SummaryThe genomic analysis of Streptococcus pneumoniae strains identified the Pneumococcal adherence and virulence factor B (PavB), whose repetitive sequences, designated Streptococcal Surface REpeats (SSURE), interact with human fibronectin. Here, we showed the gene in all tested pneumococci and identified that the observed differences in the molecular mass of PavB rely on the number of repeats, ranging from five to nine SSURE. PavB interacted with fibronectin and plasminogen in a dosedependent manner as shown by using various SSURE peptides. In addition, we identified PavB as colonization factor. Mice infected intranasally with DpavB pneumococci showed significantly increased survival times compared with wild-type bacteria. Importantly, the pavB-mutant showed a delay in transmigration to the lungs as observed in real-time using bioluminescent pneumococci and decreased colonization rates in a nasopharyngeal carriage model. In co-infection experiments the wild-type outcompeted the pavB-mutant and infections of epithelial cells demonstrated that PavB contributes to adherence to host cell. Blocking experiments suggested a function of PavB as adhesin, which was confirmed by direct binding of SSURE peptides to host cells. Finally, PavB may represent a new vaccine candidate as SSURE peptides reacted with human sera. Taken together, PavB is a surface-exposed adhesin, which contributes to pneumococcal colonization and infections of the respiratory airways.

Toll-like receptors (TLRs) are crucial pattern recognition receptors in innate immunity that are expressed in microglia, the resident macrophages of the brain. TLR2, -4, and -9 are important in the responses against Streptococcus pneumoniae, the most common agent causing bacterial meningitis beyond the neonatal period. Murine microglial cultures were stimulated with agonists for TLR1/2 (Pam 3 CSK 4 ), TLR4 (lipopolysaccharide), and TLR9 (CpG oligodeoxynucleotide) for 24 h and then exposed to either the encapsulated D39 (serotype 2) or the nonencapsulated R6 strain of S. pneumoniae. After stimulation, the levels of interleukin-6 and CCL5 (RANTES [regulated upon activation normal T-cell expressed and secreted]) were increased, confirming microglial activation. The TLR1/2, -4, and -9 agonist-stimulated microglia ingested significantly more bacteria than unstimulated cells (P < 0.05). The presence of cytochalasin D, an inhibitor of actin polymerizaton, blocked >90% of phagocytosis. Along with an increased phagocytic activity, the intracellular bacterial killing was also increased in TLR-stimulated cells compared to unstimulated cells. Together, our data suggest that microglial stimulation by these TLRs may increase the resistance of the brain against pneumococcal infections.

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Meningitis and meningoencephalitis caused by Escherichia coli are associated with high rates of mortality.When an infection occurs, Toll-like receptors (TLRs) expressed by microglial cells can recognize pathogenassociated molecular patterns and activate multiple steps in the inflammatory response that coordinate the brain's local defense, such as phagocytosis of invading pathogens. An upregulation of the phagocytic ability of reactive microglia could improve the host defense in immunocompromised patients against pathogens such as E. coli. Here, murine microglial cultures were stimulated with the TLR agonists Pam 3 CSK 4 (TLR1/TLR2), lipopolysaccharide (TLR4), and CpG oligodeoxynucleotide (TLR9) for 24 h. Upon stimulation, levels of tumor necrosis factor alpha and the neutrophil chemoattractant CXCL1 were increased, indicating microglial activation. Phagocytic activity was studied after adding either E. coli DH5␣ or E. coli K1 strains. After 60 and 90 min of bacterial exposure, the number of ingested bacteria was significantly higher in cells prestimulated with TLR agonists than in unstimulated controls (P < 0.01). Addition of cytochalasin D, an inhibitor of actin polymerization, blocked >90% of phagocytosis. We also analyzed the ability of microglia to kill the ingested E. coli strains. Intracellularly surviving bacteria were quantified at different time points (90, 150, 240, and 360 min) after 90 min of phagocytosis. The number of bacteria killed intracellularly after 6 h was higher in cells primed with the different TLR agonists than in unstimulated microglia. Our data suggest that microglial stimulation by the TLR system can increase bacterial phagocytosis and killing. This approach could improve central nervous system resistance to infections in immunocompromised patients.

The interaction of endogenous and exogenous stimulators of innate immunity was examined in primary cultures of mouse microglial cells and macrophages after application of defined Toll-like receptor (TLR) agonists [lipopolysaccharide (LPS) (TLR4), the synthetic lipopeptide Pam3Cys-Ser-Lys4 (Pam3-Cys) (TLR2) and single-stranded unmethylated CpG-DNA (CpG) (TLR9)] alone and in combination with amyloid beta peptide (Abeta) 1-40. Abeta 1-40 stimulated microglial cells and macrophages primed by interferon-c in a dose-dependent manner. Co-administration of Abeta1-40 with LPS or Pam3-Cys led to an additive release of nitric oxide (NO) and tumour necrosis factor alpha (TNF-a). This may be one reason for the clinical deterioration frequently observed in patients with Alzheimer's disease during infections. In contrast, co-application of Abeta1-40 with CpG led to a substantial decrease of NO and TNF-a release compared with stimulation with CpG alone. Abeta 1-40 and CpG did not co-localize within the same subcellular compartment, making a direct physicochemical interaction as the cause of the observed antagonism very unlikely. This suggests that not all TLR agonists enhance the stimulatory effect of Abeta on innate immunity.