Adenylate Cyclase Toxin (ACT) fromBordetella hinzii: Characterization and Differences from ACT ofBordetella pertussis

Abstract

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.

Cystic Fibrosis Mice Develop Spontaneous Chronic Bordetella Airway Infections

“…All species of Bordetella produce an adenylatecyclase toxin (ACT), dermonecrotic toxin, and tracheal cytotoxin [15]. ACT expression is thought to contribute to respiratory tract colonization [16], dermonecrotic toxin causes tissue damage [17] and tracheal cytotoxin causes ciliary paralysis [18].…”

“…The ACT produced by B. hinzii differs from other Bordetella species (such as Bordetella pertussis ) in that it has a decreased affinity for calmodulin. As a result, it does not cause the elevated cAMP levels observed in B. pertussis -infected patients [15], which is thought to contribute to the differences in virulence between the two species. B. hinzii has not been well studied, but it is plausible that though the overall virulence may be reduced compared to other well-studied Bordetella species (such as B. pertussis and Bordetella bronchospetica ), infection by B. hinzii may have similar, albeit milder effects.…”

Resemblance and divergence: the “new” members of the genus Bordetella

“…Only recently, we were able to amplify a piece of DNA from the genome encoding the bvgA gene in B. hinzii, indicating that this bacterium may in fact be endowed with virulence factors related to those of the well described Bordetellae [28]. Most interestingly, transcriptionally active cyaA genes coding for ACT could be identiWed in commensal and clinical B. hinzii isolates which, surprisingly, have identical nucleotide sequences as the orthologous gene of B. pertussis both in their coding and upstream regions [29]. This was surprising, since B. hinzii was found to be non-hemolytic on blood agar plates, while the calmodulin-activated ACT of the classical Bordetellae does not only have adenylate cyclase but also hemolytic activities.…”

“…Once more the DNA sequences of the coding and upstream regions of the cyaC genes were identical with that of B. pertussis. However, transcriptional analysis revealed transcriptional silence of this gene in the B. hinzii strains and this may explain, at least in part, the functional diVerences between ACT BH and ACT BP [29]. Despite the fact that the sequence of ACT among the members of the B. bronchiseptica complex is highly conserved, the 100% identity of the DNA sequences of the cyaA and cyaC genes of B. pertussis and B. hinzii remains puzzling, since these organisms phylogenetically are quite distantly located in the genus, and the more closely related species B. avium does not encode the cya locus at all [19].…”

“…ACT production by B. hinzii assayed by Western blotting using a polyclonal anti-ACT BP serum was comparable to that of B. pertussis. However, the comparative analysis of B. hinzii and B. pertussis ACTs revealed that despite 100% sequence conservation ACT BH lacks hemolytic activity, exhibits a reduced calmodulin binding and adenylate cyclase activity as compared to ACT BP , and cannot increase cAMP levels in host macrophages [29]. Since hemolytic and toxic activities of ACT BP require palmytoylation of its Lys-983 by the cyaC gene product, the cyaC gene of two B. hinzii strains was cloned.…”