Understanding the Evolutionary Origins and Molecular Mechanisms of Antimicrobial Peptide Resistance

The global increase in antibiotic resistance has made it difficult to treat bacterial infections, and mortality from infectious disease is rising at an alarming rate. Each year 700,000 people die from resistant bacteria such as MRSA, and some bacteria are now resistant to all available drugs. In 2014, the World Health Organisation issued its first Global Report on Antimicrobial Resistance, urging governments world-wide to join forces in tackling this health emergency. One goal is to identify new antimicrobials, but no new major class of antibiotics has been developed in 30 years. Current studies are exploring antimicrobial peptides (AMPs) for clinical use. AMPs target an essential bacterial structure (the cell wall), which cannot be easily changed by mutation, and are thus considered as "safe" regarding resistance. The same assumption was made with the introduction of vancomycin in the late 1950's, but transfer of resistance genes from environmental bacteria has resulted in one of the current "superbugs", Vancomycin Resistant Entercocci (VRE). Resistance against AMPs in environmental bacteria already exists, and many human pathogens, e.g. Staphylococci, contain related genes. To prevent a similar development as was seen for vancomycin, it is therefore imperative that we understand these AMP resistance systems and use the findings to devise strategies to counteract them. One innovative approach is to block the pathway by which bacteria detect the antibiotic and activate their resistance. A drug that interferes with this process would restore the efficacy of the antibiotic, providing a long-term solution. Similar treatments are already used in cancer therapy, but not yet to tackle antibiotic resistance. In recent years, a new type of AMP resistance has been identified in many Gram-positive bacteria, incl. human pathogens like S. aureus. These so-called Bce-like systems consist of a transporter that presumably removes the antibiotic from the cell, and a regulatory system that controls production of the transporter. Their key feature is that the transporter acts as an AMP sensor and controls the regulatory system and thus indirectly itself. Two aspects make these systems highly relevant for detailed exploration: (i) they share a conserved domain with other resistance transporters found in nearly all bacteria; (ii) their unique regulatory pathway presents a prime drug target for blocking resistance. Because pathogenic bacteria are difficult to handle, we will use the closely related bacitracin resistance Bce system of Bacillus subtilis as our experimental model. Our first aim is to determine how these systems evolved, in order to understand their relationship to other resistance systems. Bce-like transporters contain a domain, called FtsX, we expect to be important for resistance and which can be found in many disease-causing bacteria. We will use computational and experimental methods to determine the function of this domain. This will provide information on Bce-like systems as well as on the other transporters possessing an FtsX domain. The second aim addresses the question how the transporter controls the regulatory system. We will use molecular biology techniques to find out where the proteins interact, and how information is passed from the transporter to the regulatory system. Blocking this pathway will prevent activation of resistance, and we will provide the information needed to explore it as a novel drug target. The first step of the resistance pathway is detection of AMPs by the cell, yet it is unknown how Bce-like transporters accomplish this. In our third aim, we will use protein biochemistry methods to study AMP binding. Knowledge of how a drug is bound will allow the design of modifications that prevent detection and thus resistance. Our project will provide detailed understanding of AMP resistance by Bce-like systems and identify important processes to explore as drug targets in combatting resistance.