Structure and interactions of the Clostridium difficile S-layer with bacteriocins.

Clostridium difficile is an important human pathogen, causing serious illness and even death. C. difficile infection (CDI) occurs most commonly in the hospital setting, affecting patients who are already suffering ill health, but infections in the community are an increasing problem. C. difficile is naturally resistant to many common antibiotics so whilst these antibiotics kill the beneficial bacteria in the human gut they have no effect on C. difficile; indeed they actually benefit the bacterium by removing competition.

The proteins on the surface of the C. difficile bacterium are the appendages through which it interacts with its environment and the human host. The C. difficile surface is coated in a single layer of protein that forms a two-dimensional crystal that wraps around the bacterium. This surface layer (S-layer) acts as a coat of armour to protect the bacterium from attack by our immune system and is essential for the bacteria to cause disease. Despite the importance of the S-layer we know very little about how it assembles on the cell surface and what it looks like. We have recently collaborated with a biotech company who have developed new therapeutic particles that attach to the S-layer to kill the C. difficile. These particles, called Avidocins, consist of a needle inside a spring-loaded sheath. When the Avidocin binds to the cell surface the sheath contracts and drives the needle through the cell wall and membrane, killing the bacterium. Although we know that Avidocins bind to the S-layer we do not know exactly how they recognise the S-layer, where they bind, what changes they undergo while binding, how the contraction is triggered or how the needle penetrates the various layers of the cell envelope.

In this project we will combine our complementary expertise in C. difficile biology and powerful electron microscopy to:

1. Understand the structure and organisation of the S-layer. This is essential information if we want to develop new treatments that target the S-layer to treat or prevent CDI.
2. Understand the structure of the Avidocin particle both on its own and in the act of binding to and killing a C. difficile cell.

There is an urgent need to develop new therapies to combat CDI and, in particular, to develop therapies which kill C. difficile but do not cause damage to the beneficial gut bacteria. A new therapy would ideally target a distinctive part of the bacterium which is essential for its lifecycle. The S-layer is an ideal candidate for this sort of intervention because it doesn't resemble the surface of any other kind of bacteria. Avidocins are promising therapeutic agents in their own right and are also closely related to natural viruses that infect C. difficile (bacteriophage). There is a lot of interest in the potential of bacteriophage to treat infections. Many of the features of Avidocin killing that we will study are directly relevant to bacteriophage infection, including binding to the S-layer, contraction of the spring-loaded sheath and penetration of the cell envelope. Our work on S-layer will identify ways in which this important structure can be targeted to tackle CDI.

C. difficile causes gut infections that vary in severity from mild diarrhea to life threatening, inflammatory complications. Infection commonly follows antibiotic-induced intestinal dysbiois that gives C. difficile a competitive advantage. There is an urgent need for novel therapies that treat infection without causing further disruption to the microbiota.

The C. difficile cell surface is covered with a proteinaceous paracrystalline S-layer, largely comprised of the protein SlpA. Little is known about the structural organisation of the S-layer or the mechanism of cell surface assembly, although we have recently shown that it is a crucial virulence factor. We have collaborated with AvidBiotics Corp. on the development of phage-like particles (Avidocins) that efficiently kill C. difficile. These Avidocins were constructed using a naturally occurring bacteriocin chassis fused to Myoviridae receptor binding proteins (RBPs). The S-layer is the cell surface receptor for these RBPs.

Given its potential as a therapeutic target, we wish to understand more about the fundamental structural biology of the S-layer and its interactions. In this project we will determine a high resolution structure of the C. difficile S-layer by electron crystallography of frozen-hydrated natural 2D S-layer crystals. We will also determine the structure of an Avidocin both alone, using single particle CryoEM, and in complex with the S-layer, using electron cryotomography. The Avidocin structure will give key insights into the structural organisation and receptor binding of these promising therapeutics as well as the Myoviridae bacteriophage to which they are closely related. By imaging the Avidocin in pre- and post-contraction states we will also gain insights into the mechanism of cell killing. Our structure will inform development of anti-S-layer therapeutics, including bacteriophage and phage-like particles, in addition to more traditional antimicrobials that target S-layer biogenesis.

The proposed project deals with the structural characterisation of a crucial Clostridium difficile virulence factor and a novel antimicrobial, Avidocin, that targets this structure to kill the bacterium. This project will provide a number of benefits to industry and the general public in the UK and further afield.

Industry:
In the context of the current antimicrobial resistance (AMR) crisis, the development of new antimicrobials is an absolute priority. Biotech and pharmaceutical companies with an interest in the development of antimicrobials will benefit both directly and indirectly from this project. The C. difficile S-layer is an attractive target for new species-specific antimicrobials. One such antimicrobial (Avidocin) is already in development by our collaborators AvidBiotics Corp. This project will provide crucial information about the interaction between Avidocin and the C. difficile S-layer. Ultimately this will enable the rational design of Avidocins with altered specificity and the companion diagnostics necessary for successful application of this precision therapeutic. Our work will also provide fundamental knowledge of the mechanism of targeting of bacteriophage that infect C. difficile. Bacteriophage are a promising alternative to traditional antibiotics but very little is known about the mechanism of receptor binding. Through our collaboration with AvidBiotics we have identified a wide family of phage receptor binding proteins (RBPs) that all target the S-layer. In this project we will focus on one RBP, characterising its interaction with the S-layer in molecular detail. Such information will be crucial to the future development of C. difficile phage therapies.

National Health Service:
Management of C. difficile infection is a current NHS priority and is a major financial drain on the health service. Development of novel therapies to treat C. difficile associated disease, particularly those that reduce the rate of relapse, will have a major impact on the NHS.

The local community in Sheffield:
In recent years the problems of AMR and hospital-acquired infections have entered the public consciousness. The local community will benefit from our outreach activities, gaining a broader knowledge of this important scientific area, and an opportunity to engage with and contribute to the research happening within their city.

Society:
The development of a new treatment for C. difficile will have a major impact on society in general. There were over 1600 deaths involving C. difficile in England and Wales in 2012 in addition to considerable morbidity. Treatment of C. difficile infections and the resulting extended hospital stays also place a huge burden on the NHS budget.

Students and Staff:
Undergraduate and postgraduate students at the University of Sheffield will benefit from their involvement in the project, developing knowledge and skills that will contribute to successful future careers. The postdoctoral research associate employed on the project will also develop crucial research and career development skills, with opportunities for involvement in outreach, science communication and industry collaborations.