Paragraph: 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. By taking up air from the environment, the respiratory tract is regularly exposed to various microorganisms and thus has a potential risk to get infected by pathogenic bacteria or viruses 1 . Streptococci are important pathogens that may initiate respiratory tract infections 2 . They comprise a number of diverse members infecting humans and/or animals 3-5 . In general, they have evolved effective mechanisms to adhere to and colonize different mucosal surfaces 6,7 . Adherence to surface-exposed host structures often results only in asymptomatic infections 6, 8, 9 . Severe diseases occur when streptococci succeed to invade the deeper host tissues 10,11 . Disease symptoms may range from pharyngitis, scarlet fever, and toxic shock syndrome caused by S. pyogenes, also referred to as group A streptococci. Further manifestations are peumonia, meningitis, or sepsis caused by S. agalactiae (group B streptococci) and S. pneumoniae (pneumococci) [11] [12] [13] [14] . Additionally, animal streptococci may have a zoonotic potential and induce disease not only in their host but also in humans. Streptococcus suis (S. suis) is a major cause of meningitis, sepsis, arthritis and pneumonia in piglets with a worldwide distribution 3,15 . Moreover, it can cause severe infections to people that are in close contact with infected pigs. Two outbreaks of severe human infections by S. suis have occurred in China and affected several hundred persons in 1998 and 2005. The mortality rate was 56% and 18.6%, respectively 10,16 . In Vietnam, S. suis infections have been reported to be the most common cause of bacterial meningitis in adults 17 . In Thailand, S. suis also is considered as an emerging human pathogen 18 . Most cases of animal and human S. suis infections were caused by serotype 2 strains and some cases have been attributed to serotype 14 18-20 . Nevertheless, it has been only poorly studied which virulence factors of S. suis contribute to the early stages of infection, i.e. adherence to and colonization of mucosal surfaces or to crossing the mucosal epithelia to generate systemic disease. The portal of entry most often used by streptococci for invasion is the airway system. The lower respiratory tract is usually sterile because the mucociliary clearance system based on the concerted action of mucus secretion and ciliary activity provides an effective defense mechanism 6 . Although the respiratory epithelium is known to be the main target tissue of respiratory pathogens, the role of the well-differentiated airway epithelial cells has been poorly studied 21 . The well-differentiated airway epithelium is composed of different types of cells, such as mucus-producing (goblet) cells, basal cells, ciliated and non-ciliated cells. Primary culture systems for well-differentiated respiratory epithelial cells provide the closest in vitro representation of the airway epithelium and thus are the cell cultures of choice to analyze respiratory infections [21] [22] [23] [24] . Pathogenic streptococci are characterized by the release of cholesterol-dependent pore-forming cytolysins (CDC). Pneumolysin, the CDC of Streptococcus pneumoniae induces cytotoxic effects and apoptosis of macrophages and neuronal cells 25 . Furthermore, it may promote bacterial adherence and invasion 26, 27 . Suilysin, the CDC of S. suis, plays a role in the pathogenesis of invasive disease caused by this bacterial agent 28, 29 . It is responsible for cytotoxic effects and contributes to the adhesion of S. suis [30] [31] [32] [33] [34] . However, little is known about the role of suilysin in infection of the airway epithelium. Studies addressing streptococcal invasion have been performed mainly with immortalized cell lines 31,35,36 . We have recently used precision-cut lung slices to analyze the adherence of S. suis to differentiated porcine airway epithelial cells 37 . To further elucidate the question how S. suis invades the respiratory tract, in the present study air-liquid interface (ALI) cultures were used. ALI cultures of human airway epithelial cells are well established for analysis of virus infections. Porcine ALI cultures have been used only recently for infection by influenza viruses and adenoviruses 23, 38 . Compared to the human counterpart, the latter cells have the advantage that the source of cells is well-defined and reproducible as far as the age and genetic background of the animals are concerned. Here we show that infection of differentiated airway epithelial cells by S. suis at ALI conditions results in suilysin-mediated effects, such as adherence, cytotoxicity, invasion, and apoptosis. Strikingly, some of the effects are very efficient when compared to those reported in studies with conventional culture systems. 
 Title: Efficient suilysin-mediated invasion and apoptosis in porcine respiratory epithelial cells after streptococcal infection under air-liquid interface conditions OPEN
Output: False