Identification of Drosophila Yin and PEPT2 as Evolutionarily Conserved Phagosome-associated Muramyl Dipeptide Transporters

Bobby J. Cherayil,Anna Sokolovska,Neal Silverman,Laurent Boyer,Koichi S. Kobayashi,WK Eddie Ip,Michael P. Cappillino,Daniel K. Podolsky,Stéphanie Dejardin,Lynda M. Stuart,Adam Lacy-Hulbert,Guillaume M. Charrière

doi:10.1074/jbc.m110.115584

Abstract

NOD2 (nucleotide-binding oligomerization domain containing 2) is an important cytosolic pattern recognition receptor that activates NF-kappaB and other immune effector pathways such as autophagy and antigen presentation. Despite its intracellular localization, NOD2 participates in sensing of extracellular microbes such as Staphylococcus aureus. NOD2 ligands similar to the minimal synthetic ligand muramyl dipeptide (MDP) are generated by internalization and processing of bacteria in hydrolytic phagolysosomes. However, how these derived ligands exit this organelle and access the cytosol to activate NOD2 is poorly understood. Here, we address how phagosome-derived NOD2 ligands access the cytosol in human phagocytes. Drawing on data from Drosophila phagosomes, we identify an evolutionarily conserved role of SLC15A transporters, Drosophila Yin and PEPT2, as MDP transporters in fly and human phagocytes, respectively. We show that PEPT2 is highly expressed by human myeloid cells. Ectopic expression of both Yin and PEPT2 increases the sensitivity of NOD2-dependent NF-kappaB activation. Additionally, we show that PEPT2 associates with phagosome membranes. Together, these data identify Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated transporters that are likely to be of particular importance in delivery of bacteria-derived ligands generated in phagosomes to cytosolic sensors recruited to the vicinity of these organelles.

Highlights

The components of the innate immune system, including cellular defenses and inflammatory pathways, mount a rapid and tightly orchestrated response to pathogen invasion [1]
2 The abbreviations used are: Toll-like receptors (TLRs), Toll-like receptor; NLR, NOD-like receptor; MDP, muramyl dipeptide; IL, interleukin; PBS, phosphate-buffered saline; transmembrane proteins found on the cell surface and in endosomal compartments [3], and the NOD-like receptors (NLRs), which reside in the cytosol and recognize bacteria, or their derivatives, that cross the plasma membrane and enter the cell [4, 5]
Response to S. aureus Is Mediated by the Collaboration between TLR2/6 and NOD2 from the Phagosome—NF-␬B driven responses to S. aureus are mediated primarily by membrane-bound TLRs that respond to immunostimulatory components of the Gram-positive bacterial cell wall that include lipoteichoic acid, peptidoglycan, and lipopeptides [24]

Summary

To whom correspondence should be addressed

Despite the TLRs and NLRs surveying two discrete cellular compartments (the extracellular space and the cytosol), these receptors collaborate for optimal host response to many pathogens. Previous work has demonstrated that maximal activation of TLR and NOD2-dependent signaling in response to Gram-positive microbes is possible only after delivery into a phagosome and that this event is intimately associated with inflammatory cytokine induction [6, 7]. These observations reflect the multifunctional role of the phagosome during innate immune sensing of Gram-positive microbes. We show that these transporters associate with phagosome membranes and are likely to be of particular importance in delivery of ligands generated in this compartment to NOD2 recruited to the vicinity of these organelles

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION