Understanding the molecular determinants and regulation of toxin activity in bacteria

关于生物膜的研究，即我们睡觉时覆盖在牙齿上的细菌群落，已经持续了几十年。然而，即使在今天，唯一确定的去除生物膜的方法是通过机械力，即牙刷。虽然刷牙是常规的，但从船舶、管道、医疗设备等去除生物膜，是非常困难的。生物膜的形成是细菌利用的主要生存机制之一，但对其如何发生的知识是基本的。细菌持留菌是一种基因相同的静止细胞亚群，表达蛋白质毒素并表现出多药耐受性，是生物膜形成的核心，使细菌能够适应不断变化的环境条件。被称为毒素-抗毒素（TA）系统的基因对已经成为这种适应过程的关键组成部分。该项目将通过发现毒素如何抑制底物，如何被抗毒素抑制以及细菌如何去除毒素以退出持久性来确定毒素如何阻止细菌生长。除了帮助发现控制生物膜的新方法外，该项目还将通过在UA BIOTECH外展项目中实施蛋白质科学研讨会，为学生提供研究培训机会，并为高中生提供STEM教育机会。许多TA毒素，包括MqsR，这一提案的重点，都是核糖核酸酶（RNase）。为了控制生物膜的形成，必须了解核糖核酸酶毒素如何调节细菌的生长和停滞。这项研究将确定MqsR如何识别和切割mRNA底物。初步数据表明，这些信息将导致RNA酶消化mRNA的新机制。该项目还将确定抗毒素MqsA如何抑制MqsR活性，促进毒素抑制/调节新方法的开发。最后，将确定指导细菌中MqsA和MqsR蛋白水解和周转的分子决定因素。这些将定义可用于控制细胞中TA周转的蛋白质识别位点，并为细菌蛋白酶识别其内源性底物的机制提供新的见解。该奖项反映了NSF的法定使命，并被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

Research on biofilms, bacterial communities that coat our teeth when we sleep, has been ongoing for decades. However, even today, the only certain way to remove biofilms is by mechanical force, i.e. a toothbrush. While brushing teeth is routine, removal of biofilms from ships, pipes, medical devices etc., is exceedingly difficult. Formation of biofilms is one of the major survival mechanisms utilized by bacteria, but knowledge of how it occurs is rudimentary. Bacterial persisters, a genetically identical sub-population of quiescent cells that express protein toxins and exhibit multidrug tolerance, are at the core of biofilm formation and in enabling bacteria adapt to changing environmental conditions. Gene pairs known as toxin-antitoxin (TA) systems have emerged as key components of this adaptation process. This project will determine how toxins block bacterial growth by discovering how they inactivate substrates, how they are inhibited by antitoxins and how bacteria remove toxins in order to exit persistence. In addition to helping discover novel approaches for controlling biofilms, the project will provide research training opportunities for students and STEM education opportunities for high school students through implementation of the Protein Science Workshop in the UA BIOTECH outreach project.Many TA toxins, including MqsR, the focus of this proposal, are ribonucleases (RNases). In order to control biofilm formation, an understanding of how RNase toxins regulate bacterial growth and arrest must be obtained. The research will determine how MqsR recognizes and cleaves mRNA substrates. Preliminary data suggest that this information will lead to a novel mechanism used by RNases to digest mRNA. The project will also identify how antitoxin MqsA inhibits MqsR activity, facilitating development of new approaches for toxin inhibition/modulation. Finally, the molecular determinants that direct MqsA and MqsR proteolysis and turnover in bacteria will be determined. These will define protein recognition sites that can be used to control TA turnover in cells, and provide new insights into the mechanisms used by bacterial proteases to recognize their endogenous substrates. An integrated structural, biochemical and cellular approach will be utilized to address these fundamental questions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.