Sec pathway-dependent bacterial signal peptides are a major source for formyl peptide receptor 1 activation in mice and humans

Recruitment of immune cells to the site of infection is a major component of the innate immune response. Formyl peptide receptors (FPRs) promote this process by recognizing N-terminally formylated peptides that act as chemotactic stimuli on neutrophils. Formyl peptides are very characteristic for bacterial infections, because N-terminally formylated methionine residues are required in bacterial protein biosynthesis to initiate translation, whereas the majority of eukaryotic peptides are, with the exception of mitochondrial proteins, unformylated. Recent work suggests that bacterial signal peptides, which serve as signal sequences to translocate proteins across cellular membranes, may be a major source of formyl peptides.

Here we report that batch-cultured Escherichia coli BL21 (DE3) cells secrete ligands into the growth medium, which activate the FPR1 homologs of humans (hFPR1) and mice (mFpr1) in HEK293T transfected reporter systems, respectively. Notably, no relevant activation of these two FPRs was observed when the FPR1-expressing reporter cells were challenged with E. coli cell lysates. Activation of the FPR1 homologs by cell culture supernatants was growth phase-dependent and strongest with supernatants harvested from exponential growth phase E. coli cultures. Size exclusion filtration and protease treatment of the E. coli culture supernatants revealed that the FPR1-activating ligands are of proteinaceous nature and smaller than 3 kDa in size. Treatment of the E. coli cultures with the general secretory (Sec) pathway inhibitor erythrosine B almost completely abolished the secretion of the FPR1-activating ligands into the extracellular milieu, while the Sec ATPase inhibitor only marginally affected the viability of E. coli and HEK293T cell cultures, respectively.

Our results indicate that the release of formyl peptides from E. coli occurs post-translationally through the Sec pathway, supporting the hypothesis that signal peptides are a major source of bacterial N-formylated peptides.