D2.3 Report on correlation between RNAseq data analysis and toxicity assays

Although the main causes of pathogenicity for toxin-producing bacteria such as Bacillus cereus and Clostridium perfringens are well known today, the mechanisms responsible for the expression of this virulence need, however, more investigations.
The emergence of B. cereus as a foodborne pathogen and as an opportunistic pathogen has intensified the need to distinguish strains of public health concern. The pathogenic potential of B. cereus is extremely variable, with some strains being harmless and others lethal. B. cereus possesses several toxin genes located on the chromosome, such as nhe, hbl and cytK. These toxins provide an indication of the strain toxicity potential but are not sufficient, alone, to discriminate hazardous from harmless strains. Indeed, several studies have shown that Nhe production by hazardous strains is variable and that non-pathogenic strains can also produce it in large quantities. B. cereus produces other toxins such as haemolysin II (HlyII), the metalloproteases InhA1, InhA2 and the cell wall peptidase FM (CwpFM), which may also be involved in pathogenicity. To date, the above described determinants were not sufficient to completely explain the virulence of B. cereus and there are currently no markers, either alone or in combination, to differentiate pathogenic from non-pathogenic strains.
For C. perfringens, even if 17 different toxins have been described, this bacterial pathogen is currently classified in five toxinic types (A to E) only based on the production of 4 major toxins (alpha, beta, epsilon and iota). The C. perfringens type A that produces CPE toxin is a major cause of food-borne gastroenteritis (FBGE) whereas the role of other virulence factors remains poorly investigated.
Several data also indicate that, for these two pathogens, the level of toxin production could play a major role in determining the pathogenic potential of a particular strain, while the presence of specific toxin genes appears to be less discriminating. In addition, although cytotoxicity assays have already been used for classifying bacterial strains according to their toxic potency, further investigation on the toxicity mechanisms involved has been rarely carried out.
The objective of this task was to analyse the RNAseq, PCR and cytotoxic data and study potential correlations between gene presence or expression with strain toxicity and virulence.
The growth conditions of the strains, the cytotoxicity assays, the RNA extraction procedures as well as the development of RNAseq assays were defined and optimized. The data were analysed to allow gene expression and strain toxicity comparaison.