Investigations of Bacterial Biofilm Proteomes Using Artifical Amino Acids

描述（由申请人提供）：细菌生物膜直接涉及临床感染的抗菌耐药性的发展。铜绿假单胞菌的生物膜占所有医院感染的10%，是囊性纤维化患者慢性感染的主要原因。近年来，Ga3+盐已成为抗铜绿假单胞菌生物膜的有效抗生素，但其作用机制尚不清楚。铜绿假单胞菌对抗生素膜反应的蛋白质组的鉴定将对抗生素耐药机制的研究提供重要的见解；然而，鉴定新合成蛋白质的技术有限。生物正交非规范氨基酸标记技术（BONCAT）已成功地用于新合成蛋白质的高分辨率鉴定。通过代谢标记带有化学报告基因的氨基酸，只有含有新氨基酸的蛋白质才能在翻译后亲和力标记中被识别出来。虽然强大，人工氨基酸的成功结合高度依赖于细胞翻译机制。为了解决这个问题，我们建议使用l -硒代蛋氨酸（SeMet）作为蛋白质标记的替代氨基酸，因为它通过内源性翻译机制几乎完全结合到蛋白质中，并且对蛋白质功能的影响很小。翻译后氧化和消除SeMet残基形成烯烃可以与巯基化学相结合来标记新合成的蛋白质，从而消除了对化学报告的需要。该项目的长期目标是开发一种SeMet-BONCAT方法来识别低丰度蛋白质，这对于理解生物膜中随时间变化的蛋白质组变化至关重要。为了实现这一目标，我们提出了以下目标：(1)开发一种方法来促进水介质中氧化SeMet残基生成烯烃；(2)在铜绿假单胞菌Met-营养不良菌中用SeMet代谢标记蛋白质，并使用巯基化学选择性标记新合成的蛋白质；(3)在Ga3+盐暴露处理下，从铜绿假单胞菌生物膜中鉴定新的蛋白质。实验将包括使用Lewis酸表面活性剂组合催化剂（LASCs）促进SeMet残基生成烯烃，以及使用串联二维色谱和质谱法进行蛋白质鉴定。我们预计这种新的蛋白质标记和标记策略将为生物膜和其他临床相关生物体的生物学过程和分子机制提供重要的见解和理解。

DESCRIPTION (provided by applicant): Bacterial biofilms have been directly implicated in the development of antibacterial resistance of clinical infections. Biofilms of Pseudomonas aeruginosa account for 10% of all nosocomial infections and are the leading cause of chronic infections in Cystic Fibrosis patients. Ga3+ salts have recently emerged as effective antibiotic agents against P. aeruginosa biofilms, however little is known about their mechanism of action. The identification of the P. aeruginosa proteome in response to antibiofilm agents would give significant insight into the mechanism of antibiotic resistance; however, there are limited technologies to identify newly synthesized proteins. Bioorthogonal non-canonical amino acid tagging (BONCAT) has been successful in identifying newly synthesized proteins with high resolution. By metabolically labeling proteins with amino acids bearing a chemical reporter, only proteins with the new amino acid will be identified in post-translational affinity tagging. While powerful, successful incorporation of an artificial amino acid is highly dependent on the cellular translational machinery. To address this issue we propose using L-selenomethionine (SeMet) as a surrogate amino acid for protein labeling due to its near complete incorporation into proteins using endogynous translational machinery and minimal effect on protein function. Post-translational oxidation and elimination of SeMet residues to form alkenes can be coupled with thiol-ene chemistry to tag newly synthesized proteins, eliminating the need for a chemical reporter. This project's long-term objective is to develop a SeMet-BONCAT method to identify low-abundant proteins essential to understand time-dependent proteome changes in biofilms. To achieve this goal, we propose the following aims: (1) develop a methodology to promote alkene formation from oxidized SeMet residues in aqueous media, (2) metabolically label proteins with SeMet and selectively tag newly synthesized proteins using thiol-ene chemistry in Pseudomonas aeruginosa Met- auxotrophs, and (3) identify new proteins from Pseudomonas aeruginosa biofilms in response to treatment with Ga3+ salts exposure. Experiments will involve using Lewis acid surfactant-combined catalysts (LASCs) to promote alkene formation from SeMet residues and tandem 2D chromography and mass spectrometry for protein identification. We anticipate that this new protein labeling and tagging strategy will provide significant insight and understanding into the biological processes and molecular mechanisms of biofilms and other organisms of clinical relevance. PUBLIC HEALTH RELEVANCE: The relevance of the proposed research to public health is centered upon understanding the molecular mechanisms of antibiotic resistance in bacterial biofilms. By understanding these mechanisms, effective treatments can be developed to combat antibiotic resistant infections in clinical settings.