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

## TL;DR

The study shows that ExPEC bacteria rely on making their own nucleotides to survive in the bloodstream and cause severe infections, with a key regulator called PurR sensing and controlling this process.

## Contribution

The study identifies de novo nucleotide biosynthesis and PurR regulation as critical for ExPEC virulence and systemic infection.

## Key findings

- De novo nucleotide biosynthesis genes are essential for ExPEC growth in serum and systemic infection.
- PurR acts as a sensor of intracellular purine levels, regulating nucleotide biosynthesis gene expression.
- Purine supplementation inhibits PurR activity and reduces ExPEC virulence.

## Abstract

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.

Extraintestinal pathogenic Escherichia coli (ExPEC) exhibits a remarkable ability to proliferate in the bloodstream, enabling rapid dissemination throughout the host. Identifying genetic determinants that support ExPEC growth in blood is crucial for understanding its pathogenic mechanisms and discovering potential therapeutic targets. In this study, we identified the critical role of de novo nucleotide biosynthesis genes in serum growth and systemic infection by ExPEC. Moreover, we demonstrated that the de novo nucleotide biosynthesis pathway is significantly upregulated during incubation in serum or during systemic infection. PurR was identified as a key repressor of this pathway and contributed to ExPEC pathogenesis. We further provide biochemical evidence showing that purine levels modulate the DNA-binding affinity of PurR. This mechanism allows PurR to function as a molecular sensor of nucleotide availability, tuning de novo nucleotide biosynthesis gene expression to overcome nutritional constraints within the host. These findings provide valuable insights into ExPEC pathogenesis and highlight promising therapeutic targets.