Carbapenem Antibiotic Resistance in Enterobacteriaceae: Understanding Interactions of KPC Carbapenemases with Substrates and Inhibitors

beta-lactams (BLs, penicillin and its relatives) are the most used antibiotics worldwide. Carbapenems are the newest and most potent BLs, and particularly important for treating infections by so-called opportunistic Gram-negative bacteria (GNB). These are organisms, either normally present in the human body or the natural environment (soil, water), that are not considered harmful to healthy individuals but can cause infections, possibly severe and even life-threatening, in patients whose immune defences are compromised. Risk factors for such infections include wounds (surgery, burns, injury), use of medical devices (catheters, ventilators), and conditions (HIV) or treatments (cancer chemotherapy or drugs that prevent transplant rejection) that affect immune defences. Growing numbers of patients fall into these categories. GNB are a particular treatment problem as their cell structure prevents many antibiotics that kill other types of bacteria from reaching their targets; efforts to discover new antibiotics effective against GNB have been largely unsuccessful. Until recently, carbapenems were regarded as "last resort" drugs for infections by GNB unresponsive to other treatments. However, growing resistance to other antibiotics makes carbapenems increasingly a first choice when infection by a GNB is suspected. When carbapenems fail alternatives are limited and often toxic, hence carbapenem resistance is regarded as a major public health challenge. In GNB carbapenem resistance is largely due to proteins called carbapenemases that bind to and degrade carbapenems, removing their ability to kill bacteria. Carbapenemases are part of a larger group of proteins (beta-lactamases) that destroy other types of BL antibiotics, however most beta-lactamases cannot break down carbapenems. beta-lactamases can be countered by a second group of drugs (beta-lactamase inhibitors) that block their activity and enable BL antibiotics to be used to treat bacteria carrying beta-lactamases, but not all beta-lactamases can be blocked by this route and some can mutate or evolve to escape the action of inhibitors. This proposal investigates how one carbapememase from the GNB Klebsiella pneumoniae, KPC (Klebsiella pneumoniae carbapenemase) degrades carbapenems and other BL antibiotics, and interacts with one specific class of inhibitors (diazabicyclooctanes, DBOs) and how these activities are affected by mutations in KPC. Klebsiella pneumoniae is an important cause of infections (urinary and respiratory infections, sepsis) associated with healthcare, and KPC is one of the main causes of carbapenem resistance worldwide. We recently described, for the first time, how KPC binds carbapenems and other BLs (ceftazidime, an antibiotic used for healthcare-associated infections) during a key stage in their breakdown; and how KPC binds DBOs. Based on this information we will use state-of-the-art computational methods to construct detailed models of the reaction of KPC with each of these three classes of molecules, in order to identify the most likely route by which each reaction occurs. The accuracy of these models will be tested by comparing the speed predicted for each reaction with actual values measured in experiments for a range of antibiotics used in patient treatment. We will then investigate how these reactions are affected by specific alterations in KPC, seeking to understand how such changes now identified in bacteria from human patients can improve the ability of KPC to break down antibiotics and reduce the ability of DBOs to block KPC action. Finally we will use this information to design and test, in computer models and experiments, new carbapenems that resist breakdown by KPC and new DBOs that are more effective KPC inhibitors; and that in each case are not affected by KPC mutations. This provides a route by which understanding of KPC can be exploited to design new treatments effective against an important group of antibiotic resistant bacteria.