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

Streptococcus suis is an important zoonotic pathogen which can infect humans and pigs worldwide, posing a potential risk to global public health. Suilysin, a pore-forming cholesterol-dependent cytolysin, is considered to play an important role in the pathogenesis of S. suis infections. It is known that infection with influenza A viruses may favor susceptibility to secondary bacterial infection, resulting in more severe disease and increased mortality. However, the molecular mechanisms underlying these coinfections are incompletely understood. Applying highly differentiated primary porcine respiratory epithelial cells grown under air-liquid interface (ALI) conditions, we analyzed the contribution of swine influenza viruses (SIV) to the virulence of S. suis, with a special focus on its cytolytic toxin, suilysin. We found that during secondary bacterial infection, suilysin of S. suis contributed to the damage of well-differentiated respiratory epithelial cells in the early stage of infection, whereas the cytotoxic effects induced by SIV became prominent at later stages of infection. Prior infection by SIV enhanced the adherence to and colonization of porcine airway epithelial cells by a wild-type (wt) S. suis strain and a suilysin-negative S. suis mutant in a sialic acid-dependent manner. A striking difference was observed with respect to bacterial invasion. After bacterial monoinfection, only the wt S. suis strain showed an invasive phenotype, whereas the mutant remained adherent. When the epithelial cells were preinfected with SIV, the suilysin-negative mutant also showed an invasion capacity. Therefore, we propose that coinfection with SIV may compensate for the lack of suilysin in the adherence and invasion process of suilysin-negative S. suis.

Characterization of the zinc metalloprotease of Streptococcus suis serotype 2.

Streptococcus suis is a swine pathogen and zoonotic agent responsible for meningitis and septic shock. Although several putative virulence factors have been described, the initial steps of the S. suis pathogenesis remain poorly understood. While controversial results have been reported for a S. suis serotype 2 zinc metalloprotease (Zmp) regarding its IgA protease activity, recent phylogenetic analyses suggested that this protein is homologous to the ZmpC of Streptococcus pneumoniae, which is not an IgA protease. Based on the previously described functions of metalloproteases (including IgA protease and ZmpC), different experiments were carried out to study the activities of that of S. suis serotype 2. First, results showed that S. suis, as well as the recombinant Zmp, were unable to cleave human IgA1, confirming lack of IgA protease activity. Similarly, S. suis was unable to cleave P-selectin glycoprotein ligand-1 and to activate matrix metalloprotease 9, at least under the conditions tested. However, S. suis was able to partially cleave mucin 16 and syndecan-1 ectodomains. Experiments carried out with an isogenic Δzmp mutant showed that the Zmp protein was partially involved in such activities. The absence of a functional Zmp protein did not affect the ability of S. suis to adhere to porcine bronchial epithelial cells in vitro, or to colonize the upper respiratory tract of pigs in vivo. Taken together, our results show that S. suis serotype 2 Zmp is not a critical virulence factor and highlight the importance of independently confirming results on S. suis virulence by different teams.

Bordetella bronchiseptica promotes adherence, colonization, and cytotoxicity of Streptococcus suis in a porcine precision-cut lung slice model.

Bordetella (B.) bronchiseptica and Streptococcus (S.) suis are major pathogens in pigs, which are frequently isolated from co-infections in the respiratory tract and contribute to the porcine respiratory disease complex (PRDC). Despite the high impact of co-infections on respiratory diseases of swine (and other hosts), very little is known about pathogen-pathogen-host interactions and the mechanisms of pathogenesis. In the present study, we established a porcine precision-cut lung slice (PCLS) model to analyze the effects of B. bronchiseptica infection on adherence, colonization, and cytotoxic effects of S. suis. We hypothesized that induction of ciliostasis by a clinical isolate of B. bronchiseptica may promote subsequent infection with a virulent S. suis serotype 2 strain. To investigate this theory, we monitored the ciliary activity by light microscopy, measured the release of lactate dehydrogenase, and calculated the number of PCLS-associated bacteria. To study the role of the pore-forming toxin suilysin (SLY) in S. suis-induced cytotoxicity, we included a SLY-negative isogenic mutant and the complemented mutant strain. Furthermore, we analyzed infected PCLS by histopathology, immunofluorescence microscopy, and field emission scanning electron microscopy. Our results showed that pre-infection with B. bronchiseptica promoted adherence, colonization, and, as a consequence of the increased colonization, the cytotoxic effects of S. suis, probably by reduction of the ciliary activity. Moreover, cytotoxicity induced by S. suis is strictly dependent on the presence of SLY. Though the underlying molecular mechanisms remain to be fully clarified, our results clearly support the hypothesis that B. bronchiseptica paves the way for S. suis infection.

Streptococcus suis Induces Expression of Cyclooxygenase-2 in Porcine Lung Tissue.

Streptococcus suis is a common pathogen colonising the respiratory tract of pigs. It can cause meningitis, sepsis and pneumonia leading to economic losses in the pig industry worldwide. Cyclooxygenase-2 (COX-2) and its metabolites play an important regulatory role in different biological processes like inflammation modulation and immune activation. In this report we analysed the induction of COX-2 and the production of its metabolite prostaglandin E2 (PGE2) in a porcine precision-cut lung slice (PCLS) model. Using Western blot analysis, we found a time-dependent induction of COX-2 in the infected tissue resulting in increased PGE2 levels. Immunohistological analysis revealed a strong COX-2 expression in the proximity of the bronchioles between the ciliated epithelial cells and the adjacent alveolar tissue. The morphology, location and vimentin staining suggested that these cells are subepithelial bronchial fibroblasts. Furthermore, we showed that COX-2 expression as well as PGE2 production was detected following infection with two prevalent S. suis serotypes and that the pore-forming toxin suilysin played an important role in this process. Therefore, this study provides new insights in the response of porcine lung cells to S. suis infections and serves as a basis for further studies to define the role of COX-2 and its metabolites in the inflammatory response in porcine lung tissue during infections with S. suis.

Membrane Binding, Cellular Cholesterol Content and Resealing Capacity Contribute to Epithelial Cell Damage Induced by Suilysin of Streptococcus suis.

Streptococcus (S.) suis is a major cause of economic losses in the pig industry worldwide and is an emerging zoonotic pathogen. One important virulence-associated factor is suilysin (SLY), a toxin that belongs to the family of cholesterol-dependent pore-forming cytolysins (CDC). However, the precise role of SLY in host-pathogen interactions is still unclear. Here, we investigated the susceptibility of different respiratory epithelial cells to SLY, including immortalized cell lines (HEp-2 and NPTr cells), which are frequently used in in vitro studies on S. suis virulence mechanisms, as well as primary porcine respiratory cells, which represent the first line of barrier during S. suis infections. SLY-induced cell damage was determined by measuring the release of lactate dehydrogenase after infection with a virulent S. suis serotype 2 strain, its isogenic SLY-deficient mutant strain, or treatment with the recombinant protein. HEp-2 cells were most susceptible, whereas primary epithelial cells were hardly affected by the toxin. This prompted us to study possible explanations for these differences. We first investigated the binding capacity of SLY using flow cytometry analysis. Since binding and pore-formation of CDC is dependent on the membrane composition, we also determined the cellular cholesterol content of the different cell types using TLC and HPLC. Finally, we examined the ability of those cells to reseal SLY-induced pores using flow cytometry analysis. Our results indicated that the amount of membrane-bound SLY, the cholesterol content of the cells, as well as their resealing capacity all affect the susceptibility of the different cells regarding the effects of SLY. These findings underline the differences of in vitro pathogenicity models and may further help to dissect the biological role of SLY during S. suis infections.

Streptococcus suis - The "Two Faces" of a Pathobiont in the Porcine Respiratory Tract.

Streptococcus (S.) suis is a frequent early colonizer of the upper respiratory tract of pigs. In fact, it is difficult to find S. suis-free animals under natural conditions, showing the successful adaptation of this pathogen to its porcine reservoir host. On the other hand, S. suis can cause life-threatening diseases and represents the most important bacterial cause of meningitis in pigs worldwide. Notably, S. suis can also cause zoonotic infections, such as meningitis, septicemia, endocarditis, and other diseases in humans. In Asia, it is classified as an emerging zoonotic pathogen and currently considered as one of the most important causes of bacterial meningitis in adults. The "two faces" of S. suis, one of a colonizing microbe and the other of a highly invasive pathogen, have raised many questions concerning the interpretation of diagnostic detection and the definition of virulence. Thus, one major research challenge is the identification of virulence-markers which allow differentiation of commensal and virulent strains. This is complicated by the high phenotypic and genotypic diversity of S. suis, as reflected by the occurrence of (at least) 33 capsular serotypes. In this review, we present current knowledge in the context of S. suis as a highly diverse pathobiont in the porcine respiratory tract that can exploit disrupted host homeostasis to flourish and promote inflammatory processes and invasive diseases in pigs and humans.