The interaction of Streptococcus pneumoniae with plasmin mediates transmigration across endothelial and epithelial monolayers by intercellular junction cleavage.

The precise mechanisms by which Streptococcus pneumoniae overcomes epithelial and endothelial barriers to access underlying human tissues remain to be determined. The plasminogen system is highly important for the tissue barrier degradation which allows cell migration. Plasminogen is known to interact with pneumococci via enolase, glyceraldehyde-3-phosphate dehydrogenase, and choline-binding protein E. These observations prompted us to evaluate the role of this proteolytic system in the pneumococcal invasion process. We observed that coating of S. pneumoniae R6 strain with plasminogen or inactivated plasmin increased adherence to pulmonary epithelial A549 and vascular endothelial EaHy cells in vitro. This indicates that plasminogen-mediated adherence is independent of the protease activity and involves plasminogen binding to receptors on eukaryotic cell surfaces. Conversely, decreased adherence of bacterial cells coated with active plasmin was observed, indicating that the protease activity limits bacterial attachment on the cell surface. We were then interested in investigating the role of the proteolytic plasmin activity in the traversal of tissue barriers. We observed that adherence of plasmin-coated D39 (encapsulated) or R6 (unencapsulated) pneumococci induced sporadic disruptions of EaHy and A549 monolayer cell junctions. This was not observed when plasmin was inhibited by aprotinin. Endothelial junction disorganization may proceed by proteolysis of the cell junction components. This is supported by our observation of the in vitro cleavage by plasmin bound to pneumococci of recombinant vascular endothelial cadherin, the main component of endothelial adherens junctions. Finally, junction damage induced by plasmin may be related to tissue barrier traversal, as we measured an increase of S. pneumoniae transmigration across epithelial A549 and endothelial EaHy layers when active plasmin was present on the bacterial surface. Our results highlight a novel function for the plasminogen recruitment at the bacterial surface in facilitating adherence of pneumococci to endothelial and epithelial cells, while active plasmin degrades intercellular junctions. This process promotes migration of pneumococci through cell barriers by a pericellular route, a prerequisite for dissemination of S. pneumoniae in the host organism.

Streptococcus pneumoniae choline-binding protein E interaction with plasminogen/plasmin stimulates migration across the extracellular matrix.

The virulence mechanisms leading Streptococcus pneumoniae to convert from nasopharyngeal colonization to a tissue-invasive phenotype are still largely unknown. Proliferation of infection requires penetration of the extracellular matrix, which occurs by recruitment of host proteases to the bacterial cell surface. We present evidence supporting the role of choline-binding protein E (CBPE) (a member of the surface-exposed choline-binding protein family) as an important receptor for human plasminogen, the precursor of plasmin. The results of ligand overlay blot analyses, solid-phase binding assays, and surface plasmon resonance experiments support the idea of an interaction between CBPE and plasminogen. We have shown that the phosphorylcholine esterase (Pce) domain of CBPE interacts with the plasminogen kringle domains. Analysis of the crystal structure of the Pce domain, followed by site-directed mutagenesis, allowed the identification of the plasminogen-binding region composed in part by lysine residues, some of which map in a linear fashion on the surface of the Pce domain. The biological relevance of the CBPE-plasminogen interaction is supported by the fact that, compared to the wild-type strain, a mutant of pneumococcus with the cbpE gene deleted (i) displays a reduced level of plasminogen binding and plasmin activation and (ii) shows reduced ability to cross the extracellular matrix in an in vitro model. These results support the idea of a physiological role for the CBPE-plasminogen interaction in pneumococcal dissemination into human tissue.