Efficient suilysin-mediated invasion and apoptosis in porcine respiratory epithelial cells after streptococcal infection under air-liquid interface conditions

Meng, Fandan; Wu, Nai-Huei; Seitz, Maren; Herrler, Georg; Valentin-Weigand, Peter

Adherence of S. suis to well-differentiated porcine bronchial epithelial cells. Adherence to host cells is important for S. suis to cause infection and initiate disease 40, 41 . The ability of S. suis to adhere to and colonize well-differentiated epithelial cells was determined by immunofluorescence microscopy at 4 hpi. PBEC were apically infected with 1 × 10 7 CFU of S. suis per filter. After incubation at 37 °C for 4 h, cells were washed thoroughly to remove non-adherent bacteria and fixed with 3.7% formaldehyde. As shown in Fig. 3A , immunofluorescence analysis demonstrated that streptococci were able to adhere to PBEC. Bound bacteria were mainly detected on ciliated cells. As shown in Fig. 3F , quantification of adherent bacteria revealed that suilysin-positive S. suis wt strain adhered to PBEC (0.16%) with significantly higher efficiency than the suilysin-deficient mutant 10Δ sly (0.075%), respectively. The number of adherent wt bacteria was 2-fold higher than that of 10Δ sly on PBEC cells. This result is consistent with a previous report that suilysin promotes the adherence of S. suis to the immortalized respiratory epithelial cell line HEp-2 and porcine ex vivo precision-cut lung slices 37, 39 . Interestingly, adherence of the two complemented strains cW461F and cS148, respectively, was similar to that of the wt and significantly higher than that of the suilysin-deficient mutant 10Δ sly. These results suggest that macropore-formation of suilysin was not involved in promotion of S. suis adherence to PBEC. On the other hand, it has been reported that nonencapsulated S. suis strain showed higher capacity of adherence to and invasion of host cells in comparison to the parental strain 42, 43 . Therefore, we also included a suilysin-deficient nonencapsulated mutant (10Δ cpsΔ sly). As shown in Fig. 3A , lack of a capsule enhanced the adherence of the suilysin-deficient mutant to PBEC. Quantification of microscopic examinations confirmed significantly higher adherence of the nonencapsulated mutant 10Δ cpsΔ sly as compared to the encapsulated wt and the mutant 10Δ sly (Fig. 3F) . These results indicate that suilysin and the capsule can both affect the adherence of S. suis to well-differentiated epithelial cells. Suilysin shows an enhancing effect on S. suis adherence to airway epithelial cells, which is independent of macropore-formation, whereas the capsule has a reducing effect. Results are expressed as x-fold increase compared to mockinfected cells (mock). Results are expressed as means ± SEM and significance, indicated by * (P-value < 0.05), ** (P-value < 0.01), *** (P-value < 0.0001), was determined using one-way-ANOVA and Tukey multiple comparison test. Experiments were performed at least three times.