Interaction of Streptococcus pneumoniae with endothelial and epithelial cells

Streptococcus pneumoniae is a leading cause of invasive bacterial disease. Ideally, characterization of pneumococcal virulence factors should include expression during the infection process. However, analysis of in vivo gene expression has been limited by the difficulties of isolating sufficient quantities of pure and intact bacterial RNA from infected host tissues or tissue culture cells and, as a result, very little is known about the distribution of pneumococcal virulence factors in discrete body sites and their contribution to different forms of disease. This is the first study to examine the expression of S. pneumoniae genes when interacting with eukaryotic cells using whole-genome microarrays available from The Institute for Genomic Research. Total RNA was collected from pneumococci isolated from bacteria attached to a pharyngeal epithelial cell line in vitro. Microarray analysis of pneumococcal genes expressed in this model identified body site-specific patterns of expression for virulence factors, transporters, transcription factors, translation-associated proteins, metabolism, and genes with unknown function. Contributions to virulence predicted for several unknown genes with enhanced expression in vivo were confirmed by insertion duplication mutagenesis and challenge of mice with the mutants. Finally, we cross-referenced our results with previous studies that used signature-tagged mutagenesis and differential fluorescence induction to identify genes that are potentially required by a broad range of pneumococcal strains for invasive disease.; Pneumococci traverse eukaryotic cells within vacuoles without intracytoplasmic multiplication. The platelet-activating factor receptor (PAFr) has been suggested as a portal of entry. Pneumococci co-localized with PAFr on endothelial cells and PAFr-/- mice showed a substantially impaired ability to support bacterial translocation, particularly from blood to brain. Pneumococci-induced co-localization of PAR and beta-arrestin 1 at the plasma membrane of endothelial cells and PAFr-mediated pneumococcal uptake in transfected COS cells were greatly increased by co-transfection with the scaffold/adapter protein beta-arrestin 1. Activation of extracellular signal-regulated kinase kinases was required for uptake and was limited to the cytoplasmic compartment, consistent with activation by beta-arrestin rather than PAR. Uptake of the pneumococcal vacuole involved clathrin, and half the bacteria proceeded into vacuoles marked by Rab5 and later Rab7, the classical route to the lysosome. Overexpression of beta-arrestin in endothelial cells decreased co-localization with Rab7. We conclude that the association of beta-arrestin with the PAR contributes to successful translocation of pneumococci.