Deletion of genes involved in the ketogluconate metabolism, Entner-Doudoroff pathway, and glucose dehydrogenase increase local and invasive virulence phenotypes in Streptococcus pneumoniae

Streptococcus pneumoniae displays increased resistance to antibiotic therapy following bio-film formation. A genome-wide search revealed that SP 0320 and SP 0675 (respectively annotated as 5-keto-D-gluconate-5-reductase and glucose dehydrogenase) contain the highest degree of homology to CsgA of Myxococcus xanthus, a signaling factor that promotes cell aggregation and biofilm formation. Single and double SP 0320 and SP 0675 knockout mutants were created in strain BS72; however, no differences were observed in the biofilm-forming phenotypes of mutants compared to the wild type strain. Using the chin-chilla model of otitis media and invasive disease, all three mutants exhibited greatly increased virulence compared to the wild type strain (increased pus formation, tympanic membrane rupture, mortality rates). The SP 0320 gene is located in an operon with SP 0317, SP 0318 and SP 0319, which we bioinformatically annotated as being part of the Entner-Doudoroff pathway. Deletion of SP 0317 also resulted in increased mortality in chin-chillas; however, mutations in SP 0318 and SP 0319 did not alter the virulence of bacteria compared to the wild type strain. Complementing the SP 0317, SP 0320 and SP 0675 mutant strains reversed the virulence phenotype. We prepared recombinant SP 0317, SP 0318, SP 0320 and SP 0675 proteins and confirmed their functions. These data reveal that disruption of genes involved in the degradation of ketogluconate, the Entner-Doudoroff path-way, and glucose dehydrogenase significantly increase the virulence of bacteria in vivo; two hypothetical models involving virulence triggered by reduced in carbon-flux through the glycolytic pathways are presented.