CryoEM structure of a novel class of spore virulence factors on the foodborne outbreak strain Bacillus paranthracis NVH 0075-95

Bacterial endospores are remarkable examples of biological resilience, representing a dormant and heavily fortified differentiation form capable of withstanding physical and chemical stressors detrimental to vegetative cells. In pathogenic Bacillota, spores also form an infectious particle and can take up a central role in the environmental persistence and dissemination of disease. A poorly understood aspect of spore-mediated infection is the fibrous structures or ‘endospore appendages’ (ENAs) that have been seen to decorate the spores of pathogenic Bacilli and Clostridia. New methodological approaches are opening an unprecedented window on these long enigmatic structures. Using cryoID, Alphafold modelling and genetic approaches we identify a novel class of ultra-robust ENAs in the foodborne outbreak strain Bacillus paranthracis NVH 0075-95. We demonstrate that L-ENA are encoded by a rare three-gene cluster ( ena3 ) that contains all components for the self-assembly of ladder-like protein nanofibers of stacked heptameric rings, their anchoring to the exosporium, and their termination in a trimeric ‘ruffle’ made of a complement C1Q-like BclA paralogue. The role of ENA fibers in spore-spore interaction and the distribution of L-ENA operon as mobile genetic elements in predominantly pathogenic B. cereus s.l. strains, suggest that L-ENA fibers promote strain virulence by spore clustering and aggregation.