The growth and pathogenesis of <i>Citrobacter rodentium</i> are compromised when multiple mucin sugar utilization pathways are disrupted, leading to accumulation of N-acetylglucosamine 6-phosphate

Many enteric bacterial pathogens, including attaching/effacing (A/E) <i>Escherichia coli</i> strains, cause acute gastroenteritis in humans. Considering the highly competitive nature of the mammalian gastrointestinal (GI) tract, these pathogens must rely on specific metabolic adaptations to establish successful infections. We hypothesized that A/E pathogens exploit host-derived nutrients within GI mucus, including N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NeuNAc), to fuel their pathogenesis. To address this hypothesis, we disrupted both GlcNAc and NeuNAc metabolic pathways in <i>Citrobacter rodentium,</i> a murine-specific A/E pathogen, by deleting <i>nagA</i>, which encodes the GlcNAc-6-phosphate (GlcNAc-6P) deacetylase that converts GlcNAc-6P into glucosamine-6-phosphate (GlcN-6P). Compared to wild-type (WT) <i>C. rodentium</i>, the Δ<i>nagA</i> mutant showed severely attenuated colonization and pathogenesis in C57BL/6J mice. Although Δ<i>nagA</i> cannot catabolize GlcNAc and NeuNAc, its <i>in vivo</i> defect could not be explained by nutrient deprivation alone. Instead, Δ<i>nagA</i> exhibited higher levels of cytosolic GlcNAc-6P, slower growth rate when cultured <i>in vitro</i>, altered regulation of GlcN-6P synthesis, and increased susceptibility to cell wall-targeting stressors. Supplementation with glucosamine (GlcN, which can be directly converted into GlcN-6P) partially restored the growth and resistance to cell wall stress of Δ<i>nagA</i> without reducing GlcNAc-6P accumulation, indicating that dysregulated GlcN-6P synthesis rather than GlcNAc-6P toxicity underlies its phenotype. Together, these data reveal a previously unrecognized metabolic vulnerability in <i>C. rodentium</i> where the disruption of the GlcNAc and NeuNAc metabolic pathways, by inactivating NagA, creates a sugar-phosphate imbalance that compromises cell wall integrity and pathogen fitness. Hence, targeting sugar-phosphate stress responses may provide a new therapeutic strategy against GI bacterial pathogens.