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Involvement of Cellular Caveolae in Bacterial Entry into Mast Cells

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Science  04 Aug 2000:
Vol. 289, Issue 5480, pp. 785-788
DOI: 10.1126/science.289.5480.785

Abstract

Caveolae are subcellular structures implicated in the import and transcytosis of macromolecules and in transmembrane signaling. To date, evidence for the existence of caveolae in hematopoietic cells has been ambiguous. Caveolae were detected in the microvilli and intracellular vesicles of cultured mouse bone marrow–derived mast cells (BMMCs). CD48, a receptor for FimH-expressing (type 1 fimbriated)Escherichia coli, was specifically localized to plasmalemmal caveolae in BMMCs. The involvement of caveolae in bacterial entry into BMMCs was indicated because caveolae-disrupting and -usurping agents specifically blocked E. coli entry, and markers of caveolae were actively recruited to sites of bacterial entry. The formation of bacteria-encapsulating caveolar chambers in BMMCs represents a distinct mechanism of microbial entry into phagocytes.

Originally defined by their cavelike morphology (1), caveolae are pleomorphic membrane structures characterized biochemically by their buoyant density; resistance to nonionic detergents; and enrichment in cholesterol, glycolipids such as the glycosphingolipid GM1, and a specific protein, caveolin (2–4). Caveolae participate in the transcytosis of macromolecules across capillary endothelial cells (5) and in the concentration of small molecules internalized by potocytosis (6). They also serve as conduits for transmembrane signaling, containing high concentrations of signaling molecules (7, 8). Although believed to be present in virtually all cell types, the presence of caveolae in hematopoietic cells has not been clear. Cell lines of lymphocytes (9) and mast cells (10) appear to possess membrane domains consistent with caveolae but have not been shown to contain caveolin. We investigated whether mouse BMMCs form caveolae. Mast cells play a crucial role in recruiting neutrophils to sites of bacterial infection (11, 12). They can also phagocytose a wide range of bacteria (13). In antibody-deficient conditions, however, these cells and other phagocytes support the entry and subsequent survival of FimH-expressing E. coli (14,15).

Caveolar fractions were isolated from BMMCs (Fig. 1A). Immunogold microscopy of cross sections of BMMCs with caveolin-specific antibody labeled microvilli and intracellular vesicles (Fig. 1D). Thus, although BMMCs did not appear to form cavelike caveolae in their plasma membranes, vesicular and plasmalemmal forms of caveolae were present. The presence of glycosylphosphatidylinositol (GPI)-anchored molecules in native caveolae is controversial (16–18). We determined that CD48, a GPI-anchored molecule, was associated with caveolae of BMMCs (Fig. 1, A and C). Confocal microscopy of BMMCs revealed colocalization of CD48 with caveolin primarily in the plasma membrane (Fig. 1E), whereas caveolin staining in the interior of BMMCs was vesicular.

Figure 1

Localization of caveolae and CD48 in BMMCs. (Athrough C) Fractionation of BMMC lysates by means of sucrose gradient centrifugation (16, 27). BMMC lysates were subjected to sucrose gradient fractionation, and a portion of each fraction was subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with a rabbit caveolin 1–specific antibody (Transduction Labs) (A), mouse clathrin heavy chain–specific antibody (Transduction Labs) (B), and rat CD48-specific antibody (Serotec) (C). The caveolin- and CD48-enriched membrane complexes appeared in the light-density fractions (4 and 5) that corresponded to a white flocculent band in the 15 to 25% sucrose interface. These fractions exclude >95% of cellular proteins. Clathrin heavy chains, which are associated with clathrin-coated pits, were found in distinct fractions (10 and 11). (D) Localization of caveolae in BMMCs. Gold particles representing caveolin were specifically localized to the microvilli and to intracellular vesicles (arrows). (E) Confocal micrographs of a single plane (0.5 μm thick) showing colocalization of CD48 with caveolin primarily in the plasmalemmal regions of BMMCs. Antibody labels: CD48 (green), caveolin (red), and colocalization (yellow) (28).

CD48 represents the mast cell receptor for FimH-expressing bacteria (19). Binding to this receptor induces bacterial entry into BMMCs, with limited loss of bacterial viability (15). We investigated whether plasmalemmal caveolae of BMMCs, which contain CD48, were involved in the entry of type 1 fimbriated and FimH-expressing E. coli. The binding of cholera toxin B (CTXB) to GM1, clustered within cell surface caveolae, triggers caveolae-mediated internalization of these toxin subunits (20). Pretreatment of BMMCs with increasing concentrations of CTXB resulted in up to 60% inhibition of FimH-expressing bacterial entry (Fig. 2A), suggesting that the toxin had usurped available plasmalemmal caveolae and impeded subsequent uptake of FimH-expressing bacteria. However, CTXB did not have any effect on the internalization of latex beads by BMMCs (Fig. 2A). Cyclodextrin is a drug that specifically disrupts caveolar structure by eliminating cholesterol from the cell (21). In the presence of increasing concentrations of cyclodextrin, entry of FimH-expressing bacteria into BMMCs exhibited a dose-dependent inhibition (Fig. 2B). Cyclodextrin had no effect on the uptake of latex beads or of opsonized or unopsonized FimH E. coli by BMMCs (Fig. 2B). Thus, the entry of FimH-expressing bacteria into BMMCs appears to involve plasmalemmal caveolae and FimH.

Figure 2

Specific inhibition of the entry of FimH-expressing bacteria into BMMCs by a caveolae-usurping agent, methyl-β-CTXB (A), and a caveolae-disrupting agent, cyclodextrin (B). In (A), BMMCs grown on a 96-well plate or cover slips were pretreated with increasing concentrations of CTXB (0 to 80 μg/ml) for 20 min. Either E. coli ORN103(pSH2), expressing type 1 fimbriae (29) at a MOI of 100, or fluorescent latex beads (0.75 μm in diameter, Polysciences) at a MOI of 300 were added. After incubation for 20 min at 37°C, the cells were assayed (14, 30) for uptake of bacteria (black bars) or latex beads (white bars). In (B), BMMCs grown on a 96-well plate were pretreated with increasing concentrations of cyclodextrin (0 to 10 mM) for 10 min in the presence of 10 μM lovastatin. The entry of FimH-expressing E. coli(black bars) and latex beads (open bars) was examined as described above. As additional controls, the entry of opsonized FimH E. coli (hatched bars) and unopsonized FimH E. coli (gray bars) was assayed. The FimH E. coli used was E. colistrain ORN103(pUT2002) which is an isogenic derivative of ORN103(pSH2) (29). Opsonization was performed by incubation of FimH E. coli with mouse anti–E. coli antibody (1:200 dilution). The MOI of opsonized FimH E. coli was 100, whereas the MOI of the unopsonized FimH E. coli was 1000. All internalization assays were performed in serum-free RPMI-Hepes buffer. The viability of BMMCs and bacteria was not affected by the treatments with either CTXB or cyclodextrin.

Fluorescence microscopy of BMMCs with caveolin-specific antibody after exposure to bacteria revealed accumulation of caveolin around internalized bacteria, which suggested that bacterial uptake by BMMCs was accompanied by specific recruitment of cellular caveolae (Fig. 3, A and B). To verify this, we probed infected BMMCs with other markers of caveolae. We found similar recruitment patterns with GM1 (Fig. 3, C and D) and with cholesterol (Fig. 3, E and F), confirming that caveolae were being specifically mobilized to sites of bacterial entry. Immunoelectron microscopy with caveolin-specific antibody also revealed the accumulation of caveolin around the bacteria (Fig. 3K). No more than one bacterium was found in a single chamber, implying that caveolae-mediated endocytosis was a progressive process involving uptake of individual bacterium, and thus appeared distinct from the mode of entry of salmonella, in which multiple organisms are internalized in a single phagocytic event (22). No caveolar markers accumulated around BMMC-internalized latex beads (Fig. 3, H to J).

Figure 3

Specific recruitment of caveolar markers, caveolin, GM1, and cholesterol toward sites of bacterial entry. BMMCs were exposed to bacteria (A through F andK) or beads (G through J), then probed with specific markers for caveolae (31) and viewed by fluorescence (right), differential interference contrast imaging (left), or standard immunoelectron microscopy (K). Caveolin (A and B), GM1 (C and D), and cholesterol (E and F) were recruited around internalized bacteria (arrows). No accumulation of either caveolin (G and H) or GM1 (I and J) was detected around internalized beads (arrowheads point to representative beads). (K) Accumulation of caveolin (immunogold-labeled) around several intracellular bacteria (labeled B). Of the total cell-associated gold particles, 57% were around intracellular bacteria.

We further investigated whether some cellular caveolae were diverted to chambers encasing intracellular bacteria. We therefore developed a fractionation technique that allowed us to simultaneously isolate bacterium-containing chambers and cellular caveolae from the same infected BMMC. To detect bacterium-containing fractions, we prelabeled bacteria with fluorescein isothiocyanate (FITC) before exposure to BMMCs. Localization of caveolae in each fraction was achieved with the use of a caveolin-specific antibody as probe. With the modification of a previously described procedure (23,24), infected and uninfected (control) BMMCs were homogenized in detergent-free alkaline conditions and were fractionated over a discontinuous sucrose density gradient. Some cellular caveolae shifted from fractions 6 and 7 for uninfected BMMCs (Fig. 4A) to fractions 8 through 10 for infected BMMCs (Fig. 4B). Densitometric analysis of the caveolin bands indicated that fractions 8 to 10 contained 59% of total cellular caveolin. Fluorometric assays of the various fractions from infected BMMCs revealed that the majority of bacteria localized (65%) to fractions 8 through 10, with the peak residing in fraction 9 (Fig. 4E). Electron microscopic examination of thin sections of material in fraction 9 showed the presence of clumps of bacteria-containing vesicles (Fig. 4E, inset). Thus, cellular caveolae appeared to be diverted to form bacteria-encasing compartments. No caveolin was detected in the pellet where the remaining 35% of the bacteria were localized (Fig. 4, B and E). It is presumed that these bacteria were extracellular because free FITC-labeled bacteria, when subjected to fractionation in a parallel assay, localized only to the pellet. The localization of clathrin (fractions 9 and 10) remained the same in uninfected (Fig. 4C) and in infected (Fig. 4D) BMMCs. This indicated a lack of involvement of clathrin in this endocytic process and that the presence of bacteria did not alter the properties of the gradient.

Figure 4

Cofractionation of caveolar membrane complexes with intracellular bacteria in infected BMMCs. (A andB) Detection of caveolar fractions from uninfected (A) and infected (B) BMMCs. BMMC homogenates were fractionated over a 5 to 45% discontinuous sucrose gradient (24). Eleven fractions, including the pellet, were collected and subjected to SDS-PAGE and immunoblotting with antibody to caveolin. For uninfected BMMCs, a thick white flocculent band was seen at the 25 to 35% sucrose interface corresponding to fractions 6 and 7. For infected BMMCs, the white flocculent band was markedly thinner and a moderately thick yellow flocculent band was present at the 35 to 45% sucrose interface corresponding to fractions 8 to 10. (C andD) Localization of clathrin heavy chain in fractions from uninfected (C) and infected (D) BMMCs. (E) Detection of FITC-labeled bacteria in caveolae-containing fractions. The majority of bacteria were found in fractions 8 to 10, which correspond to where the displaced caveolin and the yellow flocculent band fractionated. The FITC label on the bacteria explains the yellow color of the flocculent band. (Inset) An electron micrograph of a cross section of material isolated from fraction 9.

We have shown that bacterial FimH specifically couples to its complementary receptor, CD48, clustered within plasmalemmal caveolae in mast cells. This interaction results in BMMC bacterial uptake mediated by cellular caveolae, which formed distinct intracellular bacterial containment chambers. Because caveolae do not fuse with endosomes (25), it is not surprising that E. coli contained in caveolar chambers avoid the intrinsic bactericidal activity of BMMCs and remain viable. Although the toxin subunit of Vibrio cholerae and FimH-expressing E. coli bind distinct receptors on the plasma membrane and are of markedly different sizes, entry of both into BMMCs is mediated by caveolae. These observations reveal convergence among different bacterial species in their capacity to coopt cellular trafficking pathways of the host. They also illustrate the remarkable versatility of caveolae as mediators of a broad range of endocytic processes.

  • * To whom correspondence should be addressed. E-mail: abrah006{at}mc.duke.edu

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