Nod1 and Nod2 are members of the Nod-like receptor family that detect
intracellular bacterial peptidoglycan-derived muramyl
peptides. The biological effects of muramyl
peptides have been described for over three decades, but the mechanism underlying their internalization to the
cytosol remains unclear. Using the human
epithelial cell line HEK293T as a
model system, we demonstrate here that Nod1-activating ligands entered cells through
endocytosis, most likely by the clathrin-coated pit pathway, as internalization was dynamin-dependent but not inhibited by methyl-beta-cyclodextrin. In the endocytic pathway, the
cytosolic internalization of Nod1 ligands was pH-dependent, occurred
prior to the acidification mediated by the
vacuolar ATPase, and was optimal at pH ranging from 5.5 to 6. Similarly, the Nod2 ligand MDP was internalized into host
cytosol through a similar pathway with optimal pH for internalization ranging from 5.5 to 6.5. Moreover, Nod1-activating muramyl
peptides likely required processing by endosomal
enzymes,
prior to transport into the
cytosol, suggesting the existence of a
sterically gated endosomal transporter for Nod1 ligands. In support for this, we identified a role for SLC15A4, an
oligopeptide transporter expressed in early
endosomes, in Nod1-dependent
NF-kappaB signaling. Interestingly, SLC15A4 expression was also up-regulated in
colonic biopsies from patients with
inflammatory bowel disease, a disorder associated with
mutations in Nod1 and Nod2. Together, our results shed light on the mechanisms by which muramyl
peptides get access to the host
cytosol, where they are detected by Nod1 and Nod2, and might have implications for the understanding of human diseases, such as
inflammatory bowel disease.
