Investigations of Bacterial Biofilm Proteomes Using Artifical Amino Acids

描述(由申请人提供)：细菌生物被膜直接与临床感染的抗菌素耐药性的发展有关。铜绿假单胞菌的生物被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菌剂，但对其作用机制知之甚少。鉴定铜绿假单胞菌蛋白质组对抗生素耐药的反应将有助于深入了解抗生素耐药的机制；然而，识别新合成的蛋白质的技术有限。生物正交非典型氨基酸标签(BONCAT)已成功地用于高分辨率识别新合成的蛋白质。通过用带有化学报告的氨基酸代谢性地标记蛋白质，只有含有新氨基酸的蛋白质才能在翻译后亲和标记中被识别出来。虽然有效、成功地掺入一种人造氨基酸，但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-硒蛋氨酸(SeMet)作为蛋白质标记的替代氨基酸，因为它几乎完全整合到蛋白质中，使用内源性翻译机制，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硫醇-烯化学相结合来标记新合成的蛋白质，而不需要化学报告人员。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，这是理解生物膜中依赖时间的蛋白质组变化所必需的。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的Semet残基形成烯烃；(2)在铜绿假单胞菌Met营养缺陷型细胞中用Semet代谢标记蛋白质并利用硫醇-烯化学选择性地标记新合成的蛋白质；(3)从暴露于GA3+盐处理的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，以及串联2D色谱和质谱学用于蛋白质鉴定。我们预计，这种新的蛋白质标记和标记策略将对生物膜和其他具有临床意义的生物的生物学过程和分子机制提供重要的洞察和理解。 公共卫生相关性：拟议的研究与公共健康的相关性集中在了解细菌生物膜中抗生素耐药性的分子机制上。通过了解这些机制，可以开发有效的治疗方法来对抗临床环境中的抗生素耐药感染。

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.