An alternative way of life for Bacillus thuringiensis in the late stages of an infection.
Abstract
The formation of endospores has always been considered as the unique mode of survival and transmission of sporulating Firmicutes due to the exceptional resistance and persistence of this bacterial cell form. Bacillus thuringiensis (Bt) is a Gram-positive spore-forming bacterium used as a bio-control agent against insect pests. It belongs to the Bacillus cereus group that includes the foodborne pathogen B. cereus sensu stricto and the agent of anthrax, Bacillus anthracis. It was shown that Bt was able to complete a full infectious cycle in its host by sequentially activating virulence, necrotrophism (Nec) and sporulation (Spo) genes. These studies reported that the bacterial population was heterogeneous in the host cadaver and that spores constituted only 30% of the total cell load. In this study, we investigated the behavior of a bacterial population in the late stages of an infection as well as the characteristics and the importance of the Spo- bacterial form in the Bt/Galleria mellonella infection model. Using fluorescent reporters coupled to flow cytometry as well as molecular markers, we demonstrated that the Spo- cells compose the majority of the population two weeks post-infection (pi) and that these bacteria present vitality signs. However, a protein synthesis and a growth recovery assay indicated that they are in a metabolically slowed-down state. Interestingly, they were found to be extremely resistant to the cadaver environment which proved deadly for in-vitro grown vegetative cells and, more strikingly, for spores. We were able to perform a transcriptomic analysis of this subpopulation at 7 days pi that revealed a signature profile of this state. Analysis of the expression profile of individual genes at the cell level suggested that iron homeostasis is important at all stages of the infection, whereas the oxidative stress response seems of particular importance as the survival time increases. Altogether, these data indicate that non-sporulated bacteria engage in a profound adaptation process that leads to their persistence in the host cadaver.