Exploring mechanical mechanisms of antibiotic resistance

总结 传统上，抗生素耐药性的研究集中在进化和分子水平上， 抵抗机制。然而，我们许多最好的抗生素靶向细菌细胞包膜， 它是细胞的一个机械上坚固的结构外骨骼。最终，这些抗生素会导致 细胞死亡的削弱信封足以引起爆炸的细胞由大，流体静力学 尽管细胞被膜具有重要的机械作用， 了解其中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。主 我们知识中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机能。拟议的研究将解决这一问题 通过应用创新的、高精度的、高通量的微流体技术和基于显微镜的 测定两种主要细胞包膜成分的机械性能， 细菌、外膜和细胞壁。这些检测将结合分子和 细胞生物学技术和生物物理学理论，探索一种新兴的范式， 微生物学：细菌通过自适应调整机械性能来控制抗生素耐药性 他们的细胞膜。首先，建立在最近的里程碑式的发现，外膜赋予 抗生素耐药性细菌，因为它的机械强度，依赖于外部 膜刚度和机械抗生素抗性对精细尺度的生化组成， 将系统地测量外膜。其次，外膜的机制 囊泡形成（抗生素耐药性和发病机制的潜在过程）将通过以下方法进行研究： 将囊泡形成的理论力学模型与新的显微镜测定相结合， 囊泡动力学，而遗传调谐蛋白质之间的相互作用细胞壁和 外膜最后，这些研究的范围将扩大到革兰氏阳性菌， 确定这些物种中抗生素抗性对细胞壁硬度的依赖性，特别是 重点是磷壁酸对抗性的机械贡献。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 来规避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.