Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain

Abstract

Phagocyte recognition and clearance of bacteria play essential roles in the host response to infection. In an on-going forward genetic screen, we identify the Drosophila melanogaster scavenger receptor Croquemort as a receptor for Staphylococcus aureus, implicating for the first time the CD36 family as phagocytic receptors for bacteria. In transfection assays, the mammalian Croquemort paralogue CD36 confers binding and internalization of Gram-positive and, to a lesser extent, Gram-negative bacteria. By mutational analysis, we show that internalization of S. aureus and its component lipoteichoic acid requires the COOH-terminal cytoplasmic portion of CD36, specifically Y463 and C464, which activates Toll-like receptor (TLR) 2/6 signaling. Macrophages lacking CD36 demonstrate reduced internalization of S. aureus and its component lipoteichoic acid, accompanied by a marked defect in tumor necrosis factor-alpha and IL-12 production. As a result, Cd36-/- mice fail to efficiently clear S. aureus in vivo resulting in profound bacteraemia. Thus, response to S. aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain, which initiates TLR2/6 signaling.

Figure 1.

The Drosophila protein Croquemort and its mammalian paralogue CD36 are receptors for S. aureus. (a) Croquemort and the small GTPase Rac2 are specifically required for S. aureus binding and uptake. RNAi silencing of Croquemort (Crq) and Rac2 in Drosophila S2 cells reduced phagocytosis of S. aureus (left), but not E. coli (right). *, P ≤ 0.05; **, P ≤ 0.001; significantly different from control. (b and c) Expression of CD36 confers cellular binding and internalization of S. aureus and E. coli. (b) HEK293T-CD36 expressing cells (CD36; filled bars) demonstrate a threefold increase in binding of FITC-labeled S. aureus and a twofold increase in binding of FITC-labeled E. coli over vector-transfected cells (Mock; open bars). *, P ≤ 0.05. (c) Expression of CD36 by HEK293 cells confers a threefold increase in internalization of both FITC-labeled S. aureus and E. coli, as compared with vector-transfected cells (Mock; open bars). *, P ≤ 0.05.

Figure 2.

Tyrosine 463 within the COOH-terminal cytoplasmic tail of CD36 is required to signal engulfment of S. aureus . (a) Schematic drawing of CD36 demonstrating the amino acid sequence of the COOH-terminal tail and specific mutations. COOH-terminal mutations of the murine CD36 cDNA were generated by PCR, verified by sequencing, and used to transfect HEK293T cells. (b) Before use in phagocytosis experiments, equivalent cell surface expression of wild-type CD36 (CD36^wt) and CD36 mutants (CD36^Ala and CD36^Y463F) was verified by flow cytometry analysis. (c) Transfection of CD36 COOH-terminal tail mutants fails to confer internalization (open bars), but permits binding (closed bars) of S. aureus. **, P ≤ 0.005, significantly different from wild-type CD36.

Figure 3.

CD36-deficient macrophages demonstrate impaired phagocytosis of S. aureus and its component LTA. Peritoneal macrophages from wild-type and Cd36 ^−/− mice were incubated with FITC-S. aureus or FITC-E. coli (10:1 bacteria/cell; panel a) or 10 μg/ml BODIPY-LTA (b), and phagocytosis was measured by flow cytometry in the presence of trypan blue. **, P ≤ 0.005, compared with wild-type macrophages. (c) Representative photographs of wild-type and Cd36 ^−/− macrophages incubated with FITC-S. aureus (green) and stained with DAPI (blue) to show individual nuclei. Macrophages were incubated with 10:1 bacteria/cell and allowed to internalize bacteria for 30 min at 37°C. Cells were washed 3× in PBS to remove extracellular bacteria and fixed with formaldehyde.

Figure 4.

Cd36 ^−/− macrophages demonstrate impaired early responses to whole S. aureus and, to a lesser extent, E. coli. Wild-type and Cd36 ^−/− macrophages were treated with 10:1 S. aureus (a) or E. coli (b) for 4 h, and TNFα, IL-12, and IL-6 were measured in cell culture supernatants. *, P ≤ 0.05; **, P ≤ 0.005, significantly different from wild-type macrophages.

Figure 5.

CD36 is required for LTA-induced cytokine production. Peritoneal macrophages from wild-type and Cd36 ^−/− mice were incubated with 100 ng/ml LTA or LPS for 6 h, and cytokine mRNA (a) and protein (b) expression was determined by quantitative RT-PCR and ELISA, respectively. *, P ≤ 0.05; **, P ≤ 0.005, significantly different from wild-type macrophages.

Figure 6.

CD36-mediated internalization by the COOH-terminal cytoplasmic domain is required for engagement of TLR2/6 signaling. (a) CD36 cooperates with TLR2/TLR6 to initiate NFκB activation in response to LTA. HEK293-TLR2 or -TLR9 cells expressing NFκB reporter constructs and CD36, TLR6, or both were stimulated with 1 μg/ml LTA for 5 h, and cell lysates were analyzed for luciferase activity. (b) CD36 initiates TLR2/6-mediated NFκB activation in response to S. aureus and LTA. HEK293-TLR2 cells expressing NFκB reporter constructs and CD36, TLR6, or both were stimulated with 1 μg/ml LTA or S. aureus (10:1) for 5 h, and cell lysates were analyzed for luciferase activity as described in (a). (c) CD36-mediated TLR2/6-NFκB activation requires internalization of LTA. HEK293-TLR2 cells expressing NFκB-luciferase reporter constructs and CD36, TLR6, or both were pretreated with 1 μg/ml cytochalasin D for 30 min before stimulation with LTA as described in (a). (d and e) Mutations that block CD36-mediated internalization of LTA inhibit TLR2/6 signaling. (d) HEK-293 cells were transfected with wild-type CD36 or CD36 mutant constructs and internalization of BODIPY-LTA (10 μg/ml) was measured by flow cytometry in the presence of trypan blue. (e) HEK293-TLR2 cells expressing NFκB reporter constructs and wild-type or mutant CD36 constructs with TLR6 were stimulated with LTA for 5 h, and NFκB-luciferase expression was measured. (f) Cytochalasin D treatment does not block TLR2/6 signaling by CD36 internalization-defective mutants. HEK293-TLR2 cells expressing NFκB reporter constructs, TLR6, and wild-type or mutant CD36 were pretreated with 1 μg/ml cytochalasin D for 30 min before stimulation with LTA, and cell lysates were analyzed for luciferase activity as described in (a). Data presented are the mean of triplicate samples ± SD. **, P ≤ 0.005.

Figure 7.

CD36 null mice are more susceptible to S. aureus and fail to clear circulating bacteria. (a) Wild-type and Cd36 ^−/− mice were inoculated i.v. with S. aureus (capsular strain Reynolds or the unencapsulated 3825-4 strain) or E. coli (018K) and monitored for survival. (b) Cd36^−/− mice fail to effectively clear S. aureus. Blood collected from the tail vein after 4 d was cultured on tryptic soy agar plates supplemented with 5% sheep's blood and the mean colony forming units were counted from each mouse. Representative bacterial plates are presented. (c) Cd36^−/− mice develop perinephric and pericardial abscesses. Necropsies performed on surviving mice showed abundant abscess formation in the hearts and kidneys of Cd36 ^−/− mice (absolute numbers reported in panel a). Arrowhead indicates large abscess in heart.