Quantification of the Adenylate Cyclase Toxin of Bordetella pertussisIn Vitroand during Respiratory Infection

Abstract

dWhooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ϳ10 8 CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ϳ10 8 CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ϳ60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis.

Bordetella pertussis Adenylate Cyclase Toxin Blocks Induction of Bactericidal Nitric Oxide in Macrophages through cAMP-Dependent Activation of the SHP-1 Phosphatase

“…As indeed shown in Fig. 2D, a detectable increase of intracellular cAMP concentration in RAW264.7 cells was observed already in 5 min of exposure to CyaA concentrations as low as 10 ng/ml that correspond to toxin amounts detected in nasopharyngeal fluids from B. pertussisinfected humans and primates (38). The CyaA-catalyzed cAMP production in cells then peaked within 1 h, and intracellular cAMP concentrations remained elevated for 24 h, irrespective of whether the cells were activated by the addition of E.coli LPS (100 ng/ml) or not.…”

“…Second, the calmodulin-activated catalytic domain of CyaA possesses an extremely high specific AC enzyme activity, with a turnover number of ∼2000 s 21 (58). As a result, CyaA concentrations as low as those used in this study (10 ng/ml), and detected in mucosal fluids of infected infants and experimentally challenged baboons (38), are not only sufficient for ablation of superoxide production and inhibition of neutrophil extracellular trap release by host neutrophils (8-10) but also provoke inhibition of bactericidal NO production in macrophages. This finding explains why CyaA expression is crucial for the extended survival of unopsonized B. pertussis bacteria in macrophages.…”

Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

“…With efficacy of about 2 orders of magnitude lower, the CyaA toxin can also penetrate non-myeloid cells that lack the CR3 receptor (CD11b Ϫ cells), where due to its extremely active AC enzyme it can elevate cAMP concentrations to well detectable and physiologically relevant concentrations in epithelial and other host cells (10,11). Indeed, amounts of CyaA detected in nasopharyngeal fluids and washes from diseased infants and infected olive baboons (12) indicate that CyaA may also be playing a prominent role in perturbation of barrier and innate immune defense functions of epithelia of airway mucosa. Recently, CyaAbearing outer membrane vesicles (OMVs) shed by B. pertussis were shown to deliver CyaA into epithelial cells across their apical cell surface, through which the free secreted CyaA translocates very inefficiently (13,14).…”

Differential regulation of type III secretion and virulence genes inBordetella pertussisandBordetella bronchisepticaby a secreted anti-σ factor

“…BvgAS establishes a spectrum of phenotypic phases in response to environmental cues, functioning as a rheostat that controls the infectious cycle (7,8). The Bvg + phase is characterized by high level BvgAS activity, expression of virulence factors [including pertussis toxin (9), adenylate cyclase toxin (10,11), filamentous hemagglutinin (12), fimbriae (13), the Bordetella secretion complex (Bsc) type III secretion system (T3SS Bsc ) (14)(15)(16)], and the ability to survive and replicate on respiratory epithelia (17). In the Bvg phase, the BvgAS phosphorelay is quiescent, Bvg + phase genes are silent, and "virulence-repressed genes" are maximally transcribed (4,17,18).…”