Bacillithiol and its unique drug resistance pathways in Bacilli

Low molecular weight thiol molecules play an important role in antibiotic drug resistance. There are many enzymes inside cells which facilitate the reaction of these thiol molecules with antibiotics and their subsequent detoxification and removal from the cell. For this purpose, humans (and other mammals) use a thiol molecule called glutathione, which is made from amino acids building blocks. However, not all organisms are able to produce and utilise glutathione. Recently, we have discovered a novel class of carbohydrate-like biothiol, called Bacillithiol, amongst a number of microbial pathogens. These include bacteria associated with anthrax, food poisoning, urinary tract infections and MRSA. The aim of this project is to identify and characterise the ways in which these bacteria utilise bacillithiol to detoxify various antibiotics. Understanding the differences between glutathione and bacillithiol and their respective enzymes could provide exciting opportunities to design new drugs that will selectively target antibiotic detoxification mechanisms in drug resistant bacteria without affecting the glutathione processing enzymes found in humans.

Antibiotic drug resistance is an increasing cause for concern in human and animal healthcare. Glutathione (GSH) is the principle non-protein thiol found in eukaryotes and Gram negative bacteria. GSH mediates the detoxification of xenobiotics via GS-transferase catalysed conjugation. Many Gram positive bacteria produce distinctly different low molecular weight thiols, which serve a similar role. We have recently discovered a novel carbohydrate-derived low molecular weight thiol (named bacillithiol (BSH)), in several Gram positive bacteria of medical importance, which do not produce MSH. These include Bacillus anthracis (anthrax), B. cereus (food poisoning), Staphylococcus aureus (bacterial sepsis), S. saprophyticus (urinary tract infection) and Streptococcus aglactiiae (newborn septicaemia). The essential role of low molecular weight thiols in the detoxification of many antibiotics and the fact that BSH is not found in humans warrants further investigation of its biological function(s) as potential targets for future chemotherapy. The aim of this project is to establish the role and importance of BS-conjugate amidases and BS-transferases in antibiotic resistance amongst the Bacilli . This will involved synthesis of substrates and inhibitors for kinetic and mechanistic studies. Bacillithiol affinity resins will also be developed to enable proteome-wide identification and characterisation of Bacillithiol-S-transferases as potential targets for new antibiotic chemotherapies.

WHO WILL BENEFIT FROM THIS RESEARCH Outside of academic research, this work will be of particular interest to the pharmaceutical industry. In the longer term, the outputs of this project will lead on to long term benefits in terms of improved public healthcare. HOW WILL THEY BENEFIT The identification of novel drug resistance pathways will provide the pharmaceutical industry with new directions for the development of improved antibiotics. The could be of therapeutic relevance against numerous pathogens including methicillin resistant Staphylococcus aureus (MRSA, hospital pathogen), Bacillus anthracis (biowafare agent), Bacillus cereus (food poisoning). The scope to devise new methods to reduce the threat of such pathogens will lead to improved public health on both a national and international scale. The direct outputs of this project should prompt further efforts to put these potential benefits into practice. EXPLOITATION AND APPLICATION A preliminary patent has already been filed, including a composition of matter claim for BSH and its biosynthetic intermediates. We will regularly liase with the UEA Research and Business directorate to ensure there is no premature disclosure of any exploitable results. Additional intellectual property will definitely arise from this project. This will likely include:- [1] BSH AND AFFINITY MEDIA:- We will secure intellectual property rights on the production distribution rights for materials generated in this program before we licence them out to commercial suppliers. Availability of such reagents will benefit biochemists (investigating additional functions of bacillithiol) and pharmaceutical companies (for bacillithiol-dependent enzyme inhibitor screening assays); [2] NEW DRUG TARGETS:- As these are identified/validated we will quickly seek to secure intellectual property on these discoveries. This will be of certain interest to the companies in the Pharmaceutical industry. COMMUNICATION TO THE GENERAL PUBLIC The novelty of this research project and its relevance to bacterial drug resistance (a topic of increasing public concern and awareness) offers scope to develop educational seminars/lectures pitched at a level that will be tangible to the general public. As the project matures we will consult with the University outreach coordinators to prepare and present a public engagement lecture that describes the importance of drug resistance, the challenges we face and the role that investigative research has to play (and the specific contribution of this project) in addressing these challenges. EDUCATING INDUSTRIAL RESEARCHERS Results of this work will make an excellent 'target validation' case study for Hamilton to present on the biennial SCI 'Biology for Chemists' workshop that has been running since 2006. This course gives chemists in the pharmaceutical and related industries a greater understanding of biology from a chemical perspective and show how this knowledge can be used to improve chemical design and therefore streamline the drug discovery process. 5-10 YEARS FROM NOW Now that bacillithiol has been identified, based on what is already known about the functions of glutathione (in eukaryotes and Gram negative bacteria), an extensive range of bacillithiol-dependent pathways biochemical pathways can now be explored. These include:- Bacillithiol-S-transferases, peroxidases, disulfide reductases, bacilliredoxins, S-nitrosoreductases, alcohol dehydrogenases, intracellular metal ion trafficking, regulation of transcription pathways and protein function, resistance to low pH and osmotic stress. As the field begins to mature, and the fundamental importance of bacillithiol as a multi-functional cofactor becomes more apparent, it will surely find its place in standard undergraduate biochemistry textbooks alongside glutathione.