Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel 7610001.
Proc Natl Acad Sci U S A. 2021 Mar 23;118(12). doi: 10.1073/pnas.2022012118.
is an opportunistic human pathogen that develops difficult-to-treat biofilms in immunocompromised individuals, cystic fibrosis patients, and in chronic wounds. has an arsenal of physiological attributes that enable it to evade standard antibiotic treatments, particularly in the context of biofilms where it grows slowly and becomes tolerant to many drugs. One of its survival strategies involves the production of the redox-active phenazine, pyocyanin, which promotes biofilm development. We previously identified an enzyme, PodA, that demethylated pyocyanin and disrupted biofilm development in vitro. Here, we asked if this protein could be used as a potential therapeutic for infections together with tobramycin, an antibiotic typically used in the clinic. A major roadblock to answering this question was the poor yield and stability of wild-type PodA purified from standard overexpression systems. We hypothesized that the insufficient yields were due to poor packing within PodA's obligatory homotrimeric interfaces. We therefore applied the protein design algorithm, AffiLib, to optimize the symmetric core of this interface, resulting in a design that incorporated five mutations leading to a 20-fold increase in protein yield from heterologous expression and purification and a substantial increase in stability to environmental conditions. The addition of the designed PodA with tobramycin led to increased killing of cultures under oxic and hypoxic conditions in both the planktonic and biofilm states. This study highlights the potential for targeting extracellular metabolites to assist the control of biofilms that tolerate conventional antibiotic treatment.
铜绿假单胞菌是一种机会性病原体，会在免疫功能低下的个体、囊性纤维化患者和慢性创伤中形成难以治疗的生物膜。铜绿假单胞菌拥有一系列生理特性，使其能够逃避标准抗生素治疗，尤其是在生物膜中，其生长缓慢，对许多药物产生耐受性。其生存策略之一是产生氧化还原活性的吩嗪，即绿脓菌素，促进生物膜的发展。我们之前发现一种酶 PodA 可以使绿脓菌素脱甲基，并在体外破坏铜绿假单胞菌生物膜的形成。在这里，我们想知道这种蛋白是否可以与妥布霉素一起作为铜绿假单胞菌感染的潜在治疗方法，妥布霉素是临床上常用的抗生素。回答这个问题的主要障碍是从标准过表达系统中纯化野生型 PodA 的产量和稳定性较差。我们假设产量不足是由于 PodA 必需的同源三聚体界面内的包装不良所致。因此，我们应用蛋白设计算法 AffiLib 来优化该界面的对称核心，设计出一种包含五个突变的设计，从而使异源表达和纯化的蛋白产量增加了 20 倍，并大大提高了对环境条件的稳定性。在需氧和缺氧条件下，无论是浮游状态还是生物膜状态，添加设计的 PodA 和妥布霉素都导致铜绿假单胞菌培养物的杀灭率增加。这项研究强调了靶向细胞外代谢物以协助控制耐受常规抗生素治疗的生物膜的潜力。
