SpuA-Mediated Glycogen Metabolism Modulates Acid Stress Adaptation via Formic Acid and Amino Acid Utilization in Streptococcus pneumoniae

Glycogen metabolism plays a key role in bacterial adaptation. In <i>Streptococcus pneumoniae</i>, the glycogen-degrading enzyme SpuA is widely conserved, but its physiological significance remains unclear. In this study, we investigated how SpuA affects bacterial growth and response to acid stress. We found that the <i>spuA</i> deletion strain (Δ<i>spuA</i>) produced more acidic metabolites under anaerobic conditions than the wild-type strain. In a mouse infection model, bronchoalveolar lavage fluid (BALF) from Δ<i>spuA</i>-infected mice was more acidic on day 1 post-infection, showing a lower bacterial load than wild-type infection-a finding consistent with the early growth delay observed in vitro-but the mutant later exhibited enhanced persistence at 72 h. Δ<i>spuA</i> strains also showed greater tolerance to formic acid and higher intake of serum amyloid A1 (SAA1), which may further contribute to their survival in acidic environments. Transcriptomic analysis revealed reduced utilization of certain amino acids, particularly cysteine, in Δ<i>spuA</i> strains. However, the addition of 0.05% (<i>v</i>/<i>v</i>) formic acid restored amino acid utilization in Δ<i>spuA</i> strains, and co-supplementation with formic acid and cysteine significantly enhanced Δ<i>spuA</i> growth in vitro. These findings suggest that in the absence of SpuA, <i>S. pneumoniae</i> shifts its metabolism toward formic acid production, which may act both as a metabolic signal and a stressor that influences bacterial gene expression. This shift is accompanied by increased expression of tRNAs and growth rescue, suggesting enhanced amino acid utilization capacity. Although our findings reveal a potential link between formic acid metabolism and amino acid utilization through tRNA regulation, further validation using metabolic flux analyses or targeted metabolomics will be required to confirm this relationship. These observations imply a metabolic adaptation that facilitates bacterial growth under low-oxygen, acidic conditions during infection. Our results also raise the possibility that SpuA plays a role in restraining bacterial overgrowth in the host, thereby promoting a more balanced coexistence between pathogen and host.