Role of megaplasmids in pathogen emergence: Bacillus cereus

The emergence of new pathogenic bacterial strains is often a consequence of horizontal gene transfer, (HGT) which results in the adaptation of a microorganism to a new ecological niche. Two examples of such horizontal gene transfer leading to pathogenicity are found in the Bacillus cereus group, where most strains are environmental: Bacillus anthracis is a lethal human pathogen, and emetic strains of B. cereus sensu stricto are food-borne pathogens. Both pathogens have acquired megaplasmids carrying genes clusters involved in the production of capsules or toxins, and both are adapted to specific niches: the mammal host for B. anthracis and food products for emetic strains of B. cereus. We hypothesize that pCER270, the megaplasmid that encodes the biosynthesis operon ces for the production of the highly potent emetic toxin cereulide, plays a pivotal role in this adaptation. Cereulide is increasingly recognized as a serious threat implicated in severe clinical manifestations, such as acute liver failure. A panoply of phenotypic assays, already established in the partners laboratories in Austria and France, will be applied to decipher environmental conditions and to identify plasmid encoded factors, providing the emetic B. cereus strains an adaptive advantage compared to non-emetic B. cereus strains in distinct ecological niches. Since our previous data point towards a dialog between pCER270 and the chromosome, likewise to the chromosome - toxin plasmids crosstalk in B. anthracis we will use an OMIC based approach for gaining comprehensive insights into this regulatory cross talk. The results of these complementary global approaches will allow us to draw a holistic plasmid-chromosomal crosstalk landscape for emetic B. cereus. Since plasmids belong to the mobilome of bacteria, we will further evaluate the potential risk of pCER270 dissemination within the B. cereus group by HGT under natural (insect/food) environment conditions. To investigate how the transfer of a megaplasmid can turn an environmental strain into a pathogenic, we will carry out conjugation experiments using emetic and non- emetic B. cereus group strains. Thus, the results of this project are relevant to improve our understanding of the mechanisms involved in pathogens emergence. In addition, it would provide new insights into the function of megaplasmids as part of complex regulatory networks in bacteria. Furthermore, the better knowledge of B. cereus adaptation to food products will open new perspectives to set up an efficient prevention strategy for combating this important bacterial pathogen and increasing food safety.

Megaplasmids: key to the evolution of pathogens. Horizontal gene transfer (HGT) is the main driver of new pathogenic bacterial strains. These strains have improved or newly acquired adaptive traits that enable them to conquer and colonize new ecological niches. The Bacillus cereus group, present in many different ecological niches, has two examples of such gene transfer: Bacillus anthracis is a dangerous human pathogen and emetic B. cereus can cause severe food poisoning. Both pathogens possess megaplasmids that encode toxin genes, and both representatives of the B. cereus group are adapted to specific ecological niches: B. anthracis infects the human host, while emetic B. cereus is associated with certain foods. In this project, we investigated the role and significance of the pCER270 megaplasmid. This plasmid carries the genes for the production of the potent emetic toxin cereulide and is key to the specialisation of emetic strains. We used a wide range of phenotypic assays. These allowed us to determine the specific environmental conditions and identify the plasmid-encoded genetic factors responsible for the pronounced adaptability of emetic B. cereus to specific ecological niches. Our multi-omics study demonstrated an intense crosstalk between the plasmid and the chromosome of emetic B. cereus. Notably, the loss of the pCER270 plasmid has two major consequences. Firstly, it leads to a change in the pathogenicity of emetic strains. Secondly, it significantly affects the fitness and adaptability of the bacteria. Plasmids belong to the 'mobilome' of bacteria, so we investigated the risk of the virulence megaplasmid pCER270 spreading in the B. cereus group via HGT. Our conjugation experiments in natural habitats of the bacteria proved that the plasmid pCER270 can be transferred from an emetic strain to a non-emetic B. cereus group strain. These investigations reveal the first comprehensive picture of communication between a genetically mobile element, the toxin plasmid, and the chromosome in emetic B. cereus. The results of this project are crucial for understanding how new pathotypes emerge and develop. In addition, the project has demonstrated the pivotal role of megaplasmids in complex regulatory networks in bacteria. The knowledge gained about the adaptation mechanisms of emetic strains to food will pave the way for developing novel prevention strategies in the food industry, and it is expected that the project will contribute to improving food safety. Furthermore, the project's findings emphasise the necessity to investigate uncharted territories in diagnostics and risk analysis, as conventional taxonomy-based microbial diagnostics are inadequate for evaluating the potential risks posed to health and food safety by a particular bacterium.