Identification of Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated muramyl dipeptide transporters

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.

FIGURE 1.

NOD2 participates in response to S. aureus from the phagosome. a, cytokine production in macrophages from Nod2^−/− mice stimulated with S. aureus before and after IFNγ priming. Graphs are representative of at least three independent experiments; mean ± S.D. are represented. *, p ≤ 0.05 and **, p ≤ 0.01 relative to wild-type. b, NOD2 was expressed in HEK293T cells along with TL2/TLR6 and an NF-κB reporter construct. NF-κB response to S. aureus was measured by luciferase indicate that NOD2 functions primarily by cooperation with TLR2/6. Graph are representative of at least three independent experiments; mean ± S.D. are represented. ***, p ≤ 0.001 relative to TLR2/6 + S. aureus. c, NOD2-GFP was ectopically expressed in HEK293T cells. Phagosomes were generated in those cells by challenged with latex beads. Scale bars correspond to 5 μm in the main picture and to 1 μm in magnified box. d, endogenous NOD2 surrounds phagosomes in THP1 cells. The asterisk denotes a 2 μm latex bead. Scale bar corresponds to 5 μm. e, the relationship of phagosome number to cytokine production in macrophages from Nod2^−/− mice stimulated with S. aureus after IFNγ priming demonstrates the contribution of NOD2 to both TNF-α and IL-6 production, representative of at least three independent experiments. RLU, relative light units; WT, wild-type; MFI, mean fluorescent intensity.

FIGURE 2.

Identification of Yin, a phagosome-associated MDP Drosophila transporter. a, the table summarizes all of the proteins identified form our previous proteomic analysis of the Drosophila phagosome (23) that are transporter candidates. Names and predicted molecular function were extracted from the Flybase database. b, NOD2 and an NF-κB -reporter construct were expressed in HEK293T cells with and without Drosophila Yin, a potential MDP transporter. Sensitivity to MDP was determined by luciferase activity. Graph are representative of at least three independent experiments. Mean ± S.D. are represented. **, p ≤ 0.01 relative to NOD2 + MDP. RLU, relative light units.

FIGURE 3.

PEPT2 is expressed by human phagocytic cells. a, ClustalW analysis of Drosophila Yin and the related human SLC15 oligopepetide transporters, PEPT1, PEPT2, SLC15A3, and SLC15A4. Yin shares 33–35% with identity PEPT1/2 but only 17% with SLC15A3/A4. PEPT1 and PEPT2 are 50% identical. Expression of the SLC15 family members in kidneys, THP1 (human monocyte/macrophage cell line), peripheral blood monocytes, Caco2, (colonic epithelial cell line), and HEK293T (human kidney epithelial cell line) was determined by 40 cycle RT-PCR (b) and quantitative RT-PCR (c). PEPT2, but not PEPT1, is expressed by human phagocytes, which also express SLC15A3 and SLC15A4. RLU, relative light units.

FIGURE 4.

PEPT2 is an MDP transporter. a, NOD2 and an NF-κB reporter construct were expressed in HEK293T cells with and without PEPT2, a potential phagosome-associated MDP transporter. Sensitivity to MDP was determined by luciferase activity. PEPT1, a known MDP transporter, was expressed as a positive control. b, NOD2 and an NF-κB reporter construct were expressed in HEK293T cells with and without PEPT2 or SLC15A3. Graphs are representative of at least three independent experiments. Mean ± S.D. are represented. ***, p ≤ 0.001 and *, p ≤ 0.05 relative to NOD2 + MDP. RLU, relative light units.

FIGURE 5.

Vacuolar acidification is required for response to MDP. a and b, the role of vacuolar pH in PEPT2-dependent augmentation of MDP sensitivity was determined by blocking acidification of intracellular compartments using NH₄Cl (a) or 50 μm Bafilomycin A (BafA) (b). Graphs are representative of at least three independent experiments. Mean ± S.D. are represented, **, p ≤ 0.01 relative to NOD2/PEPT2 + MDP. RLU, relative light units.

FIGURE 6.

PEPT2 localize to phagosomes. a, GFP-PEPT2 was ectopically expressed in HEK293T cells that were then allowed to internalize latex beads to generate phagosomes. PEPT2 shows strong membrane association and localization to phagosomes. Latex bead, 0.8 μm; scale bar corresponds to 5 μm. b, localization of endogenous PEPT2 in THP1 cells. PEPT2 localization was determined in resting THP1 cells (left) and during phagocytosis of latex beads (middle and right) by confocal microscopy. Left, PEPT2 localizes to the plasma membrane and intracellular compartments in resting cells. Middle, PEPT2 is enriched in the phagocytic cup. Right, PEPT2 associates with phagosomes in THP1s. An asterisk denotes 2 μm latex beads. Scale bar corresponds to 5 μm.