Infection of bovine well-differentiated airway epithelial cells by Pasteurella multocida: actions and counteractions in the bacteria-host interactions

Abstract

Pasteurella (P.) multocida is a zoonotic pathogen, which is able to cause respiratory disorder in different hosts. In cattle, P. multocida is an important microorganism involved in the bovine respiratory disease complex (BRDC) with a huge economic impact. We applied air-liquid interface (ALI) cultures of well-differentiated bovine airway epithelial cells to analyze the interaction of P. multocida with its host target cells. The bacterial pathogen grew readily on the ALI cultures. Infection resulted in a substantial loss of ciliated cells. Nevertheless, the epithelial cell layer maintained its barrier function as indicated by the transepithelial electrical resistance and the inability of dextran to get from the apical to the basolateral compartment via the paracellular route. Analysis by confocal immunofluorescence microscopy confirmed the intactness of the epithelial cell layer though it was not as thick as the uninfected control cells. Finally, we chose the bacterial neuraminidase to show that our infection model is a sustainable tool to analyze virulence factors of P. multocida. Furthermore, we provide an explanation, why this microorganism usually is a commensal and becomes pathogenic only in combination with other factors such as co-infecting microorganisms.

Keywords: Pasteurella multocida; air–liquid interface (ALI) cultures; bacterial pathogenesis; host–pathogen interactions.

Figure 1

Growth kinetics of P. multocida in well-differentiated bovine airway epithelial cell cultures. Filter-grown well-differentiated bovine bronchial epithelial cells were infected with P. multocida by inoculation with 10² or 10⁵ CFU for 1 h or 4 h. Supernatants were harvested at 4, 12 and 24hpi and used to determine the growth kinetics. The significance of the differences between the values in each group is indicated with asterisks by comparing the CFU titer vertically. Results represent the mean values of CFU ± SEM determined from three independent experiments with duplicated samples. ***P < 0.001, **P < 0.01 and*P < 0.05.

Figure 2

Cytotoxic effect of P. multocida grown on well-differentiated bovine bronchial epithelial cells. Filter-grown ALI cultures were infected by P. multocida at the indicated inoculation conditions (10² or 10⁵ CFU, 1 h or 4 h). At the times indicated, the amount of LDH released into the apical compartment was determined.

Figure 3

Effect of P. multocida infection on ciliated cells. Well-differentiated bovine airway epithelial cells were infected by P. multocida at the inoculation conditions indicated (10² or 10⁵ CFU, 1 or 4 h). At 4, 12, 24hpi, samples were immune-stained (anti-β-tubulin, DAPI) to visualize cilia (red) and nuclei (blue). A Pictures obtained by microscopy; B Quantitation of the relative red fluorescence. Results are presented as mean ± SEM compared to mock-infected samples; Scale bars-50 µm.

Figure 4

Barrier function of bovine well-differentiated airway epithelial cells after infection by P. multocida. Filter-grown ALI cultures of differentiated bovine airway epithelial cells were infected by P. multocida at the indicated inoculation conditions (10² or 10⁵ CFU; 1 or 4 h). At the times indicated, samples were analyzed for the intactness of the barrier function A by determining the transepithelial electrical resistance (TEER) and B by determining the permeability for FITC-labeled dextran.

Figure 5

Effect of infection by P. multocida on the thickness of the epithelial cell layer. Differentiated bovine airway epithelial cells were infected by P. multocida at the indicated inoculation conditions (10² or 10⁵ CFU; 1 or 4 h) and immune-stained as described in Figure 3. A Vertical sections of the immunostained samples; B relative thickness of the epithelial cell layer compared to mock infected samples. Scale bars, 50 µm.

Figure 6

Importance of the bacterial neuraminidase for P. multocida infection. Filter-grown ALI cultures were infected by P. multocida (inoculation with 10² CFU for 1 h). The importance of the bacterial neuraminidase was analyzed by pretreating some samples with neuraminidase from C. perfringens and by incubating some of the other samples in the presence of the neuraminidase inhibitor DANA. At 4, 12, 24 hpi, samples were immune-stained (anti-β-tubulin, DAPI) to visualize cilia (red) and nuclei (blue). A Pictures obtained by microscopy; B Quantitation of the relative red fluorescence. Results are presented as mean ± SEM compared to mock-infected samples.