The growth and pathogenesis of Citrobacter rodentium is compromised by disrupted mucin sugar pathways that accumulate N-acetylglucosamine 6-phosphate

Abstract

Many enteric bacterial pathogens, including the attaching/effacing (A/E) Escherichia coli 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 the monosaccharides N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NeuNAc) to fuel their pathogenesis. Using Citrobacter rodentium, a murine-specific A/E pathogen, we disrupted both GlcNAc and NeuNAc catabolism by deleting nagA, which encodes the GlcNAc-6-phosphate (GlcNAc-6P) deacetylase that converts GlcNAc-6P into glucosamine-6-phosphate (GlcN-6P). The {Delta}nagA mutant displayed dramatically impaired colonization in C57BL/6J mice and accumulated significant levels of GlcNAc-6P, unlike the {Delta}mana strain, a mutant lacking all GlcNAc and NeuNAc transporters, suggesting that the attenuation was due to sugar-phosphate stress rather than nutrient deprivation alone. Supplementation with glucosamine (GlcN) restored growth, indicating that dysregulated GlcN-6P synthesis, rather than GlcNAc-6P toxicity, underlies the defect. Furthermore, {Delta}nagA exhibited increased susceptibility to several cell wall-dependent stress conditions, in concert with compromised peptidoglycan biosynthesis due to reduced UDP-GlcNAc synthesis. These findings reveal a previously unrecognized metabolic vulnerability in C. rodentium and suggest that targeting sugar-phosphate stress responses may provide a new therapeutic strategy against GI bacterial pathogens. ImportanceEnteric pathogens like Citrobacter rodentium can exploit sugars, including N-acetylglucosamine and N-acetylneuraminic acid, derived from intestinal mucus to grow and infect their hosts. This study shows that disruption of mucin-derived sugar catabolism impairs the fitness of C. rodentium in infecting the murine intestine by causing the accumulation of a toxic intermediate of mucin sugar metabolism. Rather than impaired nutrient acquisition, the bacteria are impaired due to the buildup of N-acetylglucosamine-6-phosphate, which depletes substrates for peptidoglycan synthesis. This metabolic bottleneck weakens the bacterial cell wall, making the pathogen more sensitive to environmental stress. These findings identify a conserved metabolic stress response that could be targeted to combat enteric pathogen infections.