Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, 08544, USA.
Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, 08544, USA.
Free Radic Biol Med. 2021 Nov 20;176:286-297. doi: 10.1016/j.freeradbiomed.2021.10.005. Epub 2021 Oct 5.
Reactive nitrogen species and nutrient deprivation are two elements of the immune response used to eliminate pathogens within phagosomes. Concomitantly, pathogenic bacteria have evolved defense systems to cope with phagosomal stressors, which include enzymes that detoxify nitric oxide (NO) and respond to nutrient scarcity. A deeper understanding of how those defense systems are deployed under adverse conditions that contain key elements of phagosomes will facilitate targeting of those systems for therapeutic purposes. Here we investigated how Escherichia coli detoxifies NO in the absence of useable nitrogen, because nitrogen availability is limited in phagosomes due to the removal of nitrogenous compounds (e.g., amino acids). We hypothesized that nitrogen starvation would impair NO detoxification by E. coli because it depresses translation rates and the main E. coli defense enzyme, Hmp, is synthesized in response to NO. However, we found that E. coli detoxifies NO at the same rate regardless of whether useable nitrogen was present. We confirmed that the nitrogen in NO and its autoxidation products could not be used by E. coli under our experimental conditions, and discovered that NO eliminated differences in carbon and oxygen consumption between nitrogen-replete and nitrogen-starved cultures. Interestingly, E. coli does not consume measurable extracellular nitrogen during NO stress despite the need to translate defense enzymes. Further, we found that RelA, which responds to uncharged tRNA, was required to observe the robustness of NO detoxification to nitrogen starvation. These data demonstrate that E. coli is well poised to detoxify NO in the absence of useable nitrogen and suggest that the stringent response could be a useful target to potentiate the antibacterial activity of NO.
活性氮物种和营养剥夺是免疫反应的两个要素，用于在吞噬体中消除病原体。同时，病原细菌已经进化出防御系统来应对吞噬体应激源，其中包括解毒一氧化氮 (NO) 和应对营养缺乏的酶。更深入地了解这些防御系统在包含吞噬体关键要素的不利条件下是如何部署的，将有助于针对这些系统进行治疗。在这里，我们研究了大肠杆菌在缺乏可用氮的情况下如何解毒 NO，因为由于氮化合物（例如氨基酸）的去除，吞噬体中的氮可用性受到限制。我们假设氮饥饿会削弱大肠杆菌的 NO 解毒能力，因为它会降低翻译速率，而主要的大肠杆菌防御酶 Hmp 是响应 NO 合成的。然而，我们发现无论是否存在可用氮，大肠杆菌解毒 NO 的速度都相同。我们证实，在我们的实验条件下，NO 及其自动氧化产物中的氮不能被大肠杆菌利用，并且发现 NO 消除了氮充足和氮饥饿培养物之间在碳和氧消耗方面的差异。有趣的是，尽管需要翻译防御酶，但大肠杆菌在 NO 应激期间不会消耗可测量的细胞外氮。此外，我们发现 RelA（响应未带电 tRNA 的反应）是观察到氮饥饿对 NO 解毒稳健性所必需的。这些数据表明，大肠杆菌在缺乏可用氮的情况下能够很好地解毒 NO，并表明严格反应可能是增强 NO 抗菌活性的有用目标。
