Targeting Periplasmic Adaptor Proteins to Improve Antibiotic Efficacy

Antibiotics underpin all of modern medicine; they are used to treat bacterial infections, and to prevent infections after surgery and in patients with a suppressed immune system such as those undergoing cancer chemotherapy or organ transplantation. However, bacteria are able to employ various mechanisms to resist the action of antibiotics and the number of infections caused by bacteria that are resistant to antibiotics is increasing globally. This means that bacterial infections are becoming harder to treat. In fact, in Europe 25,000 people die every year from infections caused by drug resistant bacteria. Additionally, there is a lack of new antibiotics being developed to replace those that we can no longer use. New strategies are now needed to combat antibiotic resistance and to allow continued treatment of bacterial infections. This proposal aims to tackle this problem by investigating a novel drug target and finding molecules which inhibit it.

Bacteria become resistant to antibiotics in many ways but one important mechanism is multi-drug efflux. Bacteria have efflux pumps in their membrane which are able to pump antibiotics out of the bacterial cell. This is important because it allows bacteria to survive at higher concentrations of the drug. In addition, these pumps can export many different classes of antibiotic so the bacteria are resistant to many drugs at the same time, known as multi-drug resistant (MDR). The Resistance Nodulation Division (RND) family of efflux pumps confer antibiotic resistance to bacteria which infect humans and animals, including the foodborne pathogen Salmonella. RND pumps work with two other protein components to form a tri-partite pump system. One of the essential components of the tripartite efflux pump is called the Periplasmic Adaptor Protein (PAP) and I have previously shown that the PAPs are a good target against which to develop inhibitors. When the gene coding for the PAP was inactivated Salmonella became more susceptible to antibiotics. When the genes coding for two PAPs were inactivated, the bacteria became even more susceptible to antibiotics. This means that if drugs could be developed which inhibit the PAPs then these drugs could be given to a patient along with an antibiotic to make the bacteria susceptible to antibiotic treatment. This would allow successful treatment of otherwise resistant infections. In addition to the effect on antibiotic resistance, inactivation of the PAPs made the bacteria less able to cause infection. Therefore, inhibition of the PAPs would have the added benefit of limiting spread of an existing infection within the body or could be used to prevent colonisation of animals in the food chain.

This overall aim of this project is to identify molecules which inhibit the PAPs and could be developed into new drugs to tackle MDR. The initial tasks will use bacterial strains in which the genes for each PAP or combinations of PAPs are inactivated to work out how loss of PAPs alters the bacterial physiology. I will determine how many PAPs need to be targeted by inhibitors to give the biggest impact on antibiotic resistance and importantly, this study will also confirm that resistance to PAP inhibition is not likely to develop. Finally, potential PAP inhibitors will be identified by using high-throughput screens to test libraries of thousands of compounds.

The potential benefits from this project to society are substantial. This work will provide a greater fundamental understanding of the impact of loss of PAP function. In the longer term, development of PAP inhibitors could be used to make antibiotic resistant infections treatable by rendering bacteria susceptible to antibiotics. Furthermore, PAP inhibitors could be used to prevent food producing animals becoming colonised with bacteria to reduce the incidence of foodborne infections.

Antibiotic resistance is a current global crisis and increasing numbers of people are dying from infections because we have no drugs to effectively treat them. There is a lack of new antibiotics in development and without new strategies for treating multi-drug resistant (MDR) bacterial infections we face a return to the pre-antibiotic era. This proposal aims to tackle this problem by investigating a novel drug target and finding potential inhibitors which could be developed into drugs.
Bacteria use efflux pumps to transport antibiotics out of the bacterial cell. This reduces the internal drug concentration and confers MDR. Inhibitors of these pumps could be used in the treatment of infections in humans and animals to combat resistance and potentiate the use of previously important clinical and veterinary antimicrobials, which are no longer useful due to resistance. This is a particularly attractive strategy since it would allow many antibiotics that are already licensed to treat infections, to be utilised once more.
The RND family of efflux pumps are tri-partite systems made up of an inner membrane pump, a Periplasmic Adaptor Protein (PAP) and an outer membrane channel. I have shown that the PAP components of these tripartite MDR efflux pumps are a good target for inhibitors. Initial work will determine the biological effect of loss of PAP function on the bacteria and the impact of this on MDR. This project will validate the PAPs as a target for efflux inhibition and find the combination of PAPs to be inhibited to confer optimal susceptibility and prevent resistance to inhibitors occurring. Two large compound libraries will be screened to identify compounds which inhibit the PAPs. This project will identify PAP inhibitors while also providing fundamental understanding of the role, regulation and biology of this important family of proteins.

Every year 25,000 Europeans and 100,000 Americans die as a result of multi-drug resistant (MDR) bacterial infections. The incidence of MDR infections is rising and there is a major lack of new drugs in the pipeline to combat this problem. This proposal offers a novel approach to tackling antibiotic resistance. Therefore, the ultimate long-term (>10 years) beneficiary of this work will be the general public and public health. If compounds are identified that successfully inhibit bacterial efflux then these could be developed into novel therapeutics which could be used in combination with antibiotics to treat patients with MDR bacterial infections that would otherwise be untreatable. Importantly, this will allow renewed use of already licensed antibiotics that are not currently clinically useful due to resistance. This would have many beneficiaries. Primarily, this will benefit patients and clinicians by increasing treatment options, improving treatment outcomes, reducing length of patient stays in hospital and ultimately preventing deaths from MDR infections. In addition, PAP inhibitors could also be used to treat infected animals or to prevent colonisation of animals in food production, lowering the burden of foodborne infections. This would benefit animal health, human health and would have economic benefits for individuals and companies involved in food production.

In Europe alone MDR infections account for 600 million days of lost productivity and costs the economy in excess of 1.5 billion Euros a year in terms of added healthcare costs. In the long term, if PAP inhibitors are successful they could potentially benefit the wider economy by reducing this economic burden. In turn this would benefit the NHS and other healthcare services, policy makers, government and the tax payer could also benefit because enabling renewed use of already licensed antimicrobial drugs could be a more cost effective strategy than developing new antimicrobial drugs and getting them licensed and into the clinic.

In the short term (within the first year), this research will benefit the academic community both in the UK and abroad. It will add to the fundamental knowledge about efflux mediated multi-drug resistance and specifically the role of PAPs. In addition, novel flow cytometry assays and tools will be developed that could be adapted for diverse uses by other researchers in a variety of fields and flow cytometry and whole genome sequencing data sets will deposited for use by other academics.

The search for novel antimicrobials is also an active area of research for the pharmaceutical industry so the results from this study will impact their research. All intellectual property will be protected using Alta Innovations, the technology transfer company for the University of Birmingham which will secure patent protection and deal with licensing of any intellectual property as appropriate.

The benefits of this research to my career would be profound. This fellowship would allow me to establish my own research team within the IMI including a guaranteed PhD studentship with the first two years. The 5 year timescale of this project would allow me to produce a significant body of work which will form the basis for numerous publications and future funding opportunities. The technician and the PhD student working on the project will benefit from career development opportunities including developing new research and professional skills and the opportunity to present their research at national conferences which will benefit their ongoing career prospects and progression.