Cellular and population strategies underpinning neurotoxin production and sporulation in
Clostridium botulinum
type E cultures
Authors: Mertaoja Anna , Mascher Gerald , Nowakowska Maria B. , Korkeala Hannu , Henriques Adriano O. , Lindstrom Miia 
Published: November 2023

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.

IMPORTANCE

Toxin production and sporulation are key determinants of pathogenesis in
Clostridia
. Toxins cause the clinical manifestation of clostridial diseases, including diarrhea and colitis, tissue damage, and systemic effects on the nervous system. Spores ensure long-term survival and persistence in the environment, act as infectious agents, and initiate the host tissue colonization leading to infection. Understanding the interplay between toxin production and sporulation and their coordination in bacterial cells and cultures provides novel intervention points for controlling the public health and food safety risks caused by clostridial diseases. We demonstrate environmentally driven cellular heterogeneity in botulinum neurotoxin and spore production in
Clostridium botulinum
type E populations and discuss the biological rationale of toxin and spore production in the pathogenicity and ecology of
C. botulinum
. The results invite to reassess the epidemiology of botulism and may have important implications in the risk assessment and risk management strategies in food processing and human and animal health.