ACT was detected in the unbound void volume as the first elution fraction. Thus, almost 40% of the total B. hinzii ACT extracted never bound the CaM-Sepharose matrix.



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 4. CaM-Sepharose purification of ACT. Equal volumes of each column fraction were electrophoresed on 10% SDS-polyacrylamide gels, transferred to PVDF, and probed with polyclonal rabbit anti-ACT serum. Proteins were detected chromogenically. All BP338 and B. hinzii type fractions were processed on the same gel, while the BC-306 purification was run separately. V, void volume; W, wash; E1 to -3, elutions; Std, Precision Plus dual color protein standards with sizes in kDa to the right; arrow, full-length ACT.

 
Two distinct B. pertussis ACT structural domains bestow functionally independent activities on the toxin: hemolytic and adenylate cyclase enzymatic activities (69). The former is measured as the lysis of erythrocytes (18), while the latter represents the toxin's ability to catalyze ATP into cAMP (34, 92). The in vitro conversion of ATP to cAMP in a cell-free system is enzymatic activity. This is distinguished from the process of intoxication, also called toxin activity, whereby a portion of ACT must interact with and enter eukaryotic target cells in order to elicit increases in host intracellular cAMP pools (39). We evaluated B. hinzii-purified ACT in these established ACT biological assays (Fig. 5). The urea-containing buffer in which ACT was eluted and stored postpurification (see Materials and Methods) served as a control in each assay. ACT from B. hinzii strains exhibited a low, but consistent and reproducible, enzymatic activity in the presence of CaM that was 50-fold less than BP338 ACT (P < 0.002) (Fig. 5A). This corresponds to a specific activity of ACT from each B. hinzii strain of approximately 0.2 µmol/min/mg of toxin protein.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 5. ACT enzymatic and toxin activities. (A) Purified ACT from B. hinzii type, BC-306, and BP338 was assayed for in vitro adenylate cyclase enzymatic activity in the presence of CaM according to the procedures in Materials and Methods. Specific activity for each sample was determined by adjusting enzymatic activity by total ACT protein added. Raw buffer control enzymatic activity was 0.02 ± 0.004 pmol cAMP/min/µl. (B) J774.1 macrophages (~106 cells) were incubated with various concentrations of ACT for 1 h at 37°C, and intracellular cAMP was measured according to the intoxication procedure in Materials and Methods. Toxin activity is reported as a function of macrophage total protein. Buffer control contained 4.0 ± 0.5 pmol cAMP/mg cell protein. All data represent the mean ± standard error of the mean for two independent experiments done in duplicate and are plotted on logarithmic scales.

 
Interestingly, these B. hinzii ACT samples did not increase cAMP levels in host macrophages (Fig. 5B). Over a 1,000-fold range, BP338 ACT intoxicated in a concentration-dependent manner, whereas B. hinzii ACT activity remained equivalent to the buffer control. Increasing the length of time ACT was incubated with target cells also had no effect on B. hinzii ACT intoxication and even decreased the resultant intracellular cAMP concentrations produced by BP338 ACT (data not shown). This was consistent with previous studies (66; unpublished data) that indicated maximum intracellular cAMP generated by ACT occurs within 30 to 60 min and then readily diminishes. Unsurprisingly, these ACT preparations were also nonhemolytic. Considering the much higher concentration of ACT required to lyse erythrocytes (18) versus the amount needed to intoxicate cells and the observation that B. hinzii colonies were nonhemolytic on blood-containing plates, it was not unexpected that there was no measurable hemolytic activity in vitro.

Taken together, these results provide evidence that B. hinzii, in accordance with B. pertussis, produces a full-length ACT protein that binds CaM and retains some enzymatic activity. In contrast to B. pertussis, the amount of extractable B. hinzii ACT and its apparent affinity for CaM are lower. In addition, B. hinzii does not exhibit any toxin activity and has an approximately 50-fold reduction in adenylate cyclase enzyme specific activity relative to B. pertussis.

Calmodulin binding. Based upon the observations that (i) B. hinzii ACT exhibits decreased enzymatic activity (Fig. 5A) and (ii) a large proportion of protein is found in the unbound fraction during the calmodulin-Sepharose purification (Fig. 4) compared to ACT purified from B. pertussis, we hypothesized that one difference between the two toxins is their respective affinities for CaM. Calmodulin is the eukaryotic protein that binds within a subdomain of the N terminus of B. pertussis ACT and stimulates its adenylate cyclase activity (51, 92). To address this affinity issue, we evaluated binding of ACT to Blue-Sepharose, a reactive dye matrix that interacts with proteins through their nucleotide binding sites (54, 80). This method has been previously utilized to determine ACT binding affinities based on the differential binding ability of ACT for Blue-Sepharose in the presence and absence of CaM (50). Basically, if ACT has a high affinity for CaM then this interaction precludes the binding of ACT to Blue-Sepharose and the toxin remains in the supernatant fraction when the matrix is centrifuged. We incubated B. pertussis or B. hinzii type ACT with Blue-Sepharose with or without CaM, centrifuged the samples, and used enzyme activity as a marker for the distribution of ACT. As shown in Fig. 6, BP338 ACT had a significantly (P < 0.005) greater proportion of its measurable ACT activity complexed to CaM in the supernatant fraction (81 ± 1.4%) than B. hinzii ACT (43 ± 1.4%) in 5 h. This defect in binding capacity by B. hinzii was not overcome by increasing the length of time ACT was allowed to interact with the ligands (data not shown).



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 6. ACT-CaM interaction. ACT was incubated at 4°C with (100 nM) or without CaM in the presence of Blue-Sepharose for 5 h. The Blue-Sepharose matrix was centrifuged, and the CaM-complexed ACT remaining in the supernatant was assayed for enzymatic activity. Results are reported as a percentage of total AC activity. Data represent the mean with background subtracted ± the standard error of the mean for two independent experiments done in duplicate.

 
It is believed that calmodulin is able to activate ACT by binding to the toxin with a high affinity (0.2 nM), causing a conformational change in a flexible loop between the ATP- and CaM-binding subdomains and bringing together the amino acids necessary for ACT to bind and catalyze ATP into cAMP (23, 26). In an attempt to corroborate our Blue-Sepharose competition results, we compared the calmodulin activation between ACT from both Bordetella species. In order to accomplish that goal, we needed an alternative ACT purification method to preclude exposure of ACT to CaM prior to the AC enzymatic assay. Having determined that ACT will bind Blue-Sepharose, albeit less well than to CaM, we purified ACT from BP338 and B. hinzii type using Blue-Sepharose affinity chromatography. Each toxin was assayed for enzymatic activity both in the absence and presence of CaM. In agreement with previous studies that indicated ACT can be activated 100 to 1,000-fold upon CaM binding (92), in Table 2 it is shown that BP338 ACT displayed an approximately 450-fold stimulation in generating cAMP from ATP in the presence of CaM. However, only a 15-fold increase in enzyme activity was observed with purified ACT from B. hinzii. These combined findings suggest that B. hinzii-derived ACT may have a lower affinity for CaM, the established eukaryotic activator of B. pertussis ACT enzyme activity.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Quantitation of adenylate cyclase activity of ACT purified by Blue-Sepharose affinity chromatography

 
cyaC expression. The production of fully active ACT is dependent on the palmitoylation of the protein at Lys-983 (33). This posttranslational modification is catalyzed by the acyltransferase encoded by cyaC (4). It is well established that in the absence of CyaC, ACT is devoid of hemolytic activity and impaired in intoxication functions while retaining enzymatic activity (4, 6, 72). We investigated whether B. hinzii carries the cyaC locus by using PCR amplification of chromosomal DNA followed by DNA sequencing. As was the case with cyaA, B. hinzii type and BC-306 each possess a full-length copy of cyaC with corresponding upstream sequence that perfectly matches BP338 and Tohama I sequence (GenBank accession no. DQ141547 and DQ141548, respectively). However, unlike cyaA, when we measured cyaC mRNA transcription there was no detectable expression from either B. hinzii strain under conditions in which B. pertussis cyaC expression was visible (Fig. 7). Two cyaC primer sets, which were also used for sequencing the gene, were employed to ensure that the lack of product was not due to mRNA size or secondary structure. In addition, the same cDNA stocks used for these experiments were also used to generate the data in Fig. 2, confirming that the templates were present, competent, and able to yield detectable products in an RT-PCR assay. These data suggest that although B. hinzii maintains the cyaC DNA sequence, the lack of gene expression may be the cause of the nontoxic ACT phenotype observed.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 7. cyaA mRNA expression. An equal volume of each RT-PCR mixture was electrophoresed on the same 2% agarose gel and visualized by ethidium bromide staining. Panels were separated in order to include the sizes in kb of the Hyperladder I DNA standards (lane M). Two sets of cyaC-specific primers were utilized to yield products of 200 bp (A) and 517 bp (B). (-) RT, mock reactions with cDNA templates synthesized in the absence of reverse transcriptase were included as controls; type, B. hinzii type; 306, BC-306; Bp, B. pertussis BP338.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Given the increase in the incidence and the broadening spectrum of human diseases associated with Bordetella hinzii infection (2, 13, 15, 21, 22, 42, 84), it was startling to discover that the data on the bacterial determinants of virulence are limited to lipopolysaccharide (LPS) structure (3, 11, 85). The lack of research has probably been due, in part, to misidentification of the organism (65) and an early publication by Gerlach et al. that indicated that B. hinzii does not possess any of the virulence factors common to the other Bordetella species (25). More recently, however, these same authors have found genes in B. hinzii, B. holmesii, and B. trematum that they had previously reported to be missing (24). Additionally, in B. avium, three genes (fhaB, bvgS, and fimC) that were originally thought to be absent from the genome have also been identified (76). Thus, it was not surprising that in this study we were able to identify cyaA in the B. hinzii chromosome. Given that the G+C contents of the B. hinzii and B. pertussis genomes are similar (66 to 67% and 67.7%, respectively) (63, 83, 84), the presence of cyaA in B. hinzii may be the norm, rather than the exception, and a predictor of the existence of other Bordetella virulence genes in B. hinzii.

The DNA sequence of B. hinzii cyaA is identical to the Tohama I-derived laboratory B. pertussis strain (BP338) over the entire 5,541 bp analyzed. This includes the cyaA open reading frame as well as regulatory elements required for promoter activity. This is consistent with the fact that, unlike prn and ptx (12, 58, 59), the cyaA DNA sequence lacks polymorphisms among B. pertussis isolates (62) and is highly homologous between Bordetella species, with a greater than 97% identity between B. pertussis and B. bronchiseptica or B. parapertussis (63).

In order to characterize the properties of B. hinzii ACT, we initially purified the protein using established techniques, including urea extraction and calmodulin affinity chromatography (Fig. 4). Interestingly, when compared to BP338-derived ACT, there was much less ACT extracted from the B. hinzii strains. Our data indicate that this reduction in protein levels cannot be attributed to a decrease in gene transcription or total amount of ACT made within the bacterium. When normalized to recA, B. hinzii cyaA mRNA expression was not appreciably different than that from BP338 (Fig. 2 and Table 1). In addition, when we solubilized whole bacteria and probed for ACT in a Western blot assay (Fig. 3), the amounts of full-length ACT between BP338 and B. hinzii were comparable. Thus, there does not seem to be a difference in gene expression or mRNA stability or a defect in the translation of RNA into protein between the species.

Two other possibilities exist to explain this posttranscriptional difference in B. hinzii ACT protein amounts. First, we have recently shown that ACT localization can vary among B. pertussis strains (31), and so it is possible that in B. hinzii a majority of ACT is released into the culture medium instead of being attached to the bacterial surface and is, thereby, subsequently lost during the purification procedure. Secondly, since the purification scheme is optimized for B. pertussis, there may be points at which B. hinzii ACT is preferentially affected. Our preliminary data refute the former and support the latter theory. Although we were able to detect some ACT in the medium of growing B. hinzii cultures, it was only after concentrating the material and, even then, the quantity did not account for the observed differences during purification. On the other hand, we did observe that a significant portion of B. hinzii ACT remained in the insoluble fraction after sonication and urea extraction of the bacteria (data not shown). Given that the structure of B. hinzii ACT is presumably the same as B. pertussis ACT based on sequence data, this may reflect a difference in toxin extractability/accessibility between the strains. RTX toxins in general, and ACT in particular, are known to interact with LPS and other surface molecules, affecting both protein conformation and biological activity (5, 17, 67, 95). B. hinzii produces an LPS with an O-specific polysaccharide chain which differs significantly from other smooth-type LPS-containing Bordetella strains (such as B. bronchiseptica), as well as B. pertussis, which lacks an O-chain altogether (3, 11, 85). Thus, the association of B. hinzii ACT with its specific LPS structure, as well as the possibility that the toxin may be nonacylated, could easily alter its ability to be extracted from the membrane.

Although B. hinzii produces a full-length ACT molecule with intact active site domains and the same predicted amino acid sequence as B. pertussis, its biological activities were greatly impaired. B. hinzii ACT was nonhemolytic, unable to elevate intracellular cAMP in host macrophages, and possessed adenylate cyclase activity in the presence of CaM that was approximately 50-fold lower than BP338 (Fig. 5). The inability to lyse erythrocytes could be explained by an inadequate concentration of added toxin. Numerous previous studies have shown that oligomerization/pore formation is a cooperative process that requires high concentrations of ACT (18, 30, 55). The inability of purified B. hinzii ACT to intoxicate target cells, however, cannot simply be explained by an insufficient quantity of protein, since B. pertussis ACT can intoxicate at concentrations as low as 50 ng (Fig. 5B). However, if B. hinzii ACT was either nonacylated or differentially modified, this could explain the lack of toxin activity. It has been shown that nonacylated toxin is still capable of binding CD11b/CD18 receptors on host cells, but it is a nonproductive event eliciting either no intoxication or cytotoxicity (19) or requiring at least a 100-fold higher concentration of ACT to induce cAMP (36). Our data demonstrating the lack of cyaC expression in B. hinzii (Fig. 7) support the scenario that B. hinzii lacks the enzyme required to fully activate ACT. Examination of purified B. hinzii ACT for posttranslational modifications by mass spectrometry would address whether B. hinzii ACT can be acylated by an alternative acyltransferase and exclusively conclude that the absence of cyaC transcription directly correlates to a nonacylated toxin molecule.

In addition, three lines of evidence in this study directly point to altered affinity for binding calmodulin as a factor in B. hinzii ACT enzymatic activity. When B. hinzii lysates were passed over a CaM-Sepharose matrix, almost 40% of the ACT did not bind (Fig. 4). This was obviously not due to a matrix capacity problem, since in the same experiment an equivalently packed CaM column bound a much greater amount of ACT from B. pertussis. In Blue-Sepharose competitions, we also showed that the interaction between ACT purified from B. hinzii and the eukaryotic CaM activator was much weaker than the B. pertussis ACT-CaM binding (Fig. 6). The greatest benefit of using this assay is that one can compare toxins with different individual enzymatic activities to each other. This is because the assay is based on the ability of CaM to compete for ACT in the presence of Blue-Sepharose (50). Within a sample, the tightness of the CaM-ACT bond dictates whether ACT localizes with the CaM supernatant or Blue-Sepharose pellet fraction. Lastly, when purified by Blue-Sepharose affinity chromatography, B. pertussis ACT enzymatic activity was stimulated by CaM 30-fold more than B. hinzii ACT (Table 2). A diminished binding affinity of B. hinzii ACT for CaM would translate into a similarly reduced enzyme activation. An alternative possibility is that B. hinzii ACT requires a factor other than calmodulin for maximal activation. This is not without precedent, since ExoY, the Pseudomonas aeruginosa adenylate cyclase toxin, is dependent upon an as-yet-unidentified host protein distinct from CaM for its enhanced enzymatic activity (93). One observation that correlates with this concept is that in the absence of CaM, B. hinzii ACT activity was 10-fold higher than that seen with B. pertussis ACT (Table 2). This increased basal adenylate cyclase activity from B. hinzii may indicate a difference in the requirement for or type of activator between the species.

In conclusion, this represents the first functional characterization of a known Bordetella virulence factor now shown to be present in Bordetella hinzii. Although its direct role, if any, in pathogenesis is yet to be determined, we have shown that B. hinzii produces a full-length ACT with enzymatic activity. The variations in toxin and enzyme activities relative to the prototypical B. pertussis ACT seem to be due, at least in part, to both (i) a lack of expression of the gene (cyaC) encoding the enzyme required for activation of ACT and (ii) an altered binding affinity of ACT for host calmodulin. These data are important for defining factors utilized by this microorganism and also as a comparison to B. pertussis. With pertussis cases on the rise in recent years (37), ACT structure/function studies between the species may shed some light on the innate differences in host niches and disease states for both B. pertussis and B. hinzii.


   ACKNOWLEDGMENTS
 
We thank Brad Cookson for kindly providing us with BC-306, Mary Gray for critical reading of the manuscript, and members of the Hewlett and Hughes labs for their comments.

This work was supported by grant AI18000 from the National Institutes of Health.


   FOOTNOTES
 
* Corresponding author, Mailing address: Box 800419, University of Virginia School of Medicine, Charlottesville, VA 22908. Phone: (434) 924-5945. Fax: (434) 982-3830. E-mail: eh2v{at}virginia.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Arico, B., J. F. Miller, C. Roy, S. Stibitz, D. Monack, S. Falkow, R. Gross, and R. Rappuoli. 1989. Sequences required for expression of Bordetella pertussis virulence factors share homology with prokaryotic signal transduction proteins. Proc. Natl. Acad. Sci. USA 86:6671-6675.[Abstract/Free Full Text]
  2. Arvand, M., R. Feldhues, M. Mieth, T. Kraus, and P. Vandamme. 2004. Chronic cholangitis caused by Bordetella hinzii in a liver transplant recipient. J. Clin. Microbiol. 42:2335-2337.[Abstract/Free Full Text]
  3. Aussel, L., R. Chaby, K. Le Blay, J. Kelly, P. Thibault, M. B. Perry, and M. Caroff. 2000. Chemical and serological characterization of the Bordetella hinzii lipopolysaccharides. FEBS Lett. 485:40-46.[CrossRef][Medline]
  4. Barry, E. M., A. A. Weiss, I. E. Ehrmann, M. C. Gray, E. L. Hewlett, and M. S. Goodwin. 1991. Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation. J. Bacteriol. 173:720-726.[Abstract/Free Full Text]
  5. Bauer, M. E., and R. A. Welch. 1997. Pleiotropic effects of a mutation in rfaC on Escherichia coli hemolysin. Infect. Immun. 65:2218-2224.[Abstract]
  6. Betsou, F., P. Sebo, and N. Guiso. 1993. CyaC-mediated activation is important not only for toxic but also for protective activities of Bordetella pertussis adenylate cyclase-hemolysin. Infect. Immun. 61:3583-3589.[Abstract/Free Full Text]
  7. Boucher, P. E., K. Murakami, A. Ishihama, and S. Stibitz. 1997. Nature of DNA binding and RNA polymerase interaction of the Bordetella pertussis BvgA transcriptional activator at the fha promoter. J. Bacteriol. 179:1755-1763.[Abstract/Free Full Text]
  8. Boucher, P. E., and S. Stibitz. 1995. Synergistic binding of RNA polymerase and BvgA phosphate to the pertussis toxin promoter of Bordetella pertussis. J. Bacteriol. 177:6486-6491.[Abstract/Free Full Text]
  9. Boucher, P. E., M. S. Yang, D. M. Schmidt, and S. Stibitz. 2001. Genetic and biochemical analyses of BvgA interaction with the secondary binding region of the fha promoter of Bordetella pertussis. J. Bacteriol. 183:536-544.[Abstract/Free Full Text]
  10. Brooker, G., J. F. Harper, W. L. Terasaki, and R. D. Moylan. 1979. Radioimmunoassay of cyclic AMP and cyclic GMP. Adv. Cyclic Nucl. Res. 10:1-33.[Medline]
  11. Caroff, M., L. Aussel, H. Zarrouk, A. Martin, J. C. Richards, H. Thérisod, M. B. Perry, and D. Karibian. 2001. Structural variability and originality of the Bordetella endotoxins. J. Endotoxin Res. 7:63-68.[CrossRef]
  12. Cassiday, P., G. Sanden, K. Heuvelman, F. Mooi, K. M. Bisgard, and T. Popovic. 2000. Polymorphism in Bordetella pertussis pertactin and pertussis toxin virulence factors in the United States, 1935-1999. J. Infect. Dis. 182:1402-1408.[CrossRef][Medline]
  13. Coenye, T., J. Goris, T. Spilker, P. Vandamme, and J. J. LiPuma. 2002. Characterizations of unusual bacteria isolated from respiratory secretions of cystic fibrosis patients and decriptions of Inquilinus limosus gen. nov., sp. nov. J. Clin. Microbiol. 40:2062-2069.[Abstract/Free Full Text]
  14. Confer, D. L., and J. W. Eaton. 1982. Phagocyte impotence caused by an invasive bacterial adenylate cyclase. Science 217:948-950.[Abstract/Free Full Text]
  15. Cookson, B. T., P. Vandamme, L. C. Carlson, A. M. Larson, J. V. L. Sheffield, K. Kersters, and D. H. Spach. 1994. Bacteremia caused by a novel Bordetella species, "B. hinzii". J. Clin. Microbiol. 32:2569-2571.[Abstract/Free Full Text]
  16. Cotter, P. A., and J. F. Miller. 2001. Bordetella, p. 619-674. In E. A. Groisman (ed.), Principles of bacterial pathogenesis. Academic Press, San Diego, Calif.
  17. Czuprynski, C. J., and R. A. Welch. 1995. Biological effects of RTX toxins: the possible role of lipopolysaccharide. Trends Microbiol. 3:480-483.[CrossRef][Medline]
  18. Ehrmann, I. E., M. C. Gray, V. M. Gordon, L. S. Gray, and E. L. Hewlett. 1991. Hemolytic activity of adenylate cyclase toxin from Bordetella pertussis. FEBS Lett. 278:79-83.[CrossRef][Medline]
  19. El-Azami-El-Idrissi, M., C. Bauche, J. Loucka, R. Osicka, P. Sebo, D. Ladant, and C. Leclerc. 2003. Interaction of Bordetella pertussis adenylate cyclase with CD11b/CD18: role of toxin acylation and identification of the main integrin interaction domain. J. Biol. Chem. 278:38514-38521.[Abstract/Free Full Text]
  20. Friedman, R. L., R. L. Fiederlein, L. Glasser, and J. N. Galgiani. 1987. Bordetella pertussis adenylate cyclase: effects of affinity-purified adenylate cyclase on human polymorphonuclear leukocyte functions. Infect. Immun. 55:135-140.[Abstract/Free Full Text]
  21. Funke, G., T. Hess, and P. Vandamme. 1996. Characteristics of Bordetella hinzii strains isolated from a cystic fibrosis patient over a 3-year period. J. Clin. Microbiol. 34:966-969.[Abstract]
  22. Gadea, I., M. Cuenca-Estrella, N. Benito, A. Blanco, M. L. Fernández-Guerrero, P. L. Valero-Guillén, and F. Soriano. 2001. Bordetella hinzii, a "new" opportunistic pathogen to think about. J. Infect. Dis. 40:298-299.
  23. Gallay, J., M. Vincent, I. M. Li de la Sierra, H. Munier-Lehmann, M. Renouard, H. Sakamoto, O. Barzu, and A.-M. Gilles. 2004. Insight into the activation mechanism of Bordetella pertussis adenylate cyclase by calmodulin using fluorescence spectroscopy. Eur. J. Biochem. 271:821-833.[Medline]
  24. Gerlach, G., S. Janzen, D. Beir, and R. Gross. 2004. Functional characterization of the BvgAS two-component system of Bordetella holmesii. Microbiology 150:3715-3729.[Abstract/Free Full Text]
  25. Gerlach, G., F. von Wintzingerode, B. Middendorf, and R. Gross. 2001. Evolutionary trends in the genus Bordetella. Microbes Infect. 3:61-72.[CrossRef][Medline]
  26. Glaser, P., A. Elmaoglou-Lazaridou, E. Krin, D. Ladant, O. Barzu, and A. Danchin. 1989. Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis. EMBO J. 8:967-972.[Medline]
  27. Glaser, P., D. Ladant, O. Sezer, F. Pichot, A. Ullmann, and A. Danchin. 1988. The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coli. Mol. Microbiol. 2:19-30.[CrossRef][Medline]
  28. Glaser, P., H. Sakamoto, J. Bellalou, A. Ullmann, and A. Danchin. 1988. Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis. EMBO J. 7:3997-4004.[Medline]
  29. Goodnow, R. A. 1980. Biology of Bordetella bronchiseptica. Microbiol. Rev. 44:722-738.[Free Full Text]
  30. Gray, M., G. Szabo, A. S. Otero, L. Gray, and E. Hewlett. 1998. Distinct mechanisms for K+ efflux, intoxication, and hemolysis by Bordetella pertussis AC toxin. J. Biol. Chem. 273:18260-18267.[Abstract/Free Full Text]
  31. Gray, M. C., G. M. Donato, F. R. Jones, T. Kim, and E. L. Hewlett. 2004. Newly secreted adenylate cyclase toxin is responsible for intoxication of target cells by Bordetella pertussis. Mol. Microbiol. 53:1709-1719.[CrossRef][Medline]
  32. Gross, M. K., D. C. Au, A. L. Smith, and D. R. Storm. 1992. Targeted mutations that ablate either the adenylate cyclase or hemolysin function of the bifunctional cyaA toxin of Bordetella pertussis abolish virulence. Proc. Natl. Acad. Sci. USA 89:4898-4902.[Abstract/Free Full Text]
  33. Hackett, M., L. Guo, J. Shabanowitz, D. F. Hunt, and E. L. Hewlett. 1994. Internal lysine palmitoylation in adenylate cyclase toxin from Bordetella pertussis. Science 266:433-435.[Abstract/Free Full Text]
  34. Hanski, E. 1989. Invasive adenylate cyclase toxin of Bordetella pertussis. Trends Biochem. Sci. 14:459-463.[CrossRef][Medline]
  35. Hewlett, E. L. 1995. Bordetella species, p. 2078-2084. In G. L. Mandell, J. E. Bennett, and R. Dolin (ed.), Principles and practices of infectious diseases. Churchill Livingstone, New York, N.Y.
  36. Hewlett, E. L., G. M. Donato, M. Basler, J. Masin, P. Sebo, and M. C. Gray. 2005. Adenylate cyclse (AC) toxin from Bordetella pertussis kills J774 macrophages by multiple mechanisms: more than just cAMP. Abstr. 25th Gen. Meet. Am. Soc. Microbiol., abstract no. B-002.
  37. Hewlett, E. L., and K. M. Edwards. 2005. Pertussis—not just for kids. N. Engl. J. Med. 352:1215-1222.[Free Full Text]
  38. Hewlett, E. L., V. M. Gordon, J. D. McCaffery, W. M. Sutherland, and M. C. Gray. 1989. Adenylate cyclase toxin from Bordetella pertussis: identification and purification of the holotoxin molecule. J. Biol. Chem. 264:19379-19384.[Abstract/Free Full Text]
  39. Hewlett, E. L., L. Gray, M. Allietta, I. Ehrmann, V. M. Gordon, and M. C. Gray. 1991. Adenylate cyclase toxin from Bordetella pertussis: conformational change associated with toxin activity. J. Biol. Chem. 266:17503-17508.[Abstract/Free Full Text]
  40. Hot, D., R. Antoine, G. Renauld-Mongenie, V. Caro, B. Hennuy, E. Levillain, L. Huot, G. Wittmann, D. Poncet, F. Jacob-Dubuisson, C. Guyard, F. Rimlinger, L. Aujame, E. Godfroid, N. Guiso, M. J. Quentin-Millet, Y. Lemoine, and C. Locht. 2003. Differential modulation of Bordetella pertussis virulence genes as evidenced by DNA microarray analysis. Mol. Gen. Genomics 269:475-486.[CrossRef][Medline]
  41. Karimova, G., J. Bellalou, and A. Ullmann. 1996. Phosphorylation-dependent binding of BvgA to the upstream region of the cyaA gene of Bordetella pertussis. Mol. Microbiol. 20:489-496.[CrossRef][Medline]
  42. Kattar, M. M., J. F. Chavez, A. P. Limaye, S. L. Rassoulian-Barrett, S. L. Yarfitz, L. C. Carlson, Y. Houze, S. Swanzy, B. L. Wood, and B. T. Cookson. 2000. Application of 16S rRNA gene sequencing to identify Bordetella hinzii as the causative agent of fatal septicemia. J. Clin. Microbiol. 38:789-794.[Abstract/Free Full Text]
  43. Kersters, K., K. H. Hinz, A. Hertle, P. Segers, A. Lievens, O. Siegmann, and J. De Ley. 1984. Bordetella avium sp. nov., isolated from the respiratory tracts of turkeys and other birds. Int. J. Syst. Bacteriol. 34:56-70.
  44. Khelef, N., and N. Guiso. 1995. Induction of macrophage apoptosis by Bordetella pertussis adenylate cyclase-hemolysin. FEMS Microbiol. Lett. 134:27-32.[CrossRef][Medline]
  45. Khelef, N., H. Sakamoto, and N. Guiso. 1992. Both adenylate cyclase and hemolytic activities are required by Bordetella pertussis to initiate infection. Microb. Pathog. 12:227-235.[CrossRef][Medline]
  46. Khelef, N., A. Zychlinsky, and N. Guiso. 1993. Bordetella pertussis induces apoptosis in macrophages: role of adenylate cyclase-hemolysin. Infect. Immun. 61:4064-4071.[Abstract/Free Full Text]
  47. Kinnear, S. M., P. E. Boucher, S. Stibitz, and N. H. Carbonetti. 1999. Analysis of BvgA activation of the pertactin gene promoter in Bordetella pertussis. J. Bacteriol. 181:5234-5241.[Abstract/Free Full Text]
  48. Kinnear, S. M., R. R. Marques, and N. H. Carbonetti. 2001. Differential regulation of Bvg-activated virulence factors plays a role in Bordetella pertussis pathogenicity. Infect. Immun. 69:1983-1993.[Abstract/Free Full Text]
  49. Kuhl, S. A., R. P. McCreary, J. D. Bannan, and R. L. Friedman. 1990. Isolation and characterization of the recA gene of Bordetella pertussis. Mol. Microbiol. 4:1165-1172.[CrossRef][Medline]
  50. Ladant, D. 1988. Interaction of Bordetella pertussis adenylate cyclase with calmodulin: identification of two separated calmodulin-binding domains. J. Biol. Chem. 263:2612-2618.[Abstract/Free Full Text]
  51. Ladant, D., S. Michelson, R. Sarfati, A.-M. Gilles, R. Predeleanu, and O. Barzu. 1989. Characterization of the calmodulin-binding and of the catalytic domains of Bordetella pertussis adenylate cyclase. J. Biol. Chem. 264:4015-4020.[Abstract/Free Full Text]
  52. Ladant, D., and A. Ullmann. 1999. Bordetella pertussis adenylate cyclase: a toxin with multiple talents. Trends Microbiol. 7:172-176.[CrossRef][Medline]
  53. Laoide, B. M., and A. Ullmann. 1990. Virulence dependent and independent regulation of the Bordetella pertussis cya operon. EMBO J. 9:999-1005.[Medline]
  54. Lascu, I., H. Porumb, T. Porumb, I. Abrudan, C. Tarmure, I. Petrescu, E. Presecan, I. Proinov, and M. Telia. 1984. Ion-exchange properties of Cibacron Blue 3G-A Sepharose (Blue Sepharose) and the interaction of proteins with Cibacron Blue 3G-A. J. Chromatogr. 283:199-210.[CrossRef][Medline]
  55. Lee, S. J., M. C. Gray, K. Zu, and E. L. Hewlett. 2005. Oligomeric behavior of adenylate cyclase toxin from Bordetella pertussis in solution. Arch. Biochem. Biophys. 438:80-87.
  56. Mazengia, E., E. A. Silva, J. A. Peppe, R. Timperi, and H. George. 2000. Recovery of Bordetella holmesii from patients with pertussis-like symptoms: use of pulse-field gel electrophoresis to characterize circulating strains. J. Clin. Microbiol. 38:2330-2333.[Abstract/Free Full Text]
  57. Melton, A. R., and A. A. Weiss. 1989. Environmental regulation of expression of virulence determinants in Bordetella pertussis. J. Bacteriol. 171:6206-6212.[Abstract/Free Full Text]
  58. Mooi, F. R., Q. He, H. van Oirschot, and J. Mertsola. 1999. Variation in the Bordetella pertussis virulence factors pertussis toxin and pertactin in vaccine strains and clinical isolates in Finland. Infect. Immun. 67:3133-3134.[Abstract/Free Full Text]
  59. Mooi, F. R., H. van Oirschot, K. Heuvelman, H. G. J. van der Heide, W. Gaastra, and R. J. Willems. 1998. Polymorphism in the Bordetella pertussis virulence factors P.69/pertactin and pertussis toxin in The Netherlands: temporal trends and evidence for vaccine-driven evolution. Infect. Immun. 66:670-675.[Abstract/Free Full Text]
  60. Morris, J. T., and M. Myers. 1998. Bacteremia due to Bordetella holmesii. Clin. Infect. Dis. 27:912-913.[Medline]
  61. Njamkepo, E., F. Delisle, I. Hagege, G. Gerbaud, and N. Guiso. 2000. Bordetella holmesii isolated from a patient with sickle cell anemia: analysis and comparison with other Bordetella holmesii isolates. Clin. Microbiol. Infect. 6:131-136.[CrossRef][Medline]
  62. Packard, E. R., R. Parton, J. G. Coote, and N. K. Fry. 2004. Sequence variation and conservation in virulence-related genes of Bordetella pertussis isolates from the UK. J. Med. Microbiol. 53:355-365.[Abstract/Free Full Text]
  63. Parkhill, J., M. Sebaihia, A. Preston, L. D. Murphy, N. Thomson, D. E. Harris, M. T. Holden, C. M. Churcher, S. D. Bentley, K. L. Mungall, A. M. Cerdeno-Tarraga, L. Temple, K. James, B. Harris, M. A. Quail, M. Achtman, R. Atkin, S. Baker, D. Basham, N. Bason, I. Cherevach, T. Chillingworth, M. Collins, A. Cronin, P. Davis, J. Doggett, T. Feltwell, A. Goble, N. Hamlin, H. Hauser, S. Holroyd, K. Jagels, S. Leather, S. Moule, H. Norberczak, S. O'Neil, D. Ormond, C. Price, E. Rabbinowitsch, S. Rutter, M. Sanders, D. Saunders, K. Seeger, S. Sharp, M. Simmonds, J. Skelton, R. Squares, S. Squares, K. Stevens, L. Unwin, S. Whitehead, B. G. Barrell, and D. J. Maskell. 2003. Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Nat. Genet. 35:32-40.[CrossRef][Medline]
  64. Pearson, R. D., P. Symes, M. Conboy, A. A. Weiss, and E. L. Hewlett. 1987. Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin. J. Immunol. 139:2749-2754.[Abstract]
  65. Register, K. B., R. E. Sacco, and G. E. Nordholm. 2003. Comparison of ribotyping and restriction enzyme analysis for inter- and intraspecies discrimination of Bordetella avium and Bordetella hinzii. J. Clin. Microbiol. 41:1512-1519.[Abstract/Free Full Text]
  66. Rogel, A., R. Meller, and E. Hanski. 1991. Adenylate cyclase toxin from Bordetella pertussis. The relationship between induction of cAMP and hemolysis. J. Biol. Chem. 266:3154-3161.[Abstract/Free Full Text]
  67. Ross, P. J., E. C. Lavelle, H. G. Mills, and A. P. Boyd. 2004. Adenylate cyclase toxin from Bordelella pertussis synergizes with lipopolysaccharide to promote innate interleukin-10 production and enhances the induction of Th2 and regulatory T cells. Infect. Immun. 72:1568-1579.[Abstract/Free Full Text]
  68. Russell, F. M., J. M. Davis, M. J. Whipp, P. H. Janssen, P. B. Ward, J. R. Vyas, M. Starr, S. M. Sawyer, and N. Curtis. 2001. Severe Bordetella holmesii infection in a previously healthy adolescent confirmed by gene sequence analysis. Clin. Infect. Dis. 33:129-130.[CrossRef][Medline]
  69. Sakamoto, H., J. Bellalou, P. Sebo, and D. Ladant. 1992. Bordetella pertussis adenylate cyclase toxin: structural and functional independence of the catalytic and hemolytic activities. J. Biol. Chem. 267:13598-13602.[Abstract/Free Full Text]
  70. Salomon, Y., C. Londos, and M. Rodbell. 1974. A highly sensitive adenylate cyclase assay. Anal. Biochem. 58:541-548.[CrossRef][Medline]
  71. Scarlato, V., B. Arico, A. Prugnola, and R. Rappuoli. 1991. Sequential activation and environmental regulation of virulence genes in Bordetella pertussis. EMBO J. 10:3971-3975.[Medline]
  72. Sebo, P., P. Glaser, H. Sakamoto, and A. Ullmann. 1991. High-level synthesis of active adenylate cyclase toxin of Bordetella pertussis in a reconstructed Escherichia coli system. Gene 104:19-24.[CrossRef][Medline]
  73. Shepard, C. W., M. I. Daneshvar, R. M. Kaiser, D. A. Ashford, D. Lonsway, J. B. Patel, R. E. Morey, J. G. Jordan, R. S. Weyant, and M. Fischer. 2004. Bordetella holmesii bacteremia: a newly recognized clinical entity among asplenic patients. Clin. Infect. Dis. 38:799-804.[CrossRef][Medline]
  74. Skeeles, J. K., and L. H. Arp. 1997. Bordetellosis (turkey coryza), p. 275-288. In B. W. Calnek et al. (ed.), Diseases of poultry. Iowa State University Press, Ames.
  75. Smith, A. M., C. A. Guzman, and M. J. Walker. 2001. The virulence factors of Bordetella pertussis: a matter of control. FEMS Microbiol. Lett. 25:309-333.
  76. Spears, P. A., L. M. Temple, D. M. Miyamoto, D. J. Maskell, and P. E. Orndorff. 2003. Unexpected similarities between Bordetella avium and other pathogenic bordetellae. Infect. Immun. 71:2591-2597.[Abstract/Free Full Text]
  77. Stainer, D. W., and M. J. Scholte. 1971. A simple chemically defined medium for the production of phase I Bordetella pertussis. J. Gen. Microbiol. 63:211-220.
  78. Stibitz, S., and M.-S. Yang. 1991. Subcellular localization and immunological detection of proteins encoded by the vir locus of Bordetella pertussis. J. Bacteriol. 173:4288-4296.[Abstract/Free Full Text]
  79. Tang, Y. W., M. K. Hopkins, C. P. Kolbert, P. A. Hartley, P. J. Severance, and D. H. Persing. 1998. Bordetella holmesii-like organisms associated with septicemia, endocarditis, and respiratory failure. Clin. Infect. Dis. 26:389-392.[Medline]
  80. Thompson, S. T., and E. Stellwagen. 1976. Binding of Cibacron Blue F3GA to proteins containing the dinucleotide fold. Proc. Natl. Acad. Sci. USA 73:361-365.[Abstract/Free Full Text]
  81. Uhl, M. A., and J. F. Miller. 1994. Autophosphorylation and phosphotransfer in the Bordetella pertussis BvgAS signal transduction cascade. Proc. Natl. Acad. Sci. USA 91:1163-1167.[Abstract/Free Full Text]
  82. Uhl, M. A., and J. F. Miller. 1996. Integration of multiple domains in a two-component sensor protein: the Bordetella pertussis BvgAS phosphorelay. EMBO J. 15:1028-1036.[Medline]
  83. Vandamme, P., M. Heyndrickx, M. Vancanneyt, B. Hoste, P. De Vos, E. Falsen, K. Kersters, and K. H. Hinz. 1996. Bordetella trematum sp. nov., isolated from wounds and ear infections in humans, and reassessment of Alcaligenes denitrificans Ruger and Tan 1983. Int. J. Syst. Bacteriol. 46:849-858.[CrossRef][Medline]
  84. Vandamme, P., J. Hommez, M. Vancanneyt, M. Monsieurs, B. Hoste, B. Cookson, W. H. von Konig, K. Kersters, and P. J. Blackall. 1995. Bordetella hinzii sp. nov., isolated from poultry and humans. Int. J. Syst. Bacteriol. 45:37-45.[CrossRef][Medline]
  85. Vinogradov, E. 2002. Structure of the O-specific polysaccharide chain of the lipopolysaccharide of Bordetella hinzii. Carbohydr. Res. 337:961-963.[Medline]
  86. von Wintzingerode, F., A. Schattke, R. A. Siddiqui, U. Rosick, U. B. Göbel, and R. Gross. 2001. Bordetella petrii sp. nov., isolated from an anaerobic bioreactor and emended description of the genus Bordetella. Int. J. Syst. Evol. Microbiol. 51:1257-1265.[Abstract]
  87. Weingart, C. L., P. S. Mobberley-Schuman, E. L. Hewlett, M. C. Gray, and A. A. Weiss. 2000. Neutralizing antibodies to adenylate cyclase toxin promote phagocytosis of Bordetella pertussi