Proteome Wide Profiling of <i>N</i>‑ε-Lysine Acetylation Reveals a Novel Mechanism of Regulation of the Chitinase Activity in Francisella novicida

Francisella tularensis is a Gram-negative bacterium that causes the zoonotic disease tularemia. The historical development of tularemia as a biological weapon has led to it being characterized by the CDC as a category A biothreat agent. Neither posttranslational modification (PTM) of proteins, in particular lysine acetylation, in Francisella nor its subsequent regulation of the protein activity has been well studied. In this work, we analyze N-ε-lysine acetylation of the F. tularensis ssp. novicida proteome by mass spectrometry for the first time. To create a comprehensive acetylation profile, we enriched protein acetylation using two approaches: (1) the addition of glucose or acetate into the culture medium and (2) direct chemical acetylation of N-ε-lysines with acetyl phosphate. We discovered 280 acetylated proteins with 1178 acetylation sites in the F. tularensis ssp. novicida strain U112. Lysine acetylation is an important PTM that regulates multiple cellular processes in bacteria, including metabolism, transcription, translation, stress response, and protein folding. We discovered that Francisella chitinases A and B are acetylated naturally and when chemically induced by acetyl phosphate. Moreover, chemical overacetylation of chitinases results in silencing of the enzymatic activity. Our findings suggest a novel mechanism of posttranslational regulation of the chitinase activity and that acetylation may play a role in Francisella’s regulation of the protein activity.土拉弗朗西斯菌（Francisella tularensis）是一种革兰氏阴性（Gram-negative）细菌，可引发人畜共患病土拉菌病（tularemia）。土拉菌病作为生物武器的历史应用，使其被美国疾病控制与预防中心（Centers for Disease Control and Prevention, CDC）列为A类生物威胁因子。目前针对土拉弗朗西斯菌体内蛋白质的翻译后修饰（posttranslational modification, PTM）——尤其是赖氨酸乙酰化——及其后续对蛋白质活性的调控机制的研究仍较为匮乏。 本研究首次利用质谱（mass spectrometry）技术分析了土拉弗朗西斯菌新凶手亚种（F. tularensis ssp. novicida）的蛋白质组（proteome）N-ε-赖氨酸乙酰化修饰情况。为构建全面的乙酰化修饰图谱，我们通过两种策略富集蛋白质乙酰化修饰：（1）向培养基中添加葡萄糖或乙酸盐；（2）利用乙酰磷酸（acetyl phosphate）对N-ε-赖氨酸进行直接化学乙酰化。在U112株土拉弗朗西斯菌新凶手亚种中，我们共鉴定到280个乙酰化蛋白质及1178个乙酰化修饰位点。 赖氨酸乙酰化是一类重要的翻译后修饰，可调控细菌体内包括代谢、转录、翻译、应激响应及蛋白质折叠在内的多种细胞进程。我们发现土拉弗朗西斯菌的几丁质酶（chitinase）A与B既可发生天然乙酰化，也可在乙酰磷酸的化学诱导下发生乙酰化修饰。此外，几丁质酶的过度化学乙酰化会导致其酶活性丧失。 本研究结果揭示了一种调控几丁质酶活性的新型翻译后修饰机制，并提示乙酰化修饰可能在土拉弗朗西斯菌的蛋白质活性调控中发挥重要作用。