(710b) Inhibition of Escherichia coli Nitric Oxide Defenses By Amino Acids

Within the phagosomes of immune cells bacteria are presented with a battery of antimicrobial stresses, such as acidic pH, superoxide, nitric oxide (NO), and nutrient deprivation (e.g., carbon sources and amino acids). Bacteria, which include uropathogenic Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Pseudomonas aeruginosa, have evolved defenses against phagosomal stresses that allow them to survive and cause illness. These defenses constitute virulence factors that could serve as targets for novel anti-infectives. A deeper understanding of these defense systems and their interactions under physiological conditions could illuminate avenues for therapeutic intervention. Because nutritional and nitrosative stresses imposed by phagosomes overlap, we investigated the effect of amino acid (AA) availability on E. coli NO defenses. NO can inactivate enzymes by binding to heme and iron-sulfur clusters, and it has been shown to disrupt the activity of several AA synthesis enzymes. Since the main NO defenses are induced by stress and need AAs for translation of the detoxification enzymes, our initial hypothesis was that bacteria not supplemented with AAs would be less equipped to deal with NO. Surprisingly, we found that the presence of AAs greatly impairs the ability of E. coli to detoxify NO, and that the impairment was due to physiological differences before and during stress rather than chemical differences in the environment during NO exposure. Further experiments and analyses revealed that AA suppression of NO defenses originated from impaired synthesis of Hmp, the dominant enzyme for NO detoxification in aerobic and microaerobic conditions. Additional experiments showed that the stringent response, which is governed by the metabolite regulator (p)ppGpp, is crucial for the regulation of E. coli NO defenses. These data establish that E. coli depends on stringent control to respond to two concurrent stresses imposed by phagosomes, NO and amino acid deprivation, and suggest that targeting the stringent response could be an effective means to impair bacterial NO detoxification.