Functional BvgAS Virulence Control System in Bordetella bronchiseptica Is Necessary for Induction of Ca²⁺ 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 Ca²⁺ signaling. Wild-type B. bronchiseptica (RB50) preferentially adhered to cilia and induced ciliated host cell Ca²⁺ transients within 2 min of coincubation, whereas coincubation with an avirulent strain (RB57) resulted in limited binding and Ca²⁺ 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⁺ phase is necessary to establish infection, whereas mutants locked in a Bvg⁻ 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% CO₂ 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 μl of HBSS for microscopic observation (Fig. 1A). Wild-type B. bronchiseptica (RB50) or a Bvg⁻ 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 10⁸ to 10⁹ CFU/ml. Binding assays were performed in an open cell chamber by video microscopy on an Olympus IX70 inverted microscope with a ×100 phase contrast or a ×60 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 consecutive additions of 250-μl bacterial suspensions. Cultures were subsequently washed with 1 ml of HBSS at 3 min and 5 min to remove nonadherent bacteria. Single images were collected every 3 s for a total of 5 min and later analyzed frame by frame to determine the numbers of bacteria attached to ciliated and aciliated RTEC at 75 s, 90 s, and every 30 s thereafter. Host intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were determined by using published equations (9) after digital imaging of RTEC loaded with fura-2 by fura-2AM incubation. Fura-2 fluorescence was observed with an Olympus IX70 microscope after alternating excitation at 340 and 380 nm by a 75-W Xenon lamp linked to a Delta Ram illuminator (Photon Technologies Incorporated [PTI], Monmouth Junction, N.J.) and a fiber optic line. Images of emitted fluorescence above 510 nm were recorded by an ICCD camera (PTI) and simultaneously displayed on a 21-inch Vivitron color monitor. The imaging system was under software control (ImageMaster; PTI) on an IBM clone computer. Comparisons of samples were done by Student paired t tests; differences were considered significant for P values of <0.05.

FIG. 1.

Differential interference contrast image of RTEC explant cultures. RTEC before (A) and after (B) 4 min of coincubation with RB50. Cells lose their native cuboidal shape as they migrate from the explant, and cilia tend to occupy only a portion of the apical membrane. Individual ciliated and aciliated cells are marked (c and ac, respectively); arrows in panel B show representative bacteria bound to individual cilia.

Wild-type B. bronchiseptica (RB50) preferentially bound to ciliated RTEC within seconds of coincubation, and binding increased over the 5-min incubation period (6.35 ± 0.55 bacteria/cell). Coincubation of RTEC with an avirulent Bvg⁻ strain (RB57) resulted in limited adherence to ciliated cells (peak value of 1.5 ± 0.35 bacteria/cell at 5 min) and no adherence to aciliated cells (Fig. 2). Within 2 min of coincubation, adherence of RB50 to ciliated cells (2.24 ± 0.36 bacteria/cell) was significantly greater than adherence of RB57 to ciliated cells (1.35 ± 0.27 bacteria/cell). At the 2-min time point, neither bacterial strain displayed adherence to aciliated cells. This suggested a BvgAS⁻ and ciliated host cell-specific binding in initial Bordetella-host cell interactions. This preferential binding is in agreement with adhesion studies of B. bronchiseptica and swine nasal ciliated epithelial cells (5); however, our ability to quantify early attachments in live cells via microscopy resulted in a consistently higher number of bacteria attachments at earlier time points (i.e., 2 min versus 15 min). We considered that our assay might be limited by objective focal depth (e.g., not all bacterial binding could be simultaneously recorded at the tip and the base of the cilia). However, electron microscopy studies of bordetellae and ciliated cells of the rabbit airway have shown that the pathogen preferentially binds to the upper third of the cilia (10). Additionally, we repeated the coincubations with a Bvg⁺, green fluorescent protein-expressing strain of B. bronchiseptica under confocal microscopy in “Z” scan mode and found that initial interactions of bacteria and host cells were at the top third of the cilia and within the focal plane (data not shown).

FIG. 2.

Binding of B. bronchiseptica to RTEC cultures. The numbers of bacteria bound to individual RTEC are plotted over time (seconds [Sec]) for the following: RB50 to ciliated cells (black columns); RB50 to aciliated cells (grey columns); and RB57 to ciliated cells (white columns). There was no detectable binding between RB57 and aciliated cells in this time frame. Bacteria were considered bound to the cell after frame-by-frame analysis revealed that they remained in the same position over 15 s. Clumps of bound bacteria were scored as a single bound bacterium. Within 2 min of coincubation, the number of RB50 bacteria bound to ciliated cells was significantly higher than that for aciliated cells or for RB57 bacteria bound to ciliated or aciliated cells (P < 0.05). RB50 binding remained significantly higher throughout the experiment. Error bars represent standard errors; numbers of experiments (n) are shown in the box.

In previous coincubation experiments with B. bronchiseptica and a canine explant model, ciliary beat was altered by virulent (but not avirulent) strains within 5 min, with complete ciliostasis as early as 30 min (3). In the canine explant model studies, heat-killed virulent strains bound to cilia but did not cause ciliostasis, indicating that bacterial effects on ciliated cells extended beyond physical attachment. Ciliostasis did not occur within the time frame of our experiments. However, ciliary dyskinesia was observed in the RB50 coincubation experiments following bacterial attachment, and ciliostasis followed within hours of coincubation. Similar changes were absent from the RB57 coincubation experiments.

Because ion transport, ciliary beat frequency, and mucin release, all key components of mucociliary clearance, can be activated via Ca²⁺ and protein kinase C-dependent mechanisms (13, 15), we examined host cell [Ca²⁺]_i during coincubation with B. bronchiseptica. Ciliated RTEC displayed at least one transient increase in [Ca²⁺]_i in 30% ± 5% of cells between 2 and 8 min of coincubation with RB50 (Fig. 3). In contrast, transient increases in [Ca²⁺]_i for aciliated cells during RB50 coincubation (9.5% ± 6.8%) or RTEC coincubated with RB57 (4.8% ± 2.0% for ciliated and 5.0% ± 1.4% for aciliated cells) were not significantly different from that observed in RTEC washed with HBSS alone (7.5% ± 6.3%). To determine if a lack of initial binding of RB57 to host cells prevented transient increases in [Ca²⁺]_i in host cells, RB57 mutants were centrifuged onto RTEC to force host cell binding, and digital imaging was repeated. Under these conditions, host cells were associated with 15 to 40 bacteria but did not display transient increases in Ca²⁺, further suggesting a role for BvgAS-dependent gene products in host cell signaling.

FIG. 3.

RTEC intracellular Ca²⁺ signaling in response to coincubation with B. bronchiseptica. The percentages of RTEC that displayed at least one transient increase in [Ca²⁺]_i during coincubation with bacteria are plotted as follows: RB50 with ciliated cells (black columns); RB50 with aciliated cells (grey column); RB57 with ciliated cells (white column); RB57 with aciliated cells (vertically striped column); and cells washed with HBSS alone (horizontally striped column). Transient increases in Ca²⁺ were defined as changes in [Ca²⁺]_i of at least 150 nM (i.e., a two- to threefold increase over resting [Ca²⁺]_i) within 2 to 8 min of coincubation of RTEC and B. bronchiseptica. RB50 induced a significantly larger amount of cells displaying Ca²⁺ transients (P < 0.05; *) in ciliated RTEC (30.5%) than in aciliated RTEC or than that for RB57 coincubation with ciliated cells (4.8%) or aciliated cells (5.5%) or in RTEC washed with HBSS alone (7.5%).

Here we assess initial interactions between the respiratory pathogen B. bronchiseptica and its complementary host, ciliated airway epithelial cells, using a ciliated cell culture model. In agreement with previous studies, we found that virulent B. bronchiseptica preferentially bound to ciliated host cells, and this interaction can occur within minutes (3, 5). Moreover, we demonstrated that significant differences in ciliary attachment between virulent and avirulent bacteria occurred within 2 min. B. bronchiseptica strains with an active BvgAS virulence control system additionally induced host cell Ca²⁺ responses in ciliated cells as part of the initial pathogen-host interaction. We have not determined whether the observed [Ca²⁺]_i changes are beneficial to host defense mechanisms (e.g., to temporarily increase ciliary beat frequency and dislodge bacterium) or to bacterial cell pathogenesis (e.g., to alter local mucin concentration to reduce the effectiveness of mucociliary defense). However, this experimental system can be used in conjunction with bacterial genetics to assess the role(s) for bacterial gene loci or individual gene products that contribute to initial pathogen-host interactions that influence host defense mechanisms by altering host cell signaling pathways.