SPORE NANOFIBER: Exploring novel extremely heat and chemically resilient nanofibers expressed on bacterial spores

Spores are bacteria's response to hostile environments, functioning as small survival capsules capable of enduring for years under extreme conditions such as heat, cold, drought, and even disinfectants. The ability of spores from the Bacillus cereus (B. cereus) group to survive food processing poses a significant challenge to the food industry and presents risks in medical and defense-related contexts. These are the spores we are studying in the SPORE NANOFIBER project. Specifically, we are focusing on the fibers that cover the surface of the spores, called ENA fibers (Endospore Appendages). ENA fibers are microscopic structures—so small they are invisible to the naked eye, with a width measured in nanometers and a length in micrometers. Despite their modest size, they have a significant impact on the spores' ability to survive and adapt to their environment. Previously, only one type of ENA fiber was known, called S-ENA, which is present in all species within the B. cereus group. In 2023, we began a systematic investigation to understand these fibers in more detail. Using cryo-electron microscopy—a technique that freezes the fibers in their natural state before analysis—we were able to reveal new structural details of the fibers. We also discovered an entirely new type of ENA fiber, which we named L-ENA (Ladder-like ENA) due to their unique ladder-like structure. L-ENA fibers not only look different from S-ENA, but our findings suggest they also play a critical role in spore aggregation, the process by which spores cluster into tight groups. This is particularly relevant when studying B. cereus spores isolated from sick individuals, where L-ENA fibers are more prominent. We believe these fibers may be crucial in infection processes, possibly explaining why some variants of B. cereus are more aggressive than others. To understand how ENA fibers function, we have employed a range of advanced techniques. Optical tweezers allow us to manipulate individual fibers and observe their behavior, while genetic engineering enables us to create mutant spores where we have removed or altered certain proteins linked to the ENA fibers. By combining these techniques, we have made some exciting discoveries. It turns out that L-ENA fibers have a small tip fibril, called L-BclA, that is essential for their aggregation function. When we removed L-BclA from the spores, the L-ENA fibers lost their ability to connect the spores. Even more surprisingly, L-BclA is also present on S-ENA fibers, suggesting that these fibers share functions. We have also investigated how ENA fibers affect the spores’ ability to adhere to various surfaces. In the food industry, this is particularly relevant, as spores can stick to production equipment and survive cleaning processes, increasing the risk of contamination. We found that ENA fibers are especially effective at helping spores adhere to materials like polypropylene and stainless steel—common materials in production environments—while they are less able to stick to surfaces like polystyrene and glass. This provides valuable insights that can be used to improve hygiene protocols in food production facilities. By understanding which materials spores more easily adhere to and how to prevent them from sticking, the industry can make better choices to reduce the risk of B. cereus contamination, which is crucial for ensuring food safety and improving product quality. This project has already given us valuable insights, but we are still at the beginning of understanding the full scope of what ENA fibers can do. Our findings have been presented at national and international conferences and have already been published in two peer-reviewed articles in respected scientific journals. We also have several exciting new discoveries. For instance, we are now working to understand the role of ENA fibers in biofilm formation—a phenomenon where bacteria form protective layers on surfaces, increasing their resistance to disinfection. We have also identified a third type of fiber, which we expect to characterize and publish findings on in 2025. These studies will contribute to our understanding of how to combat B. cereus in industry while also opening new possibilities for controlling bacterial infections in medical contexts. ENA fibers may even inspire the development of new resistant materials. Our research on ENA fibers provides a unique insight into B. cereus' ability to survive, adapt, and spread in various environments. This is not just an academic interest—it has practical implications for how we can improve food safety, protect against bacterial infections, and possibly even develop new methods to prevent contamination across multiple industries in the future. We are still in the early stages of exploration, but the results we have achieved so far show that we are on the right track.

Bacillus cereus sensu lato (s.l.) is a large group of bacteria whose endospores are of food safety, industrial, medical and biodefense importance. Their endospores are decorated with multiple micrometres-long, a few nanometres wide fibers (endospore appendages (Enas)). The team behind this proposal has recently forced a major breakthrough by identifying the protein subunits that build the Enas and the genes encoding them. Notably, they represent a completely novel type of proteinaceous nanofibers, with unique structural properties and self-assembly mechanisms, that have never been described before. They are the third type of pili ever described in Gram-positive species and the first spore pili that have ever been structurally and genetically characterized. We have so far identified two major structural types of Enas that are widely distributed among species belonging to the large B. cereus group of bacteria. The SPORE NANOFIBER will use state-of the art cryo-EM, lazer-tweezer technology, 3D-modelling, gene-knockouts, recombinant genes, various functional analyses and an insect larvae infection model to generate knowlede on Enas composition, 3D structure, assembly mechanisms and biophysiochemical and mechanical properties. We will examine the role of Enas in various spore-related functions such as colonization of abiotic surfaces and biofilm formation as well as in spore binding to intestinal mucosal surfaces of humans, animals and insect larvae. The Enas are also of high interest for bio-nanotechnology as they are highly flexible, exhibit an extreme heat-, chemical- and enzymatic resistance, and can be produced in large quantities in vitro. This project has a great potential to generate knowledge that can be used to invent more efficient strategies to prevent or reduce spore attachment-related problems in the food industry and in medicine. Altogether, the proposed project fits well with the call for proposal for “Project for Scientific Renewal”.