Expression of staphylococcal enterotoxins SEB and SEC under stress conditions relevant to food production

Staphylococcal food poisoning (SFP) is one of the most prevalent food intoxications. It is caused by enterotoxins of Staphylococcus aureus that are preformed in food during bacterial growth. Two staphylococcal enterotoxins (SEs) of relevance in the food con- text are enterotoxins SEB and SEC since they are highly expressed. S. aureus benefits from a growth advantage over competing microflora when hurdles such as low pH, salting, drying, or curing are applied. To date, there is little data investigating the effect of these stress conditions that are relevant to food production on SEB and SEC expres- sion. SEB and SEC have been suggested to be regulated by the accessory gene regu- lator (Agr), staphylococcal accessory regulator (SarA), and alternative sigma factor B (σB or sigB). However, results from previous studies mainly relied on unprecise quan- tification methods like western- and northern blots and used few strains with strain selection not taking different SEC variants into account. The aims of this thesis were to i) investigate the influence of food related stress conditions (lactic acid, NaCl, glucose, nitrite) on SEC expression, ii) identify regulatory elements controlling SEC expres- sion under food related stress conditions, and iii) develop a tool to monitor single cell heterogeneity of SEB producing S. aureus under stress conditions. To determine the influence of food related stress conditions on SEC expression, I developed an sec RT- qPCR and an in-house ELISA assay for SEC quantification. I could show that glucose had a higher inhibitory effect on toxin production than lactic acid and NaCl stress. Ni- trite on the other hand even led to higher toxin production in some strains. To identify relevant regulatory elements under nitrite stress agr, sigB, and sarA deletion mutants were used to monitor sec mRNA expression. ∆agr mutants generally expressed less sec than wildtype (wt) strains. However, this effect was not stress dependent. Loss of sarA did not significantly influence sec transcription. σB however, seemed to re- act specifically to nitrite stress and might outweigh the influence of agr. To monitor single cell heterogeneity of SEB, a plasmid transcriptionally fusing an seb promoter to an unstable GFP variant was generated and is currently being chromosomally inser- ted in different wild type strains. In conclusion, these findings indicate that stressors encountered in food production and preservation influence sec expression in a way that cannot be predicted based exclusively on viable cell counts. In addition, data on sec regulation suggests that σB plays a role under nitrite stress conditions. The im- portance of Agr may have been overestimated in the past as this regulator could be bypassed in our experiments. Because effects seen at mRNA level do not always carry over to the protein level it is of importance to combine transcriptional and transla- tional data. The observed effects of food related stressors are an important step in improving our understanding of SE expression under stress conditions and could in a next step be validated in the food matrix with the overarching goal of optimizing food production parameters to minimize SE production