The effect of bacterial co-infection on the infection of well-differentiated porcine respiratory epithelial cells by swine influenza viruses

Swine influenza virus (SIV) and Streptococcus (S.) suis are common pathogens of the respiratory tract in pigs, both being associated with pneumonia. However, the interactions of these two pathogens with well-differentiated respiratory epithelial cells and the contribution to the pathogenesis of co-infection are only poorly understood. Here my colleagues and I established two primary cell culture systems for well-differentiated airway epithelial cells, porcine precision-cut lung slices (PCLS) and porcine airway air-liquid interface (ALI) cultures, to analyze the infection of SIV and/or S. suis. First I analyzed the viral or bacterial mono-infection of PCLS, on the one hand by five swine influenza A viruses of different subtypes and on the other hand by the parental S. suis serotype 2 as well as by different mutant streptococci, respectively. Infection of PCLS by SIV revealed that a higher ciliostatic effect and virus titer was obtained in PCLS infected by the H3N2 subtype viruses in comparison to the H1N1/2006 strain; these virulence properties of the different viruses correspond to the pathogenicity properties determined in animal experiments. Concerning the infection of S. suis, both the parental strain of S. suis and the mutants analyzed were able to adhere to and to efficiently colonize ciliated cells and the mucus-producing cells of the bronchiolar epithelium. These data indicate that PCLS provide a model that is able to assess the virulence of influenza A viruses and to study S. suis adherence and colonization. Bacterial co-infection often aggravates the clinical outcome both in humans and animals which is associated with a high risk of developing more complicated diseases. Hence, I further established the PCLS co-infection model for analyzing the effect of secondary S. suis infection after prior infection by SIV to address the interaction between both pathogens and their contribution to co-infection. I found that SIV promoted adherence, colonization, and invasion of S. suis in a two-step process. First, in the initial stages, the α-2,6-linked sialic acid present in the capsular polysaccharide of S. suis mediated the adherence of encapsulated, but not nonencapsulated, S. suis to SIV-infected cells, as a result of the direct interaction of the hemagglutinin of SIV with the α-2,6-linked sialic acid of S. suis. Second, at a later stage of infection, high-virulent SIV promoted S. suis adherence and invasion into deeper tissues by damaging ciliated epithelial cells. On the other hand, secondary bacterial infection had a negative effect on the replication of SIV. My finding revealed that at least two different mechanisms contribute to the beneficial effects of SIV on S. suis infection, including sialic acid-mediated bacterial attachment to SIV-infected cells and virus-mediated damage of ciliated epithelial cells. Primary airway epithelial cells maintained under ALI conditions allow the analysis of a wide variety of respiratory pathogens but few studies has been reported about bacterial infection on ALI so far. My colleague and I established porcine airway ALI cultures, for porcine tracheal epithelial cells (PTEC) as well as for porcine bronchial epithelial cells (PBEC), to analyze the contribution of suilysin, a bacterial cytolysin, to the virulence properties of S. suis infection. I found that the suilysin-positive S. suis serotype 2 wt showed not only higher adherence but also a substantially higher number of intracellular bacteria in both PTEC and PBEC as compared to a suilysin-deficient mutant (10Δsly). In addition, a decreased amount of ciliated cells was observed as a result of localized lesions. Furthermore, increased apoptosis was detected on PBEC infected with suilysin-positive wt streptococci. My findings suggest that the soluble suilysin plays a crucial role in mediating invasion of S. suis into well-differentiated porcine respiratory epithelial cells and that most likely suilysin-dependent apoptosis is responsible for respiratory epithelial cell death.