More surprising was the finding that deletions in genes putatively coding for (co-)chaperones

lead to an enhanced survival in human serum. One of those, namely 4_G12 (ΔdjlA), is a member of the J-domain protein family. DjlA can substitute for DnaJ co-chaperone [22] and seems to have multiple functions. However, it has also been described that DjlA negatively Copanlisib regulates the response of the two component RcsCDB signaling system to envelope stress. The Rcs signal transduction system positively regulates the expression of many different genes among those are the ones forming the capsular polysaccharide synthesis operon (cps)[23]. The expression of capsules may provide protection from serum killing components (see above). In a study by Shiba et al. [24] it was demonstrated that djlA deletion resulted in increased activation of the Rcs system. This might positively regulate cps transcription. Mutant 21_G1 (ΔybaJ) exhibiting an enhanced serum tolerance was shown to be affected in a gene coding for the YbaJ protein. It has been proposed that YbaJ EPZ5676 molecular weight and its adjacent protein Hha may form a so called toxin-antitoxin pair where YbaJ (antitoxin) negatively regulates the expression of Hha (toxin), the BIBW2992 latter one (among other functions) serving as a repressor for type 1 fimbriae [25]. Type 1 fimbriae are highly immunogenic,

Thymidine kinase thus a strain not expressing these structures may have an advantage in survival during exposure in human serum [26]. In the present study we further examined the hypothesis that the disruption of the regulatory gene ybaJ may lead to an activation of the Hha protein which in turn would negatively influence transcription of the key fimbrial structural gene fimA. RT-qPCR experiments were performed in order to quantify hha and fimA mRNA levels in the C. sakazakii ES5 wt and mutant 21_G1(ΔybaJ) strains, before and after exposure to human serum. The levels of fimA mRNA were more

than 4.5 log lower in the mutant 21_G1(ΔybaJ) strain compared to the C. sakazakii ES5 wt strain. The hha mRNA levels were for the mutant compared to the wt 5 log lower and not like expected higher, suggesting that the deletion of the ybaJ gene did not result directly in a de-repression/ activation of the hha gene in our experimental set up (Figure 3). Our results rather suggest that ybaJ itself may be involved in the regulation/activation of the expression of the type 1 fimbriae in C. sakazakii. Figure 3 Relative levels of hha and fimA mRNA in control (T 0 ) and serum treated (T 120 ) C. sakazakii ES5 wt and mutant 21_G1 (Δ ybaJ ) cells. RNA was isolated from mid exponential growth stage cells prior (T0) and after (T120) human serum exposure. Values were normalized using 16S rRNA as a reference gene.