Meningitis is the most serious of invasive
infections caused by the Gram-positive bacterium Streptococcus pneumoniae.
Vaccines protect only against a limited number of serotypes, and evolving bacterial resistance to antimicrobials impedes treatment. Further insight into the molecular pathogenesis of invasive
pneumococcal disease is required in order to enable the development of new or adjunctive treatments and/or
pneumococcal vaccines that are efficient across serotypes. We applied genomic array footprinting (GAF) in the search for S. pneumoniae genes that are essential during experimental
meningitis. A total of 6,000 independent TIGR4 marinerT7 transposon mutants distributed over four libraries were injected intracisternally into rabbits, and cerebrospinal fluid (CSF) was collected after 3, 9, and 15 h. Microarray analysis of mutant-specific probes from CSF samples and inocula identified 82 and 11 genes mutants of which had become attenuated or enriched, respectively, during
infection. The results point to essential roles for capsular
polysaccharides, nutrient uptake, and
amino acid biosynthesis in bacterial replication during experimental
meningitis. The GAF phenotype of a subset of identified targets was followed up by detailed studies of directed mutants in competitive and noncompetitive
infection models of experimental rat
meningitis. It appeared that
adenylosuccinate synthetase,
flavodoxin, and LivJ, the substrate
binding protein of a
branched-chain amino acid ABC transporter, are relevant as targets for future
therapy and prevention of
pneumococcal meningitis, since their mutants were attenuated in both models of
infection as well as in competitive growth in human cerebrospinal fluid in vitro.