De novo nucleotide biosynthesis and its dynamic regulation are crucial for systemic infection by extraintestinal Escherichia coli

Extraintestinal Escherichia coli (ExPEC) proliferates rapidly in the bloodstream, causing life-threatening systemic infections with high mortality rates. Understanding how ExPEC adapts to the blood environment is essential for elucidating its pathogenesis and identifying potential antimicrobial targets. Using transposon mutagenesis-based high-throughput screening, we systematically identified genes critical for ExPEC growth in serum. Our findings demonstrate that de novo nucleotide biosynthesis genes are indispensable for serum proliferation, and animal infection assays further confirm their vital role in ExPEC virulence. Genetic disruption of this pathway did not affect bacterial stress response or adhesion capacity but severely impaired ExPEC's intracellular survival in macrophages. Transcriptomic profiling combined with a luminescent reporter system revealed significant upregulation of de novo nucleotide biosynthesis genes during both serum incubation and systemic infection. This upregulation was mediated by the transcriptional repressor PurR, and could be inhibited by purine supplementation. Furthermore, enhanced in vivo fitness was observed for the purR deletion strain, whereas the opposite was seen for the purR overexpression strain, indicating a crucial role for PurR in ExPEC pathogenesis. Quantitative analyses showed that serum incubation significantly depletes intracellular purine pools in ExPEC. Electrophoretic mobility shift assays (EMSA) demonstrated that specific purines modulate PurR-DNA binding affinity. These results suggest that PurR acts as a sensor of intracellular purine concentration changes, regulating de novo nucleotide biosynthesis genes to facilitate host environment adaptation. This study reveals the essential role of de novo nucleotide biosynthesis in ExPEC virulence and describes a pathogenesis mechanism involving nucleotide metabolism regulation to overcome nutritional immunity, offering a foundation for developing therapies against systemic ExPEC infections.