# Catabolism of serine enantiomers represses enterohemorrhagic Escherichia coli virulence factors via modulation of the nitrogen stress response

## TL;DR

This study reveals how the breakdown of serine enantiomers in EHEC represses virulence factors by modulating the nitrogen stress response.

## Contribution

The study identifies that ammonia from serine catabolism, not intact serine, mediates T3SS repression in EHEC.

## Key findings

- Both D- and L-serine repress the T3SS by modulating the nitrogen stress response.
- Ammonia release from serine breakdown is essential for T3SS repression in EHEC.
- EHEC uses oxidative deamination to produce regulatory signals from D-serine.

## Abstract

To optimize within-host fitness, bacterial pathogens exploit intricate sensory mechanisms to precisely modulate gene expression in response to host-associated cues. D-serine, a host-produced metabolite enriched in urine inhibits the type 3 secretion system (T3SS) of enterohemorrhagic Escherichia coli (EHEC). However, the molecular mechanism linking D-serine exposure to virulence repression remains incomplete. Here, we show that multiple amino acids, including L-serine, converge on this same regulatory pathway and repress the T3SS. A combination of transcriptomics, metabolomics, and targeted deletions reveal that this regulation is mediated by the release of ammonia, the nitrogenous product of serine breakdown, rather than by sensing of intact serine. Our data suggest that distal intestinal colonization by EHEC is facilitated by adaptation to amino acid-depleted environments.

Attaching and effacing pathogens, including enterohemorrhagic Escherichia coli (EHEC), colonize their preferred intestinal niche by sensing diverse host-, diet-, and microbiota-derived signals and coordinating the expression of virulence factors. D-serine, a host metabolite abundant in urine but scarce in the intestine, restricts EHEC colonization by transcriptionally repressing the type 3 secretion system (T3SS) while activating the SOS stress response. However, the mechanism underlying virulence regulation by D-serine remains unestablished. Here, we show that multiple amino acids, including L-serine converge on this pathway, repressing the T3SS without inducing the SOS response. Transcriptomic analyses showed a common response to D- and L-serine dominated by repression of nitrogen stress response genes. Mutational analysis identified the response regulators NtrC and Nac as essential mediators of T3SS repression by both serine enantiomers. Disruption of L-serine deaminase enzymes crucially revealed that T3SS repression depends on cytoplasmic ammonia/ammonium release rather than sensing of intact serine. While EHEC lacks canonical D-serine catabolic capacity, through metabolomics we provide evidence of oxidative deamination activity, capable of producing this regulatory signal. Together, these findings establish a mechanistic link between amino acid catabolism, nitrogen stress signaling, and virulence regulation in EHEC, highlighting how metabolic flux fine-tunes pathogen adaptation to intestinal niches.