Uncovering the antimicrobial and antibiotic potentiating mechanism of acesulfame-K and maximising its topical therapeutic potential.

Infectious diseases were once the leading cause of death amongst men and women in almost all age demographics in the UK. However, the discovery of antibiotics revolutionised our ability to treat bacterial infections and, as a result, saved millions of lives. Bacteria inhabit almost every corner of our planet due to their incredible ability to adapt to different environmental niches. This capacity to evolve and survive even in the most inhospitable environments means that, following the introduction of a new antibiotic to our healthcare systems, resistant bacterial strains rapidly appear. This cycle has kept repeating until the emergence, in some instances, of infections that cannot be effectively treated with any currently available antibiotics. This is creating a dangerous situation where a "post-antibiotic" era is now becoming a reality, threatening all aspects of healthcare from cancer treatment to dental work. At the forefront of pathogens that can evolve multidrug resistance is Acinetobacter baumannii. This pathogen can infect individuals who are already sick or have a supressed immune system, leading to a variety of life-threatening clinical complications and, potentially, death. This creates a problem particularly in hospitals where most A. baumannii outbreaks occur. Prior to the 2000s, A. baumannii infections were relatively infrequent and, typically, very treatable. However, there has been a rapid increase in the number of these infections, such that this bacterium now accounts for 20% of all infections seen in Intensive Care Units (ICUs) worldwide. These infections are incredibly difficult to treat, with up to 75% of A. baumannii isolated from these patients being resistant to more than 3 types of antibiotic. Previously, we have shown that the artificial sweetener acesulfame K (ace-K), a compound is consumed by millions of people around the world every day in "sugar free" or "calorie free" food and drinks, has a remarkable ability to tackle this pathogen. We demonstrated that not only can ace-K inhibit this pathogens growth. It can also inhibit a range of virulent processes that it uses to establish infection, including the ability to move from the initial site of infection and the capacity of this bacteria to form communities called biofilms which help it overcome antibiotic therapy. Remarkably, we also demonstrated that this compound will make A. baumannii vulnerable to antibiotics that it has previously evolved resistance to. We now want to explore what exactly ace-K is doing to the cell to stop it growing and to increase its sensitivity to antibiotics. We will use a range of cutting-edge fluorescent microscopy, proteomics and molecular biology techniques to uncover exactly how ace-k effects the bacterial cell and resensitises it to antibiotics. We will develop, characterise and assess novel ace-K loaded wound dressings to tackle acute and long-term, difficult to treat infections and test them in a porcine ex vivo wound model. We will also test these loaded wound dressings in a mouse wound model to determine their capacity to treat infection. As ace-k is approved for consumption by every international regulatory body including the Food and Drug Administration, it means it has been extensively tested for safety. Therefore, there is significant potential that the use of ace-K as a therapeutic to tackle infection could be fast tracked to clinical trials and into hospitals. This would overcome one of the main barriers delaying the introduction of new antimicrobials drugs which is that all the safety testing and trials required before final approval can take over 15 years on average to complete.

A central facet to the pathogenic success of Acinetobacter baumannii is in part attributable to its ability to overcome antibiotic therapy. Recent work from the McCarthy Lab has identified a compound, acesulfame K (ace-K), with previously unrecognised antimicrobial and antibiotic potentiating activity. This proposal takes a highly interdisciplinary and disruptive approach to characterising the mechanism of action of this compound and determining its therapeutic potential. This includes the use of cutting-edge technologies such as thermal proteomic profiling and mouse models of chronic wound infection.

For aim 1 this proposal will use a combination of live cell microscopy, and state-of-the-art molecular genetics tools that have been developed by the McCarthy lab to define the consequences of ace-K exposure on peptidoglycan in the cell envelope, and the role the DnaK/GrpE/GroSL system in influencing cell envelope disturbances. We will also use cutting-edge Proteome Integral Solubility Alteration assays integrated with Expression Proteomics (PISA-Express) to identify direct protein-ace-K interactions and impacts on the total proteome. We will also repeat this assay to understand the fate of meropenem within the cell in the presence and absence of ace-K. For aim 2 we will develop and characterise novel ace-K, and ace-K + antibiotic loaded aerogel and hydrogel wound therapies to target acute and chronic wound infections. We will then assess the developed formulations using an ex vivo porcine skin model of wound infection. Finally, in aim 3, we will rigorously assess the efficacy of the developed wound therapies in a world leading mouse acute and chronic wound model to demonstrate the therapeutic potential of ace-K.