CAREER: Metabolic Engineering to Potentiate Immunity and Discover Novel Antivirulence Therapies

CBET - 1453325 Brynildsen，Mark P.普林斯顿大学抗生素耐药性是一个日益严重的公共卫生威胁，由于抗生素管道的减少而恶化。对“最后一道防线”抗生素产生耐药性的菌株已经出现，我们的抗生素库被淘汰的危险是真实的。由于传统抗生素的发现未能跟上耐药性的上升，因此需要新的方法来解决这一迫在眉睫的危机。抗病毒疗法包括靶向感染所需的宿主-病原体相互作用的新型抗感染药。由于抗生素施加选择性压力而不管它们的位置（例如，在人类、牲畜、污水、土壤内）和抗病力疗法仅在宿主内的感染部位施加选择性压力，抗病力疗法预计比抗生素更不容易产生耐药性。此外，由于它们的病原体特异性，它们也被预测比抗生素对肠道细菌的危害更小。一氧化氮（NO）是由免疫细胞产生的抗微生物剂，并且其抑制发病机制的重要性通过许多需要NO防御系统来建立或维持感染的细菌而突出。禁用病原体NO防御构成了一个有前途的抗毒力策略;然而，这些系统的已知元素的抑制剂要么对人类有毒，要么遭受到细菌细胞的运输不良。该项目的总体目标是对多种细菌病原体中的NO应激进行详细的定量了解，以确定针对NO防御系统的新型抗病力策略。增强病原体中NO毒性的挑战将作为一个代谢工程问题来处理，这是一个从其他抗病毒研究的范式转变，这项工作将填补基本知识空白，了解细菌NO压力下很少研究，但生理上重要的条件。预计拟议研究的结果将导致抗病毒疗法，解决抗生素耐药性的公共卫生危机，代谢工程方法应用于各种基于NO的现象（例如，共生），并在不同群体的个人（4 - 12年级学生，社区学院和本科生，代表性不足的少数民族学生，和一般公众）对生物工程的兴趣的刺激。 由于NO暴露的生物学结果是由一个复杂的动力学竞争，增强NO毒性的挑战可以减少到一个代谢工程问题：如何才能NO流量被引导远离解毒系统和破坏性反应？在这项提议中，代谢工程技术将被应用于识别和理解生理条件下细菌NO防御的弱点（例如，微需氧，酸性pH）。首先，定量，实验验证的动力学模型NO应力在三种细菌（一个模式生物，两种病原体）在生理条件下将开发。第二，将通过使用基因组规模的突变体库、竞争测定和下一代DNA测序来鉴定在这些条件下NO防御的新参与者。由于机制的洞察力可以阐明增加NO毒性的紧急策略（例如，协同效应），新的参与者改变NO防御的机制将通过使用整体建模方法来识别。为了补充这些研究活动，并激励个人追求生物工程的职业生涯，NO模型之一将形成主机病原体网络游戏的基础;拟议研究的元素将由社区学院，高中和本科生进行;研究结果将被纳入代谢工程选修课的创新活动套件。此外，社区学院和高中学生将在公共论坛上展示他们的作品，以教育和激励4 - 12年级学生，代表性不足的少数民族和公众。这些目标的成功实现将为抗病毒疗法的发展提供目标，提高对生理条件下细菌发病机制和NO应激的理解，并激励学生和公众追求生物工程的职业生涯。CBET部门生物技术和生物化学工程项目的职业生涯奖是共同的，由分子和细胞生物学系的系统和合成生物学项目资助。

CBET - 1453325 Brynildsen, Mark P.Princeton University Antibiotic resistance is a growing public health threat that is worsened by a declining antibiotic pipeline. Strains resistant to 'last line of defense' antibiotics have appeared and the danger of our antibiotic arsenal becoming obsolete is quite real. Since conventional antibiotic discovery has failed to keep pace with the rise of resistance, novel methodologies are required to address this looming crisis. Antivirulence therapies comprise a novel class of anti-infectives that target host-pathogen interactions required for infection. Since antibiotics exert selective pressure regardless of their location (e.g., inside humans, livestock, sewage, soil) and antivirulence therapies exert selective pressure only at infection sites within the host, antivirulence therapies are projected to be less prone to resistance development than are antibiotics. Further, due to their pathogen-specific nature, they are also predicted to be less harmful to commensal bacteria than are antibiotics. Nitric oxide (NO) is an antimicrobial generated by immune cells, and its importance to inhibiting pathogenesis is highlighted by the many bacteria that require NO defense systems to establish or sustain an infection. Disabling pathogen NO defenses constitutes a promising antivirulence strategy; however, inhibitors of the known elements of these systems are either toxic to humans or suffer from poor transport into bacterial cells. The overall goal of this project is to develop a detailed, quantitative understanding of NO stress in multiple bacterial pathogens in order to identify novel antivirulence strategies that target NO defense systems. The challenge of potentiating NO toxicity in pathogens will be approached as a metabolic engineering problem, which is a paradigm shift from other antivirulence research, and this work will fill fundamental knowledge gaps in understanding of bacterial NO stress under rarely studied, but physiologically important conditions. Results from the proposed research are expected to lead to antivirulence therapies that address the public health crisis of antibiotic resistance, application of metabolic engineering approaches to diverse NO-based phenomena (e.g., symbiosis), and stimulation of interest in bioengineering in a diverse group of individuals (4th-12th graders, community college and undergraduate students, under-represented minority students, and the general public). Since the biological outcome of NO exposure is dictated by a complex kinetic competition, the challenge of potentiating NO toxicity can be reduced to a metabolic engineering problem: How can NO flux be directed away from detoxification systems and toward damaging reactions? In this proposal, metabolic engineering techniques will be applied to identify and understand weaknesses within bacterial NO defenses under physiological conditions (e.g., microaerobic, acidic pH). First, quantitative, experimentally-validated kinetic models of NO stress in three bacterial species (one model organism, two pathogens) under physiological conditions will be developed. Second, novel participants in NO defense under those conditions will be identified through the use of genome-scale mutant libraries, competition assays, and next-generation DNA sequencing. Since mechanistic insight can illuminate emergent strategies to increase NO toxicity (e.g., synergistic effects), the mechanisms by which novel participants alter NO defenses will be identified with the use of an ensemble modeling approach. To complement these research activities and inspire individuals to pursue careers in bioengineering, one of the NO models will form the basis of a host-pathogen web-game; elements of the proposed research will be performed by community college, high school, and undergraduate students; and results from the research will be incorporated into an innovative suite of activities in a metabolic engineering elective. Further, community college and high school students will present their work at public forums to educate and inspire 4th-12th graders, under-represented minorities, and the general public. Successful achievement of these goals will provide targets for the development of antivirulence therapies, improve understanding of both bacterial pathogenesis and NO stress under physiological conditions, and motivate students and the general public to pursue careers in bioengineering.Due to the interdisciplinary nature of the project, this CAREER award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.