Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P., 243122, India.
Division of Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P., 243122, India.
Mol Biol Rep. 2020 Mar;47(3):2231-2242. doi: 10.1007/s11033-020-05328-3. Epub 2020 Feb 19.
The human gut pathogen, Salmonella Typhimurium (S. Typhimurium) not only survives but also replicates inside the phagocytic cells. Bacterial proteins are the primary targets of phagocyte generated oxidants. Because of the different amino acid composition, some proteins are more prone to oxidation than others. Many oxidant induced modifications to amino acids have been described. Introduction of carbonyl group is one of such modifications, which takes place quite early following exposure of proteins to oxidants and is quite stable. Therefore, carbonyl groups can be exploited to identify oxidant susceptible proteins. Hypochlorous acid (HOCl) is one of the most potent oxidants produced by phagocytes. Incubation of S. Typhimurium with 3 mM HOCl resulted in more than 150 folds loss of bacterial viability. Proteins extracted from HOCl exposed S. Typhimurium cells showed about 60 folds (p < 0.001) more carbonyl levels as compared to unexposed cells. Similarly, 2, 4-Dinitrophenylhydrazine (2, 4-DNPH) derivatized proteins of HOCl treated S. Typhimurium cultures reacted strongly with anti-DNP antibodies as compared to buffer treated counterpart. Next, we have derivatized carbonyl groups on the proteins with biotin hydrazide. The derivatized proteins were then isolated by avidin affinity chromatography. Mass spectrometry based analysis revealed the presence of 204 proteins.
人类肠道病原体沙门氏菌（Salmonella Typhimurium，S. Typhimurium）不仅能在吞噬细胞内存活，还能在其内进行复制。细菌蛋白是吞噬细胞产生的氧化剂的主要靶标。由于氨基酸组成不同，一些蛋白比其他蛋白更容易氧化。已经描述了许多氧化剂诱导的氨基酸修饰。羰基的引入是其中一种修饰，它在蛋白质暴露于氧化剂后相当早地发生，并且非常稳定。因此，羰基基团可用于鉴定易受氧化剂影响的蛋白质。次氯酸（HOCl）是吞噬细胞产生的最有效的氧化剂之一。将 S. Typhimurium 与 3 mM HOCl 孵育会导致细菌活力丧失超过 150 倍。从暴露于 HOCl 的 S. Typhimurium 细胞中提取的蛋白质显示出比未暴露细胞高约 60 倍（p < 0.001）的羰基水平。同样，与缓冲液处理的对照相比，用 2,4-二硝基苯肼（2,4-DNPH）衍生化 HOCl 处理的 S. Typhimurium 培养物的蛋白质与抗-DNP 抗体强烈反应。接下来，我们用生物素酰肼将羰基基团衍生化到蛋白质上。然后通过亲和层析法分离衍生化的蛋白质。基于质谱的分析揭示了存在 204 种蛋白质。
