Catalytic Mechanism, Regulation and Drug-Target Potential of Bacterial Alarmone Hydrolases

About This Grant

Catalytic Mechanism, Regulation and Drug-Target Potential of Alarmone Hydrolases The research program in our laboratory focuses on understanding bacterial signaling mechanisms through the molecule (p)ppGpp, a key mediator in bacterial stress responses. Often called the "alarmone," (p)ppGpp helps bacteria survive adverse conditions such as nutrient starvation and antibiotic exposure. By halting cell growth and shifting bacteria into a persistent state, (p)ppGpp enables tolerance to chemotherapies without requiring genetic resistance, contributing to recurring infections. (p)ppGpp level is controlled by both its synthesis and degradation. The current paradigm of alarmone signaling, however, primarily focuses on the sensory role of alarmone synthetases, while hydrolases have been largely overlooked. Our goal over the next five years is to elucidate the catalytic mechanisms and regulatory roles of (p)ppGpp hydrolases. X-ray crystallography will be used to determine a hydrolase structure bound to a transition-state analog, revealing how the enzyme organizes its active site for catalysis. Literature and our preliminary data both suggest that alarmone hydrolases may also sense transition metals like manganese and iron. During infections, host defenses cause metal starvation or expose pathogens to toxic levels of zinc or reactive oxygen/nitrogen species. We hypothesize these stresses inactivates (p)ppGpp hydrolases, promoting alarmone accumulation and bacterial persistence. To investigate how metal stress impacts alarmone degradation, we will use structural biology, biochemistry and bacterial genetics to study these enzymes in vitro and in vivo. Additionally, we will explore the potential of hydrolases as antibiotic targets. While most antibiotics target essential genes, (p)ppGpp hydrolases have been overlooked due to the assumption that alarmone accumulation caused by inhibiting them would be pro-survival and bacteriostatic. However, our previous work with a related alarmone, (p)ppApp, suggests that unchecked alarmone accumulation without hydrolase activity can be lethal. We will assess the viability of a “synthetase+/hydrolase-” state for (p)ppGpp in Gram-negative pathogens to evaluate the therapeutic potential of hydrolase inhibition. Our research will significantly expand the understanding of bacterial persistence and provide new insights into how bacteria sense metal stress via alarmone signaling. The knowledge gained from this work could open new avenues for therapeutic developments by targeting alarmone hydrolases. Our long-term vision is to identify bacterial vulnerabilities that can be exploited to combat antibiotic persistence, improving clinical outcomes in the treatment of chronic infections. This research is poised to make foundational insights to bacterial stress response and resilience and advance our understanding of the communication between human hosts and bacterial symbionts.