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.

To combat the alarming rate of antibiotic resistance, antimicrobial peptides (AMPs) are seen as promising new drugs, as they are considered low-risk for resistance. However, AMP resistance already exists in many bacteria, with homologous genes present in important pathogens. Our work is focussed on Bce-like systems that consist of a two-component regulatory system that controls production of a resistance transporter. Importantly, the transporter has a second role as sensory unit controlling the activity of the regulatory system. Bce-like systems are found in several clinically relevant Gram-positive bacteria. We have established the bacitracin resistance system, Bce, of Bacillus subtilis as an experimentally tractable and highly relevant model.

Bce-like transporters share a conserved FtsX domain of unknown function with other resistance transporters found in most bacterial phyla. We will (i) use bioinformatics to study the phylogeny of these transporters and combine the computational results with subcellular protein localisation and mutagenesis of the FtsX-motif to identify the function of this domain, which will be of relevance to a range of drug resistance systems.

We previously showed that the transporter forms a sensory complex with the system's kinase, but the mechanism by which it controls kinase activity is unknown. We will use mutagenesis and cysteine accessibility scans to identify (ii) sites of protein interactions and (iii) conformational changes in the kinase caused by the transporter. Exploiting the presence of two paralogous systems in B. subtilis, we will (iv) analyse the establishment of signalling specificity. To examine how the Bce system detects its substrate, we will (v) study ligand binding using protein biochemical and structural analyses.

Molecular level insights into Bce-like systems, particularly their signalling process, will allow development of innovative treatment strategies that directly target resistance.

This project contributes directly to the BBSRC's priority area "Combatting antimicrobial resistance", which is part of the "UK five year antimicrobial resistance strategy". Our study will address the "fundamental microbiology" of resistant bacteria, by determining the signalling processes by which the bacteria respond to the presence of antibiotics. Our phylogenetic comparison of different resistance transporters that share a conserved FtsX domain will further address the question "how resistance develops and is maintained". In the medium to long term, our findings will allow the development of "mitigation strategies" and "novel antimicrobials and alternatives to antimicrobials" designed to bypass or block resistance. Thus, our research simultaneously addresses many aspects of the priority area's scientific scope.

Our work will provide information on the evolutionary origins of antimicrobial peptide resistance and on the molecular mechanisms underpinning drug perception and signal transduction, which will be important for developing new treatment strategies and, in the long-term, new policies for antibiotic use in therapy. Its most immediate impact will be through knowledge transfer to other researchers. In the academic sector, we expect researchers with an interest in antibiotic resistance, signalling networks, evolutionary biology, microbial cell biology and drug discovery to directly benefit from our study. Our results will further allow researchers in the biomedical commercial sector, e.g. Novacta, Novabiotics or Cantab Anti-Infectives, to identify novel strategies to combat resistance.

Pharmaceutical and biomedical companies will further benefit from our research through knowledge of the molecular mechanisms of drug perception and of the signalling pathway that leads to activation of resistance. We have already established links to Cantab Anti-Infectives, which will be consolidated during the funding period and are intended to produce long-term collaborations. Our research has strong potential to influence the development of new drugs or drug modifications designed to bypass detection. It is further expected to lead to new treatment strategies aimed at directly blocking the signalling pathway to prevent activation of resistance. Our research will identify promising new drug targets and thus ultimately influence both public health and the economy of the UK.

By dissemination of our results we will further reach national and international policymakers, such as the UK's Review on Antimicrobial Resistance or the World Medical Association. These authorities are expected to encourage governments to create incentives for companies to invest in research and development not only of new antibiotics, but of entirely novel treatment strategies. Our project will identify targets for such innovative therapies and is thus expected to influence policy, ultimately contributing to a reduction of morbidity and hospitalisation (health benefit) and the costs of treatment (economic benefit) of infectious diseases.

We will reach out to the public to improve the understanding of (micro-)biology in general and antibiotic resistance in particular. Through media coverage and personal experience, most members of the public have a strong awareness of the phenomenon of antibiotic resistance. However, many misconceptions exist and create worry and uncertainty, which we will help to alleviate through explanations of our research.

This project will further have a strong impact on training of young scientists. The PDRA will receive training in state-of-the-art molecular and biochemical techniques. Together with opportunities to establish contacts to biomedical companies, this will make the PDRA highly competitive for a future career in academia or industry. Moreover, training of undergraduate and project students in new approaches to tackle antibiotic resistance will have a strong impact on the next generation of researchers.