Mechanisms of spore engulfment in C. difficile

Clostridioides difficile, commonly known as C. diff, is a bacterium that can cause diarrhoea, often with repeat episodes, and, in more severe cases, death. It is resistant to most commonly used antibiotics and using these drugs creates the conditions for infection to occur, as the normal, health-promoting bacteria in the gut is disrupted. Continued use of antimicrobials leads to new resistant and multi-resistant strains including against several commonly used antibiotics. Intense antibiotic use across the world during the current pandemic has made this risk higher and the search for more species-specific drugs even more important.
The life cycle of C. diff involves growth, formation of dormant cells called spores, and germination of the spores when the conditions are favourable. Spores provide C. diff with a formidable capacity to remain in the environment and in the host, offering resistance to radiation, heat and most commonly used cleaning agents. Due to these properties, spores are responsible for transmission of C. diff infections (CDI) and repeat episodes. Highly resistant spores produced by antibiotic-resistant/multi-resistant strains may be one of the most serious challenges we face in terms of control of C. diff. Despite its importance, the exact mechanisms of spore formation are still poorly studied.

In this project we propose to combine our expertise to study a key aspect of spore formation. As spores are dormant cells, the potential for antimicrobial resistance to arise is diminished, making this an innovative way to reduce and control CDI to be explored in the future.

Spore formation starts with the bacterial cell dividing into a smaller cell - the forespore - and a bigger, mother cell. The forespore is then surrounded by the mother cell in a process called engulfment. We have previously shown that two key protein machineries - the DP and Q:AH - are essential for this process and started revealing some of their key features. In this project we will build upon this work and answer three key questions:

1. How is the engulfment machinery organised?
We will characterise the interactions between these proteins, their role and their organisation. We will also determine the structures of these proteins to better understand their mode of action.

2. Which forces drive the process?
We will investigate what drives engulfment by characterising the composition of the main cell envelope - the peptidoglycan - how it varies and any other proteins involved in the process.

3. How does engulfment and sporulation impact disease?
We will investigate the role of these machineries in disease using disease, transmission and infection models.

This project will enhance our knowledge of a fundamental process in CDI and open new therapeutic avenues to control infection.

C. difficile infections (CDIs) are a major cause of morbidity and mortality in UK hospitals, with recent outbreaks of more virulent strains posing an increasing challenge to healthcare. There is an urgent need for new, effective methods for infection control and treatment. The current pandemic has led to intense use of antimicrobials worldwide, further enhancing the risks of antimicrobial resistance in the near future.
Spores are the infective agents in CDI and are also involved in disease recurrence and persistence. After asymmetric cell division, the smaller cell - the forespore - is engulfed by the mother cell, followed by maturation and release of the spore. We have shown (MRC NIRG) that 2 protein machineries - DP and Q:AH - are essential for sporulation. We will build on this work to further our understanding of the complex network required for engulfment - the engulfasome - and its role in disease, transmission and recurrence.

We aim to:

1. Define the components and organisation of the engulfasome
We will characterise interactions of DP and Q:AH in C. difficile and other potential engulfasome proteins and their three-dimensional organisation throughout engulfment. We will determine the structure of these complexes and the individual proteins to elucidate the molecular details of their mode of action.
2. Reveal the driving forces of engulfment
We will reveal the composition and architecture of PG at different stages of sporulation and study the enzymatic activities involved in the process. This will reveal how PG remodelling drives engulfment.
3. Elucidate the role of spore engulfment in disease
We will investigate the role of the engulfasome machineries in CDI using in vitro models as well as their impact on pathogenicity in acute and persistent animal disease.

This project will enhance our knowledge of a fundamental process in CDI and allow a better understanding of how sporulation impacts CDI.