Lead


Lay summary
Background. Recognition of pathogens is commonly based on identification of microbial molecular patterns, such as bacteria-derived peptidoglycans and peptides, by specific cellular receptors. Subsequent activation of signalling pathways initiate defence responses to fend off the invading microorganisms. Two mammalian nucleotide-binding oligomerization domain proteins (Nod1 and Nod2) were recently identified as intracellular sensors of bacterial products.1-3 The Nod2 gene was found to be mutated in 20-30% of Crohn’s disease patients, suggesting its involvement in pathophysiology of inflammatory bowel disease (IBD).4,5 A component of the peptidoglycan derived from bacterial cell walls, muramyl dipeptide (MDP), is known to act as a ligand for Nod2. MDP and other bacteria-derived peptides and peptidoglycans, such as muramyl tripeptide (MTP) and n-formyl peptides (such as fMLP) can induce apoptosis, regulate inflammatory responses of epithelial cells, and are involved in innate recognition ofmicroorganisms.6-12 Apart from Nod2, other genes involved in the genetic basis of IBD encode intestinal gut transporters such as the organic cation transporter 1 and 2 (OCTN1/2), the multidrug resistance gene 1 (MDR1), and the human peptide transporter (hPepT1).13-16 Recently, the intestinal di- and tripeptide transporter hPepT1 has been identified to be responsible for MDP and fMLP uptake into epithelial cells.8,17,18
Working Hypothesis. Bacteria-derived components such as MDP, MTP, and fMLP are taken up by transport proteins in epithelial cells. These bacterial products may alert the innate system in the presence of potentially harmful invaders. They activate signalling pathways in epithelial cells, thereby defending the organism against infection by pathogens.Peptidoglycans derived from enteric commensal, probiotic and pathogenic microorganisms may be important as modulators of inflammation, both in healthy subjects and in patients with inflammatory bowel disease. They may also induce expression of intestinal epithelial heat shock proteins (Hsp) and promote stability of tight junctions, thus protecting epithelial integrity in the colon.

Specific Aims.
A.Intestinal Transporters. To study mRNA and protein expression of avariety of transporters (hPepT1, OCTN1/2, MDR1, OATP-A, -B, -E, OCT1, OAT2, CNTs, ENTs, and others) potentially responsible for uptake of bacterial products in healthy intestinal tissue and in tissue of patients with inflammatory bowel disease (Crohn’s disease and ulcerative colitis) Furthermore, I aim to investigate the effect of cytokines and corticosteroids on the regulation of these transporters in cell lines that endogenously express these genes, as well as in fresh human biopsies.

B.Bacterial Peptidoglycan and Peptide Uptake. To investigate whichintestinal transporters are involved in the cellular uptake of bacterial peptidoglycans and peptides.

C.Effects of Bacteria-Drived Peptides and Peptidoglycans in theIntestine. To investigate the cytoprotective response (up-regulation of heat shock proteins, stabilization of tight junctions, and suppression of NF-B activity) in the epithelial Caco2 cells and intestinal biopsies treated with bacteria-derived peptides, such as fMLP, MDP, and MTP.

Expected Value of the Proposed Project. The proposed work should enhance our understanding of intestinal physiology in man and form a basis for further studies on the pathogenesis of inflammatory bowel disease and the removal of infectious agents by the host. Further characterization of bacteria-derived peptides is necessary to clearly define the role of these proteins in inflammation and/or infectious processes. Studies on regulation and expression of the different transporters involved in bacterial product uptake in human intestinal tissue will yield essential information about the usefulness of these carriers in potential drug-targeting strategies.