Essential role of proline synthesis and the one-carbon metabolism pathways for systemic virulence of <i>Streptococcus pneumoniae</i>

Virulence screens have indicated potential roles during <i>Streptococcus pneumoniae</i> infection for the one-carbon metabolism pathway component Fhs and proline synthesis mediated by ProABC. To define how these metabolic pathways affect <i>S. pneumoniae</i> virulence, we have investigated the phenotypes, transcription, and metabolic profiles of Δ<i>fhs</i> and Δ<i>proABC</i> mutants. <i>S. pneumoniae</i> capsular serotype 6B BHN418 Δ<i>fhs</i> and Δ<i>proABC</i> mutant strains had strongly reduced virulence in mouse sepsis and pneumonia models but could colonize the nasopharynx. Both mutant strains grew normally in complete media but had markedly impaired growth in chemically defined medium, human serum, and human cerebrospinal fluid. The BHN418 Δ<i>proABC</i> strain also had impaired growth under conditions of osmotic and oxidative stress. The virulence role of <i>proABC</i> was strain specific, as the D39 Δ<i>proABC</i> strain could still cause septicemia and grow in serum. Compared to culture in broth, in serum, the BHN418 Δ<i>fhs</i> and Δ<i>proABC</i> strains showed considerable derangement in global gene transcription that affected multiple but different metabolic pathways for each mutant strain. Metabolic data suggested that Δ<i>fhs</i> had an impaired stringent response, and when cultured in sera, BHN418 Δ<i>fhs</i> and Δ<i>proABC</i> were under increased oxidative stress and had altered lipid profiles. Loss of <i>proABC</i> also affected carbohydrate metabolism and the accumulation of peptidoglycan synthesis precursors in the BHN418 but not the D39 background, linking this phenotype to the conditional virulence phenotype. These data identify the <i>S. pneumoniae</i> metabolic functions affected by <i>S. pneumoniae</i> one-carbon metabolism and proline biosynthesis, and the role of these genetic loci for establishing systemic infection.IMPORTANCERapid adaptation to grow within the physiological conditions found in the host environment is an essential but poorly understood virulence requirement for systemic pathogens such as <i>Streptococcus pneumoniae</i>. We have now demonstrated an essential role for the one-carbon metabolism pathway and a conditional role depending on strain background for proline biosynthesis for <i>S. pneumoniae</i> growth in serum or cerebrospinal fluid, and therefore for systemic virulence. RNAseq and metabolomic data demonstrated that the loss of one-carbon metabolism or proline biosynthesis has profound but differing effects on <i>S. pneumoniae</i> metabolism in human serum, identifying the metabolic processes dependent on each pathway during systemic infection. These data provide a more detailed understanding of the adaptations required by systemic bacterial pathogens in order to cause infection and demonstrate that the requirement for some of these adaptations varies between strains from the same species and could therefore underpin strain variations in virulence potential.