Stress responses of Bacillus subtilis to membrane-active cationic antimicrobial peptides were studied. Global analysis of gene expression by DNA macroarray showed that peptides at a subinhibitory concentration activated numerous genes. A prominent pattern was the activation of two extracytoplasmic function sigma factor regulons, SigW and SigM. Two natural antimicrobial peptides, LL-37 and PG-1, were weak activators of SigW regulon genes, whereas their synthetic analogue poly-L-lysine was clearly a stronger activator of SigW. It was demonstrated for the first time that LL-37 is a strong and specific activator of the YxdJK two-component systems, one of the three highly homologous two-component systems sensing antimicrobial compounds. YxdJK regulates the expression of the YxdLM ABC transporter. The LiaRS (YvqCE) TCS was also strongly activated by LL-37, but its activation is not LL-37 specific, as was demonstrated by its activation with PG-1 and Triton X-100. Other strongly LL-37-induced genes included yrhH and yhcGHI. Taken together, the responses to cationic antimicrobial peptides revealed highly complex regulatory patterns and induction of several signal transduction pathways. The results suggest significant overlap between different stress regulons and interdependence of signal transduction pathways mediating stress responses.

Interactions between selectins and their oligosaccharide-decorated counter-receptors play an important role in the initiation of leukocyte extravasation in inflammation. L-selectin ligands are O-glycosylated with sulphated sialyl Lewis X epitopes (sulpho-sLex). Synthetic sLex oligosaccharides have been shown to inhibit adhesion of lymphocytes to endothelium at sites of inflammation. Thus, they could be used to prevent undesirable inflammatory reactions such as rejection of organ transplants. In vitro synthesis of sLex glycans is dependent on the availability of recombinant glycosyltransferases. Here we expressed the catalytic domain of human alpha-1,3-fucosyltransferase VII in the yeasts Saccharomyces cerevisiae and Pichia pastoris. To promote proper folding and secretion competence of this catalytic domain in yeast, it was fused to the Hsp150 delta carrier, which is an N-terminal fragment of a secretory glycoprotein of S. cerevisiae. In both yeasts, the catalytic domain acquired an active conformation and the fusion protein was externalised, but remained mostly attached to the cell wall in a non-covalent fashion. Incubation of intact S. cerevisiae or P. pastoris cells with GDP-[14C]fucose and sialyl-alpha-2,3-N-acetyllactosamine resulted in synthesis of radioactive sLex, which diffused to the medium. Finally, we constructed an S. cerevisiae strain co-expressing the catalytic domains of alpha-2,3-sialyltransferase and alpha-1,3-fucosyltransferase VII, which were targeted to the cell wall. When these cells were provided with N-acetyllactosamine, CMP-sialic acid and GDP-[14C]fucose, radioactive sLex was produced to the medium. These data imply that yeast cells can provide a self-perpetuating source of fucosyltransferase activity immobilized in the cell wall, useful for the in vitro synthesis of sLex.