Award details

Structural transitions and cellular remodelling in spore germination

Reference	BB/W015072/1
Principal Investigator / Supervisor	Professor Per Bullough
Co-Investigators / Co-Supervisors	Dr Robert Fagan, Professor Jamie Hobbs
Institution	University of Sheffield
Department	School of Biosciences
Funding type	Research
Value (£)	887,142
Status	Current
Type	Research Grant
Start date	23/12/2022
End date	22/12/2026
Duration	48 months

Abstract

Firmicutes form long-lived, resistant spores. In response to germinants, they metamorphose into active vegetative cells. The demands of resistance/dormancy versus rapid germination must be reconciled, but how? The radical structural changes taking place in germination will be our focus, specifically the spore-cellular envelope transition. These proteinaceous envelopes are resistant assemblies, so how are they breached to allow the cell to emerge? If we can explain in structural terms how the spore-vegetative cell transition takes place we can exploit vulnerabilities. We will use CryoEM, AFM, fluorescence microscopy & molecular biology to understand how this remarkable metamorphosis takes place. We will test our ability to perform structural work on an anaerobe- C. sporogenes. We will develop a workflow of analysis from germination to the final mature vegetative state. We will then apply our gained expertise to C. difficile. (1) We will determine 'baseline' structures from which we can follow changes & test hypotheses. We will reveal for the first time the 3D structure of a Clostridial spore in molecular detail. (2) We will follow germinating spores in a time-resolved manner initially as snapshots by CryoEM but then dynamically by AFM. We will establish early surface changes that occur in germination/outgrowth. (3) We will map envelope domains involved in cell exit, by CryoEM, AFM, fluorescence microscopy and mutagenesis. AFM will map mechanical properties. We will generate the first 3D picture simultaneously revealing the architectural, mechanical & chemical properties of the spore envelope. (4) We will test whether the envelope breach is enzymatic or mechanical. We will knock out structural proteins & proteases. We will complement these studies by time-resolved AFM. (5) By fluorescence microscopy, CryoEM and AFM we will visualise the timing & localisation of appearance of new protein subunits on the emerging vegetative cell surface.

Summary

Bacteria such as those causing botulism and anthrax survive harsh conditions and spread disease as spores. However for these spores to cause disease symptoms they must germinate into so-called vegetative cells. Toxins produced by the vegetative cells are most often the main factor in making infected animal or human hosts sick. We wish to understand how this germination takes place and how the active vegetative cell emerges from the dormant spore. This is a remarkable metamorphosis where one intricate type of cell structure is completely transformed into a radically different structure- it is analogous to the metamorphosis of a seed into a seedling and arguably as complex! The process is also interesting because vegetative cells are much more vulnerable to attack, for example by antibiotics or disinfectants, than spores are. Thus if we could work out how to 'germinate to exterminate', we could develop new weapons against a number of diseases and food-spoilage organisms. We will study germination in two selected organisms- Clostridium sporogenes (C. sporogenes) and Clostridioides difficile (C. difficile). C. sporogenes is non-pathogenic but very closely related to C. botulinum which causes botulism, a potentially lethal paralysis. Spores are extremely heat resistant so eradication from food requires costly heat treatment that lowers nutritional value & makes the product less appealing, yet there is increasing demand for minimally processed, safe foods. C. botulinum is also listed as a potential bio-weapon (UK Anti-terrorism, Crime & Security Act 2001). The advantage of using the harmless C. sporogenes is that we can learn a lot about C. botulinum without the need for highly specialised containment facilities. C. difficile is the leading cause of antibiotic-associated diarrhoea, especially in hospital patients and the elderly. Disease occurs when ingested spores germinate in response to bile acids in the gut. The toxins are the major cause of disease & in many cases,death. Ungerminated spores remaining in the gut can be the source of recurrent infections even after treatment. Our multidisciplinary project will apply state of the art techniques of structural and molecular biology to investigate the way these spores germinate. A crucial aspect of this work will involve mapping the proteins that make up spores in 3D molecular detail. To achieve this, we will exploit exciting new developments in microscopy - such as cryo-electron tomography - that allow us to visualise spores and cells in unprecedented detail. We will create 3D images of the spore where we can even identify and locate individual molecules. We will then go on to visualise the structures of spores as they transform into active cells, as might happen when contaminated food is ingested. The project will exploit new developments in imaging methods that allow us to see the structures more clearly. It will involve combinations of molecular genetics, use and development of electron light and scanning probe imaging approaches and computational image processing. Ultimately we aim to make a 3D movie that explains the mechanical and biochemical changes that take place when the active vegetative cell emerges out of the spore.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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