S. pneumoniae continues to be responsible for high global morbidity and mortality from pneumonia, bacteremia, and meningitis.
While major efforts have recently been directed toward the development of cheap and effective vaccines against pneumococcal pneumonia and bacteremia, similar measures toward the prevention of pneumococcal meningitis are lacking. This could in part be because protection against meningitis is assumed for vaccines that prevent bacteremia. Nevertheless, pneumococcus is now the commonest cause of bacterial meningitis in children, the elderly, and adults with specific underlying medical conditions and accounts for the most severe form of bacterial meningitis in terms of the degree of morbidity and mortality (33
). Indeed, in spite of the widespread use of the 7-valent pneumococcal conjugate vaccine (Prevnar), S. pneumoniae
is still the commonest cause of bacterial meningitis in children in the USA, mostly due to nonvaccine serotypes (3
). Therefore, the development of effective, multivalent vaccines providing non–serotype-dependent protection represents the best prospect for managing pneumococcal diseases, including meningitis, in the 21st century. However, the critical determinants that enable certain strains of pneumococci to cross the BBB to cause meningitis are still largely unknown. We hypothesized that progression from the blood to the brain will require niche-specific alterations in virulence gene expression and that surface-exposed virulence factors that are upregulated during this transition could be protective immunogens against meningitis.
Until recently, progress on the systematic examination of pneumococcal gene expression patterns in distinct host niches was hampered by technical difficulties associated with isolating sufficient quantities of pure and intact bacterial RNA from infected tissues to perform accurate, quantitative mRNA analyses. These difficulties might explain why previous studies had used suboptimal in vivo models or in vitro surrogates to estimate pneumococcal gene expression patterns in distinct host niches. However, in this study, the technical difficulties of in vivo–derived RNA extraction, enrichment, and linear amplification were overcome and further refined, permitting the first microarray comparison and analysis of transcription kinetics between distinct host niches of the same animal, including sites such as the nasopharynx, where pneumococci exist in very low numbers. This provides a superior model system for analysis of pneumococcal gene expression during pathogenesis of invasive disease.
The present study focused on identifying S. pneumoniae proteins that are important for the development of meningitis. First, we compared in vivo–derived transcriptomes of pneumococci simultaneously harvested from the blood and brain of infected mice. Second, the role of key genes was investigated at the molecular level, using targeted mutagenesis, mouse models of disease, and functional assays. Using this approach, several genes encoding proteins that could potentially contribute to meningitis were identified, foremost among which was formate-tetrahydrofolate ligase (Fhs). Bioinformatic analysis showed that Fhs is a metabolic enzyme that is not predicted to be surface exposed or secreted and hence is a suboptimal vaccine target. Nevertheless, the mechanism or mechanisms whereby this protein contributes to virulence is the subject of a separate investigation.
Another gene of interest identified in our screen was glpO
, encoding GlpO, which catalyzes the oxidation of α-glycerophosphate (an intermediate in glycerol metabolism), to dihydroxyacetone phosphate, with the concomitant reduction of oxygen to H2
). Since the pneumococcus lacks catalase, it produces excessive amounts of H2
as a by-product of aerobic metabolism, with deleterious effects on both the host and competing nasopharyngeal microflora (35
). In M. mycoides
SC, GlpO is involved in production and translocation of toxic H2
into host cells, causing inflammation and cell death (32
). Our in vitro studies using a ΔglpO
mutant as well as purified protein have shown that pneumococcal GlpO has a potent cytotoxic effect on HBMECs and that this is a consequence of H2
generation from glycerol metabolism. The ΔglpO
mutant also exhibited markedly reduced adherence to HBMECs. Previous studies have shown that generation of H2
by, or in response to, pneumococci has major deleterious effects on the brain, triggering apoptosis in microglia and neurons (37
), and may also mediate peroxidation of brain lipids (39
). However, GlpO is not the only pneumococcal enzyme capable of generating H2
. The others are SpxB, which has been implicated in pneumococcal meningitis (albeit using a rat intrathecal challenge model that bypasses the BBB) (40
), and the NADH oxidase (Nox), shown to be important in respiratory tract and otitis media infection models (41
). It is unlikely that SpxB plays the major role in H2
-mediated damage in pneumococcal meningitis. The spxB
gene is not upregulated in the brain, as its substrate is pyruvate produced from glucose metabolism. Our in vitro studies showed that a ΔspxB
mutant produced significant amounts of H2
in the presence of glycerol, but not glucose, whereas the ΔglpO
mutant produced high H2
in the presence of glucose, but not glycerol. Glucose is more abundant in blood than in the brain, while glycerol is more abundant in the brain than in the blood (42
). Thus, although SpxB may play a significant role in colonization and sepsis, as has been suggested by others (10
), GlpO is expected to be the major pneumococcal generator of H2
in the brain.
Pneumococcal GlpO clearly plays a significant role in the pathogenesis of pneumococcal meningitis. In an i.n. challenge model that mimics the natural route and course of infection in humans, numbers of the ΔglpO
mutant in the brain compartment were significantly lower than the WT in both separate challenge and competition experiments, whereas numbers in the blood were not significantly different. Moreover, passive immunization with anti-GlpO reduced the numbers of WT WCH43 entering the brain, but did not affect the level of bacteremia. There was also markedly lower meningeal inflammation and histological damage in the brains of ΔglpO
-challenged mice, compared with those challenged with WT WCH43. The increased pathology in the latter would be expected to increase BBB permeability, and this is consistent with the apparent increase in extravasation of EB dye in mice infected with WCH43 versus ΔglpO
. However, even though the ΔglpO
mutant was less virulent than the isogenic WT strain, there was still residual tissue damage, suggesting that other factors also play an important role. A strong candidate is Ply, which has been shown to be cytotoxic to a wide range of eukaryotic cells and also elicits host inflammatory responses capable of generating ROS production (46
To our knowledge, this study provides the first direct evidence that GlpO plays a significant role in pneumococcal meningitis. The underlying mechanism most likely involves in situ generation of H2O2 from glycerol metabolism as well as promotion of adherence to the cerebrovascular endothelium, collectively promoting penetration of the BBB and generation of meningeal inflammation. GlpO was also an effective vaccine antigen in its own right and provided additive protection when coadministered with Ply toxoid. We showed that anti-GlpO serum significantly neutralizes H2O2 production and the associated cytotoxic activity of GlpO. We also used flow cytometry to show that incubation of WT WCH43 with anti-GlpO promotes deposition of C3 on the bacterial surface. Thus, the mechanism underlying the protection elicited by immunization with GlpO is likely to include direct antibody-mediated neutralization of GlpO activity in vivo (thereby attenuating penetration of the BBB) as well as promotion of opsonophagocytic clearance.
This study also identified other proteins that could potentially contribute substantially to the disease process, and their roles are currently under investigation. Nevertheless, the demonstration by active and passive immunization-challenge experiments that GlpO is protective against invasive pneumococcal disease and contributes to the development of meningitis strongly suggest that it might be a suitable candidate for future neuroimmunoprotective strategies against pneumococcal meningitis. Moreover, the in vivo transcriptomic strategy described in this paper can be adapted to the discovery of virulence genes that contribute to the development of meningitis caused by other pathogens such as Haemophilus influenzae type b and Neisseria meningitidis.