Cellular and population strategies underpinning neurotoxin production and sporulation in Clostridium botulinum type E cultures

Abstract

Toxin production and sporulation are key determinants of pathogenesis in Clostridia. Clostridium botulinum produces the most potent toxin known, the botulinum neurotoxin (BoNT), which blocks neurotransmission and causes a life-threatening paralysis called botulism. BoNT production and sporulation share a common regulator Spo0A, which suggests coordination of the two traits. Describing the relationship between toxin production and sporulation is fundamental toward understanding the evolutionary and mechanistic logic and further control of clostridial pathogenesis. Here, we provide the first single-cell resolution analysis of BoNT production and sporulation in C. botulinum type E cultures by using a fluorescent reporter to follow the activation of the BoNT gene promoter. BoNT was expressed by a subpopulation of cells and was released through Spo0A-mediated autolysis of vegetative cells or upon release of mature spores. All possible combinations of toxin production and sporulation resided in wild-type C. botulinum type E cultures, indicating neither tight co-regulation nor strict independence of the two traits. The population structure and the degree of overall heterogeneity were affected by growth phase and environmental conditions, with cold temperature inducing large diversity and cultural stability, in line with adaptation to fluctuating temperatures that C. botulinum type E strains likely encounter in nature. We also observed Spo0A-independent BoNT production by a small cell subpopulation of the spo0A-null strain. Our observation of toxin gene activation in the forespore invites speculation on possible alternative biological roles for toxin production by vegetative and sporulating cells and reflection on the evolutionary rationale of toxin production with respect to the ecology of spore-forming pathogens.