Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pflA genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.Streptococcus pneumoniae is the leading cause of pneumonia in children and adults, and it is a major cause of otitis media, meningitis, and septicemia (22). Despite considerable progress in pneumococcal vaccine development, the serotype specificity and the genomic plasticity of the pneumococcus will hamper its success (36). In addition, the increasing frequency of antibiotic resistance (14) makes it important to understand the mechanisms of pathogenesis of pneumococcal disease.Probably one of the most understudied fields of pneumococcal biology is that of how the pathogen generates its metabolic energy. The pneumococcus is strictly fermentative, and sugars are the major sources of energy for biosynthesis and growth (19,52). Therefore, in vivo fitness is determined to a large extent by the processes governed by sugar metabolism. Accumulating evidence suggests that there is a direct link between pneumococcal virulence and sugar metabolism (6,16,20,21,50). This is not limited to proteins involved in polysaccharide degradation, transport, and regulation (16, 31) but also includes those involved in redox metabolism, such as NADH oxidase (Nox) (6), and in pyruvate metabolism (42,50). For example, mutation of nox caused diminished virulence in a systemic infec...