Functional BvgAS Virulence Control System in Bordetella bronchiseptica Is Necessary for Induction of Ca ²⁺ Transients in Ciliated Tracheal Epithelial Cells

Abstract

To study initial Bordetella bronchiseptica-tracheal epithelial cell interactions, we coincubated B. bronchiseptica with rabbit tracheal explant cultures and assayed bacterial adherence and host cell Ca 2؉ signaling. Wild-type B. bronchiseptica (RB50) preferentially adhered to cilia and induced ciliated host cell Ca 2؉ transients within 2 min of coincubation, whereas coincubation with an avirulent strain (RB57) resulted in limited binding and Ca 2؉ signaling. The described cell system allows for assessment of initial B. bronchiseptica-host cell interactions that can contribute to pathogenicity or to host cell defense.The tracheal epithelium creates a physical barrier between respired air and the underlying tissue of the upper respiratory tract. In addition to the barrier, the epithelium generates a "mucociliary escalator" to clear particulate materials, including pathogenic bacteria, from the airway and keep them from reaching the lungs (15). Local environmental changes can influence coordinated ciliary movement through second messenger signaling pathways (12). Such changes elicited by respiratory pathogens through toxins and adhesins can help to overcome mucociliary defense and establish infection (16). Bordetella bronchiseptica is a gram-negative bacterium that colonizes the airways of a variety of animals. Similar to other bordetellae, B. bronchiseptica responds to environmental conditions and switches between virulent and avirulent phases via a two-component signal transduction system termed BvgAS (1, 2, 4, 11). In animal studies the Bvg ϩ phase is necessary to establish infection, whereas mutants locked in a Bvg Ϫ state (e.g., RB57) are unable to establish infection (6, 7). It is accepted that establishment of infection involves factors under BvgAS control that allow for adherence of B. bronchiseptica to cilia in the upper airway (3,5,7,10).To investigate initial Bordetella-host cell interactions, we monitored B. bronchiseptica and explant cultures of rabbit tracheal epithelial cells (RTEC) with video and digital imaging microscopy. RTEC were grown at 37°C in 5% CO 2 on collagen-coated glass coverslips in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as described previously (8). Cultures were washed extensively with Hanks' balanced salt solution (HBSS) and covered with 250 l of HBSS for microscopic observation (Fig. 1A). Wild-type B. bronchiseptica (RB50) or a Bvg Ϫ avirulent strain (RB57) was grown to log phase under constant shaking at 37°C in supplemented stainer Scholte broth (14). Bacterial cultures were resuspended in HBSS to 10 8 to 10 9 CFU/ml. Binding assays were performed in an open cell chamber by video microscopy on an Olympus IX70 inverted microscope with a ϫ100 phase contrast or a ϫ60 differential interference contrast objective and time lapse video capability. RTEC cultures were washed with HBSS and imaged with a DAGE 300T charge-coupled-device camera (Fig. 1B). To initiate interactions, RTEC cultures were initially exposed to bacteria by replacing the HBSS with four con...

Bordetellafilamentous hemagglutinin plays a critical role in immunomodulation, suggesting a mechanism for host specificity

“…Although our data are consistent with this hypothesis, proof will require identification of a specific receptor(s) that can distinguish FHA Bb from FHA Bp . If such a receptor exists, the fact that both FHA Bb and FHA Bp can mediate adherence to a variety of epithelial and macrophage-like cell lines in vitro suggests that this receptor may be expressed only on cilia, to which Bordetella has been shown to display a distinct predilection (52,53), or nonciliated cells within the context of the live respiratory mucosa. Furthermore, because B. bronchiseptica displays a broad host range that includes humans, it is possible that, whereas FHA Bp may be able to bind a receptor or a form of a receptor that is present only in humans, FHA Bb may display less stringent binding properties and be capable of binding both human-specific and non-human-specific receptors.…”

Bordetella bronchisepticaAdherence to Cilia Is Mediated by Multiple Adhesin Factors and Blocked by Surfactant Protein A

“…RTEC were mounted onto an open chamber on an Olympus IX70 microscope stage and adjusted to monitor actively beating ciliated cells. A 60ϫ oil immersion objective with differential interference contrast optics and an additional 1.5ϫ magnification zoom lens allowed for the visualization of both bacteria and cilia (14). RTEC cultures were washed thoroughly with HBSS prior to experimentation.…”

“…Bordetella binding assay. Binding studies were adapted from previously described methods (14). To control for the binding effects of conditioned growth medium that could be different between bacterial strains, B. bronchiseptica strains from log phase growth, described above, were collected by centrifugation and resuspended into HBSS to an optical density at 600 nm of between 0.20 and 0.34.…”

“…We have previously shown that a wild-type strain of B. bronchiseptica (RB50) grown under Bvg ϩ conditions preferentially adhered to the cilia of RTEC cultures within seconds of coincubation, whereas a nonmotile, phase-locked Bvg Ϫ mutant derivative of RB50 (RB57) showed limited binding over a 5-min coincubation (14). Since RB50 can modulate between Bvg ϩ and Bvg Ϫ phases in response to environmental conditions and because RB57 cells lack the flagella normally associated with the Bvg Ϫ phase, we repeated our binding studies with phase-locked Bvg ϩ (RB53) and phase-locked Bvg Ϫ (RB54) strains (Fig.…”

“…In this study, we used primary cultured rabbit tracheal epithelial cells (RTEC) as a model for ciliated respiratory epithelium from a natural host of B. bronchiseptica and measured the ability of B. bronchiseptica strains deficient in individual adhesins and/or toxins to directly bind to cilia on a time scale (Ͻ5 min) consistent with overcoming mucociliary defense (14). We show that loss of known individual adhesins (FHA, pertactin, or fimbriae) or toxins (CyaA, DNT) reduced ciliary attachment compared to Bvg ϩ strains but remained significantly higher than Bvg Ϫ strains.…”