Structural characterization of the bacterial flagellum basal body

Many bacteria utilize a long, rotating filament on their surface, called the flagellum, to swim in their surrounding environment, and for adherence to solid surfaces. In particular, these features are essential for many pathogenic bacteria such as E.coli, Salmonella, Shigella etc.. to cause disease. This is because the flagellum allows them to spread in the host's body, and to adhere to their cells, as well as to medical devices such as catheters. As a consequence, understanding the bacterial flagellum at the molecular level could have numerous medical implication, and disrupting its rotation and adherence properties would largely reduce infection in a clinical setting. However, the flagellum is a very sophisticated nano-machine, and studying its mechanism of action at the molecular level is particularly challenging. A central region of the flagellum, called the "basal body", serves as an anchoring point around which the complete machinery forms. Despite the importance of this region, we know very little about its molecular details, and about how its constituting components come together to form a stable platform around which the flagellum assembles. The objective of the proposed research is to use advanced biophysics methods, including the most cutting-edge electron microscopy techniques, to generate a molecular "map" of the flagellum basal body. We will then exploit this map to determine how its constituting components come together to form such a sophisticated structure.

These results could be exploited to generate new therapies that block the formation or function of the flagellum. This would be of particular interest because the flagellum is widespread amongst bacteria, and therefore such therapy could target infections from a wide range of bacteria. In addition, a molecular map of the basal body will provide clues of how bacteria were able to "evolve" structures such as the flagellum, by comparison to other known nano-machines. Finally, understanding how the flagellum forms could be used to develop motile drug delivery systems, which could be exploited for a wide range of medication such as anticancer treatment or gene therapies.

The flagellum is a macromolecular nano-machine that plays essential roles in bacterial cell organization and motility. It is also a virulence factor in many bacterial pathogens, responsible for dissemination and adherence to surfaces. However, its size, complexity, dynamics, and membrane embedding renders is very challenging for structural characterization. In particular, our current understanding of the membrane-embedded basal body region is limited to low-resolution data, despite its central role on flagellum assembly and function.

The flagellum basal body is primarily composed of the inner-membrane protein FliF and the outer membrane-associated proteins FlgH and FlgI. We propose to characterize its architecture and assembly, using a hybrid structural approach, combining near-atomic resolution structures of assembled complexes by cryo-EM, with atomic structures and interaction studies of isolated domains. To that end, we will purify these proteins in isolation, and obtain their atomic structures by X-ray crystallography, NMR or single-particle cryo-EM. We will employ a range of biochemical and biophysical techniques to characterize their oligomerization and/or interaction in vitro. Finally, we will use cryo-EM to obtain structural information on the intact flagellum basal body. These data will allow us to obtain an atomic model of the flagellar basal body, and propose a molecular mechanism for its assembly.

This hybrid approach will allow us to understand the molecular details of basal body architecture and assembly, and to formulate comparisons with the related type III secretion system basal body. Ultimately, we plan to exploit these results to develop inhibitors of flagellum assembly, which could be used as antibiotics in a clinical setting. The proposed research could also be exploited in order to design flagellum-propelled drug delivery systems.

This proposal focuses on the characterization of the bacterial flagellum, a major component of the bacterial cell architecture and a potential target for new antibacterial therapeutics. This project will provide a number of benefits to the pharmaceutical industry and the general public in the UK and abroad:

Industry:
As a potential antibiotic target and antigenic determinants, structural characterization of the flagellum could be exploited for the design of novel therapeutics. In addition, understanding the flagellum at the molecular level will facilitate its exploitation for the development of new drug delivery systems. Through collaborations with the Sheffield Science Gateway, we will develop collaborations with pharmaceutical companies that could lead to new antibiotics and drug delivery systems.

National Health Services:
Antibiotic resistance it a global health challenge, and a major priority for the NIH. It also poses a significant economic burden on health services. In particular, multi-drug resistant Pseudomonas infections in hospital environments are increasing, and the flagellum is directly involved in these infections. By providing potential new avenues for the development of antibacterial drugs, this project will contribute to helping the NHS in its mission to improve the health and wellbeing of patients and communities.

Sheffield and the North Yorkshire:
One of the major cause for antibiotic resistance is the over-consumption of antibiotics. Raising awareness of this problem among the general public is essential in the fight against excess antibiotics prescription. The outreach activities associated with this project will contribute to raise awareness of this important issue in the communities at Sheffield and the region.

Students and staff at the University of Sheffield:
The flagellum is a visually impressive nano-machine, and will enhance the teaching experience for undergraduate students. In addition, a number of undergraduate and postgraduate students will benefit from being involved with the project, and acquire cutting-edge structural biology techniques. Finally, the PDRA associated with the project will receive many opportunities for scientific training, and well as for acquiring transferable skills.