Bacterial Peptide Recognition and Immune Activation Facilitated by Human Peptide Transporter PEPT2

Abstract

Microbial detection requires the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) that are distributed on the cell surface and within the cytosol. The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family functions as an intracellular PRR that triggers the innate immune response. The mechanism by which PAMPs enter the cytosol to interact with NLRs, particularly muropeptides derived from the bacterial proteoglycan cell wall, is poorly understood. PEPT2 is a proton-dependent transporter that mediates the active translocation of di-and tripeptides across epithelial tissues, including the lung. Using computational tools, we initially established that bacterial dipeptides, particularly g-D-glutamyl-meso-diaminopimelic acid (g-iE-DAP), are suitable substrates for PEPT2. We then determined in primary cultures of human upper airway epithelia and transiently transfected CHO-PEPT2 cell lines that g-iE-DAP uptake was mediated by PEPT2 with an affinity constant of approximately 193 mM, whereas muramyl dipeptide was not transported. Exposure to g-iE-DAP at the apical surface of differentiated, polarized cultures resulted in activation of the innate immune response in an NOD1-and RIP2-dependent manner, resulting in release of IL-6 and IL-8. Based on these findings we report that PEPT2 plays a vital role in microbial recognition by NLR proteins, particularly with regard to airborne pathogens, thereby participating in host defense in the lung.Keywords: human; lung; bacterial; cell surface molecules; acute phase reactants Pathogen-associated molecular patterns (PAMPs) are recognized by cell surface or cytosolic pattern recognition receptors (PRRs) (1). One family of intracellular PRRs include the nucleotide-binding oligomerization domain (NOD)-containing proteins (NODs or NOD-like receptors [NLRs]) that sense intracellular pathogen invasion and trigger signaling cascades, thereby activating the host immune response (2-4). NOD1 and NOD2 mediate intracellular recognition of bacterial proteoglycan (PGN)-derived molecules through recognition by their carboxy-terminal leucine-rich repeat region (5). This results in recruitment of the downstream kinase protein RIP2 and initiation of NF-kB-mediated transcription and elaboration of newly synthesized cytokines and chemokines (6). Biochemical and functional analyses have identified g-D-glutamyl-mesodiaminopimelic acid (g-iE-DAP) and muramyl dipeptide (MDP) as the minimal bacterial muropeptide epitopes recognized by NOD1 and NOD2, respectively, resulting in immune activation (5, 7).Enzymes that are naturally present in the epithelial lining fluid (e.g., lysozyme) are believed to act in concert to degrade the bacterial cell wall as a front-line defense mechanism, thereby liberating muropeptide fragments that include both g-iE-DAP and MDP. The dipeptides may also be derived as byproducts of bacterial PGN biosynthesis, cell growth, and division; therefore, it is plausible that these particular PAMPs reside in the airway...

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