Investigations of Bacterial Biofilm Proteomes Using Artifical Amino Acids

描述（由申请人提供）：细菌生物膜直接参与临床感染的抗菌药物耐药性的发展。铜绿假单胞菌的生物膜占所有医院感染的10%，并且是囊性纤维化患者慢性感染的主要原因。Ga 3+盐最近已成为对抗铜绿假单胞菌生物膜的有效抗生素剂，然而对其作用机制知之甚少。鉴定铜绿假单胞菌蛋白质组对抗菌膜试剂的响应将对抗生素耐药性的机制提供重要的见解;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签（BONCAT）已成功地鉴定新合成的蛋白质与高分辨率。通过用带有化学报告物的氨基酸代谢标记蛋白质，只有具有新氨基酸的蛋白质将在翻译后亲和标记中被鉴定。虽然强大，但人工氨基酸的成功掺入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，由于其接近完全纳入蛋白质使用内源性翻译机制和对蛋白质功能的影响最小。SeMet残基的翻译后氧化和消除以形成烯烃可以与硫醇-烯化学偶联以标记新合成的蛋白质，从而消除对化学报告物的需要。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，这对于了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在水性介质中从氧化的SeMet残基形成烯烃，（2）在铜绿假单胞菌Met营养缺陷型中用SeMet代谢标记蛋白质并使用硫醇-烯化学选择性地标记新合成的蛋白质，以及（3）鉴定来自铜绿假单胞菌生物膜的响应于用Ga 3+盐暴露处理的新蛋白质。实验将涉及使用刘易斯酸表面活性剂结合的催化剂（LASC），以促进烯烃形成的SeMet残基和串联的2D色谱和质谱蛋白质鉴定。我们预计，这种新的蛋白质标记和标签策略将提供重要的洞察力和理解的生物膜和其他生物体的临床相关的生物过程和分子机制。 公共卫生相关性：拟议的研究与公共卫生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性感染。

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.