Investigating structure, dynamics and mechanism of the bacterial type IV pilus machinery

Type IV pili (T4P) are protein filaments assembled on the surface of many bacteria, used for cell motility, adherence, communication, biofilm formation, virulence, as well as environmental DNA uptake. T4P are thus crucial for bacterial survival, enabling them to respond to changing environments, facilitating genome plasticity, and the spread of antimicrobial resistance (AMR) genes. Due to their importance in microbiology, T4P are well-studied, yet it is not clear how they can mediate such diverse functions. In this project, we aim to build a molecular picture of the interplay between T4P structure, properties and function through a combination of microbial genetics, molecular dynamics simulations, structural biology, and a catalogue of functional assays. By uncovering how the structure and properties of T4P relate to their diverse roles, this research has the potential to lead to new approaches for targeting bacterial infections and controlling the transmission of resistance genes, ultimately contributing to the development of more effective antimicrobial therapies. Historically, the multifunctional nature of T4P has made it challenging to identify specific properties responsible for function. Our research aims to address this challenge by investigating two distinct types of T4P within a single organism. In previous BBSRC-funded research (NIRG to Project-lead VG), we discovered that Thermus thermophilus assembles two T4P with different properties and functions. This presents a unique opportunity to unravel the fundamental mechanisms and underlying principles governing the functions of these essential microbial filaments. Our objectives are to: Examine how the structural properties of different T4P relate to their specific function Understand how T4P are assembled and retracted through the co-ordinated action of ATPases Elucidate how the T4P machinery facilitates DNA uptake To achieve these objectives, we aim to foster interdisciplinary frontier bioscience to advance knowledge and drive significant discoveries through complementary expertise, collaboration and the development of new tools. We will capitalise on a newly funded cryo-transmission electron microscope (BBSRC ALERT23 bid led by Project-lead VG), crucial to the success and impact of this proposal. The potential applications and benefits of this work are extensive and varied. Specifically, the project will contribute molecular and mechanistic knowledge about T4P that will facilitate a deeper understanding of bacterial physiology and survival mechanisms. Additionally, the findings will inform a wide range of downstream applications. For example, T4P are promising targets for anti-virulence strategies; in the longer-term, we can develop novel approaches to disrupt T4P function or inhibit biofilm formation in priority pathogens, thereby improving public health and economic outcomes. In biotechnology, insights into DNA uptake mechanisms can be harnessed to enhance the genetic engineering of organisms that are typically difficult to transform, leveraging their natural DNA uptake capabilities. By uncovering the mechanism behind these essential microbial structures, we have the potential to drive innovative solutions for pressing medical and industrial challenges in the future. More broadly, we will demonstrate the benefit of interdisciplinary research in addressing fundamental research problems and contribute to the training in key skills for talented researchers. Taken together, this research proposal addresses the BBSRC high-level objective “Advancing the frontiers of bioscience discovery” and within that the priority area “Understanding the rules of life”, and links to the BBSRC areas for investment and support “Antimicrobial resistance” and “Transformative Technologies”.