Exploring mechanical mechanisms of antibiotic resistance

摘要 传统上，对抗生素耐药性的研究主要集中在进化和分子方面。 抗性机制。然而，我们的许多最好的抗生素都是针对细菌的细胞膜， 这是一种机械上坚固的细胞外骨骼结构。最终，这些抗生素会导致 通过使包膜变弱到足以导致细胞被大的流体静力学爆炸而导致的细胞死亡 里面有压力。尽管细胞膜具有重要的机械作用，但我们几乎没有 了解其内部的哪些分子和部分对其承载能力至关重要。 解决这个问题将改变我们对抗生素耐药性的理解。一个主要的 我们知识上存在这种差距的原因是将机械力应用于 单个细菌细胞，同时监测它们的生理。拟议的研究将解决这一问题 通过应用创新、高精度、高通量的微流体和基于显微镜的 用于测量两种主要细胞包膜成分的机械性能的方法 细菌、外膜和细胞壁。这些检测将与分子和 细胞生物技术和生物物理理论，以探索一种新兴的 微生物学：细菌通过自适应地调整机械性能来控制抗生素耐药性 他们的细胞包膜。首先，基于最近的里程碑式的发现，外膜赋予 细菌对抗生素的抗药性是因为其机械强度大，对外源的依赖 膜硬度和机械抗菌性对细菌细微生化成分的影响 将对外膜进行系统测量。其次，外膜的作用机制。 囊泡形成(抗生素耐药性和致病机制的一个过程)将由 将囊泡形成的理论力学模型与新的显微镜分析相结合来量化 囊泡形成动力学，同时遗传调节细胞壁和细胞壁之间的蛋白质相互作用 外膜。最后，这些研究的范围将扩大到革兰氏阳性细菌 确定抗生素耐药性对这些物种细胞壁硬度的依赖性，特别是 重点关注磷壁酸对抗性的机械贡献。总而言之，这些研究将 改变我们对细菌病原体生存和生长的理解，并指出新的策略 以避免抗生素耐药性和治疗细菌感染。

Summary Traditionally, studies of antibiotic resistance have focused on evolutionary and molecular mechanisms of resistance. However, many of our best antibiotics target the bacterial cell envelope, which is a mechanically robust, structural exoskeleton for the cell. Ultimately, these antibiotics cause cell death by weakening the envelope enough to cause explosion of the cell by the large, hydrostatic pressure within it. Despite the central mechanical importance of the cell envelope, we have little understanding of which molecules and moieties within it are critical for its load-bearing capacity. Addressing this question would transform our understanding of antibiotic resistance. A primary reason for this gap in our knowledge is the formidable challenge of applying mechanical forces to single bacterial cells while monitoring their physiology. The proposed research will address this obstacle by applying innovative, highly precise, high-throughput microfluidics and microscopy-based assays to measure the mechanical properties of two of the major cell envelope components in bacteria, the outer membrane and the cell wall. These assays will be combined with molecular and cell biological techniques, and biophysical theory, to explore an emerging paradigm within microbiology: that bacteria control antibiotic resistance by adaptively tuning the mechanical properties of their cell envelope. First, building on the recent landmark finding that the outer membrane confers antibiotic resistance to bacteria because of its mechanical strength, the dependence of outer membrane stiffness and mechanical antibiotic resistance on the fine-scale biochemical composition of the outer membrane will be systematically measured. Next, the mechanism of outer membrane vesiculation (a process underlying antibiotic resistance and pathogenesis) will be investigated by combining a theoretical mechanical model of vesiculation with novel microscopy assays to quantify vesiculation dynamics, while genetically tuning protein-protein interactions between the cell wall and outer membrane. Finally, the scope of these studies will be extended to Gram-positive bacteria by determining the dependence of antibiotic resistance on cell wall stiffness in these species, specifically focusing on the mechanical contributions of teichoic acids to resistance. Together, these studies will transform our understanding of bacterial pathogen survival and growth, and point to fresh strategies to circumvent antibiotic resistance and treat bacterial infections.