Pneumolysin, neuraminidases A and B, and hyaluronidase are virulence factors of Streptococcus pneumoniae that appear to be involved in the pathogenesis of meningitis. In a murine model of meningitis after intracerebral infection using mutants of S. pneumoniae D39, only mice infected with a pneumolysin-deficient strain were healthier at 32 and 36 h, had lower bacterial titers in blood at 36 h, and survived longer than the D39 parent strain. Cerebellar and spleen bacterial titers, meningeal inflammation, and neuronal damage scores remained uninfluenced by the lack of any of the virulence factors.Streptococcus pneumoniae meningitis frequently causes severe neurological sequelae and death (9,15,23). A number of pneumococcal proteins have been characterized as putative virulence factors, among them pneumolysin, the neuraminidases A and B, and hyaluronidase (7,8,16,17,21,22,27). Pneumolysin, a cytoplasmic protein, is released during autolysis of the bacterium and probably also via an autolysis-independent mechanism (3, 21). It interacts with cholesterol in the cell wall of host cells and forms transmembrane pores by oligomerization, leading to loss of membrane integrity of host cells. In sublytic concentrations it is capable of inhibiting respiratory burst, chemotaxis, and bactericidal activity of polymorphonuclear leukocytes (20). Furthermore, it leads to complement consumption (6), thereby reducing serum opsonic activity (1, 2). Neuraminidase activity has been indirectly linked to virulence in human pneumococcal meningitis on the basis of elevated cerebrospinal fluid concentrations of N-acetylneuraminic acid in patients with coma and bacteremia (19). A deficiency of neuraminidase A led to decreased virulence in a model of pneumococcal pneumonia (17). The role of hyaluronidase has not been studied extensively. Strains causing meningitis showed a higher in vitro expression of hyaluronidase (16). Intranasal instillation of pneumococci with addition of hyaluronidase to the inoculum was followed by meningitis in a model of pneumococcal pneumonia (28). We used a mouse model based on intracerebral infection (14, 26) to assess the role of pneumolysin, neuraminidases A and B, and hyaluronidase in meningitis by using mutants of an S. pneumoniae type 2 strain deficient in these putative virulence factors.(This work was presented, in part, at the 40th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 17 to 20 September 2000 [abstr. 433]).Bacteria. Mutant strains of S. pneumoniae D39 were generated by insertion duplication mutagenesis as described in detail before (30). In brief, internal gene fragments amplified from chromosomal DNA were ligated with the insertion vector pJDC9 by standard DNA techniques (12). The insertion was performed at position 547 of the 1,416-bp ply gene, position 605 of the 3,108-bp nanA gene, position 735 of the 2,094-bp nanB gene, and position 534 of the 2,850-bp hyl gene. Erythromycin-resistant transformants were selected with 1 g of erythromycin/ml on Luria-Bertani ag...