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

Abstract

Streptococci may colonize the epithelium in the airways and other entry sites. While local infection often remains asymptomatic, severe or even fatal diseases occur when streptococci become invasive and spread to different sites in the infected host. We have established porcine respiratory air-liquid interface cultures (ALI) from the porcine lung to analyze the interaction of streptococci with their primary target cells. As representative of the streptococcal family we chose Streptococcus suis (S. suis) that is not only a major swine respiratory pathogen but can also infect humans. Suilysin, a cholesterol-dependent cytolysin (CDC), is an important virulence factor. By comparing a S. suis wt strain with a suilysin-deficient mutant, we demonstrate that suilysin contributes to (i) adherence to airway cells (ii) loss of ciliated cells (iii) apoptosis, and (iv) invasion. Furthermore, we show that cytolytic activity of suilysin is crucial for these effects. A striking result of our analysis was the high efficiency of S. suis-induced apoptosis and invasion upon infection under ALI conditions. These properties have been reported to be less efficient when analyzed with immortalized cells. We hypothesize that soluble effectors such as suilysin are present at higher concentrations in cells kept at ALI conditions and thus more effective. These results should be relevant also for infection of the respiratory tract by other respiratory pathogens.

Viral Coinfection Replaces Effects of Suilysin on Streptococcus suis Adherence to and Invasion of Respiratory Epithelial Cells Grown under Air-Liquid Interface Conditions

“…1, at 4 hpi the release of LDH was significantly increased in PBEC infected with the wt S. suis strain but not after monoinfection with the suilysin-deficient mutant. This finding is consistent with the results of our previous study on S. suis monoinfection of well-differentiated airway epithelial cells (26). Viral-bacterial coinfec-tion, in any of the combinations analyzed, did not result in an increase in the cytotoxic effect compared to that in virus-monoinfected PBEC.…”

“…2A and B. The S. suis wt strain was more efficient in adherence to the airway cells than the 10Δsly mutant, which is consistent with the findings of previous studies (25,26,32). In the case of respiratory epithelial cells preinfected with SIV, bacterial adherence was enhanced for both the wt and suilysin-deficient mutant 10Δsly.…”

“…The monoinfected samples (Fig. 4B, top) showed the difference in invasion efficiency between wt and mutant S. suis that has been reported recently (26). While the mutant bacteria were mainly detectable on top of the epithelial cells, wt streptococci invaded into deeper areas of the epithelial cell layer.…”

“…Differences in the time kinetics of S. suis-and SIV-induced cytotoxicity. Both S. suis and influenza virus have a cytotoxic effect on epithelial cells (26,30). To find out how the two pathogens contribute to the cytotoxic effect in a coinfection scenario, the release of cellular lactate dehydrogenase (LDH) was determined during the course of a coinfection with SIV and S. suis.…”

“…Suilysin (SLY), the yet only known cytotoxic protein secreted by S. suis, has been studied for its contribution to pathogenesis at different stages in the development of systemic disease induced by S. suis (24). It has been suggested that SLY facilitates the colonization and the establishment of the initial stages of infection in pigs by promoting host cell lysis and the invasion of S. suis through the epithelium when bacteria have colonized the upper respiratory tract (25,26). Furthermore, S. suis isolates lacking the suilysin gene have been shown to be avirulent in a mouse infection model when applied via the intraperitoneal route but virulent in pigs after intravenous injection, which indicates that suilysin is not essential for S. suis to cause systemic infection in pigs once the bacteria have entered the blood vessels (27).…”