Expanding the genetic code in Salmonella

 描述(申请人提供)：沙门氏菌感染是一种常见的细菌性疾病，影响肠道。据估计，它每年在美国造成约120万人患病，约23,000人住院，450人死亡。由于多重耐药沙门氏菌菌株的出现，对其生理和发病机制的深入了解对于确定新的靶点以开发新的抗菌剂具有重要意义。目前，遗传密码扩展策略已被广泛应用于将各种非天然氨基酸(UAA)整合到靶蛋白中，以解决由于20种天然氨基酸的化学多样性有限而难以或不可能用大多数经典方法解决的问题。然而，很少有研究报道在沙门氏菌等人类病原体中加入尿酸。在这里，我们将通过引入一种正交翻译系统(OTS)来扩展沙门氏菌的遗传密码，该系统可以有效地将各种UAA整合到目标蛋白质中，形成标记和探针，用于共聚焦显微镜、顺磁共振光谱(EPR)、傅立叶变换红外光谱(FTIR)或荧光共振能量转移(FRET)(目标1)。为了概念验证，我们将使用这个OTS来研究沙门氏菌中细菌微室(BMC)的空间组织，这是以前从未研究过的(目标2)。骨髓基质细胞是一种大的蛋白质复合体，含有病毒衣壳和包埋酶，参与多种代谢途径。研究表明，发生在骨髓细胞内的1，2-丙二醇和乙醇胺的降解与沙门氏菌的发病有关。我们将在沙门氏菌中将对叠氮苯丙氨酸引入骨髓细胞的外壳蛋白中，然后进行无铜点击反应，形成用于动态成像的小尺寸荧光探针，以克服常用荧光蛋白标签对骨髓细胞组装和功能的潜在干扰。将确定骨髓基质细胞的空间分布，细胞分裂过程中骨髓基质细胞的分离，骨髓基质细胞负责空间组织的关键蛋白，以及骨髓基质细胞蛋白与细胞骨架组件之间的相互作用。这些研究将提供新的信息，并可能揭示我们对骨髓基质细胞的知识的新范式。综上所述，我们将展示遗传密码扩展策略在细菌研究中的实际应用。由于沙门氏菌是一种重要的模式病原体，我们在这项建议中开发的OTS可以被许多实验室广泛用于促进微生物发病机制的研究。

 DESCRIPTION (provided by applicant): Salmonella infection is a common bacterial disease that affects the intestinal tract. It is estimated to cause about 1.2 million illnesses in the Unitd States each year, with about 23,000 hospitalizations and 450 deaths. A deep knowledge of its physiology and pathogenesis is important for identifying novel targets to develop new antimicrobial agents due to the emergence of multidrug-resistant Salmonella strains. Currently, the genetic code expansion strategy has been widely used to incorporate various unnatural amino acids (UAAs) into target proteins to solve the problems which are difficult or impossible to address by most classical methods due to the limited chemical diversity of the 20 natural amino acids. However, very few studies have been reported to incorporate UAAs in human pathogens such as Salmonella. Here, we will expand the genetic code in Salmonella by introducing an orthogonal translation system (OTS) which can efficiently incorporate various UAAs into target proteins to form labels and probes for confocal microscopy, paramagnetic resonance spectroscopy (EPR), Fourier transform infrared spectroscopy (FTIR), or fluorescence resonance energy transfer (FRET) (Aim 1). For proof-of-concept, we will use this OTS to investigate the spatial organization of bacterial microcompartments (BMCs) in Salmonella which has not been studied before (Aim 2). BMCs are large protein complexes containing viral capsid-like shells and encapsulated enzymes involved in various metabolic pathways. Studies have implicated 1,2-propanediol and ethanolamine degradations, which occur within BMCs, in Salmonella pathogenesis. We will incorporate p-azido-phenylalanine into the shell proteins of BMCs in Salmonella, followed by a Cu-free click reaction to form small-sized fluorescent probes for dynamic imaging to overcome the potential interference with the assemblies and functions of BMCs from the commonly used fluorescent protein tags. The spatial distribution of BMCs, the segregation of BMCs during cell divisions, key proteins of BMCs responsible for the spatial organization, and the interactions between BMC proteins and cytoskeletal components will be determined. These studies will provide new information and may reveal new paradigms in our knowledge of BMCs. In summary, we will show a practical application of the genetic code expansion strategy in studies of bacteria. Since Salmonella is an important model pathogen, the OTS we developed in this proposal could be broadly used by many laboratories to facilitate studies of microbial pathogenesis.