Tetraspanin CD81 is required for Listeria monocytogenes invasion

Abstract

Listeria monocytogenes is an intracellular bacterial pathogen that invades epithelial cells by subverting two cellular receptors, E-cadherin and Met. We recently identified type II phosphatidylinositol 4-kinases alpha and beta (PI4KIIalpha and PI4KIIbeta) as being required for bacterial entry downstream of Met. In this work, we investigated whether tetraspanins CD9, CD63, and CD81, which figure among the few described molecular partners of PI4KIIalpha, function as molecular adaptors recruiting PI4KIIalpha to the bacterial entry site. We observed by fluorescence microscopy that CD9, CD63, and CD81 are expressed and detected at the cellular surface and also within intracellular compartments, particularly in the case of CD63. In resting cells, colocalization of tetraspanins and PI4KIIalpha is detectable only in restricted areas of the perinuclear region. Upon infection with Listeria, endogenous CD9, CD63, and CD81 were recruited to the bacterial entry site but did not colocalize strictly with endogenous PI4KIIalpha. Live-cell imaging confirmed that tetraspanins and PI4KIIalpha do not follow the same recruitment dynamics to the Listeria entry site. Depletion of CD9, CD63, and CD81 levels by small interfering RNA demonstrated that CD81 is required for bacterial internalization, identifying for the first time a role for a member of the tetraspanin family in the entry of Listeria into target cells. Moreover, depletion of CD81 inhibits the recruitment of PI4KIIalpha but not that of the Met receptor to the bacterial entry site, suggesting that CD81 may act as a membrane organizer required for the integrity of signaling events occurring at Listeria entry sites.

FIG. 1.

Expression of CD9, CD63, and CD81 in HeLa cells. Cell extracts from wild-type cells or cells treated with siRNAs were run on 10% polyacrylamide gels, samples were transferred onto nitrocellulose membranes, and Western blot analyses were performed under nonreducing conditions using specific anti-CD9, anti-CD63, or anti-CD81 antibodies. Left lanes show the detection of CD9 as a major band of 24 kDa, and a secondary minor band can be observed at 26 kDa. CD63 is detected as a smear due to the high levels of glycosylation of the protein. CD81 is detected as a band of 26 kDa. Right lanes show the absence of protein expression after inactivation with specific siRNAs.

FIG. 2.

Distribution of endogenous CD9, CD63, CD81, and PI4KIIα in HeLa cells infected with Listeria. Cells were infected for 10 min with L. monocytogenes strain BUG 1641; cells were then fixed and processed for immunofluorescence analyses using anti-CD9, anti-CD63, or anti-CD81 antibodies and anti-PI4KIIα immune serum. The colocalization of Listeria with tetraspanins is indicated by large arrows, the colocalization of Listeria with PI4KIIα is indicated by arrowheads, and the colocalization of Listeria with both markers is indicated by small arrows. Boxes labeled a to f in the merge panels have been enlarged at the top of the figure. Bar, 3 μm; phase, phase-contrast micrograph.

FIG. 3.

Distribution of Met, CD9, CD63, CD81, and PI4KIIα in HeLa cells infected with Listeria. Cells were transfected with CD9-GFP, CD63-GFP, CD81-GFP, and PI4KIIα-CFP constructs for 24 h, and then cells were infected for 10 min with wild-type L. monocytogenes strain BUG 1641; cells were then fixed and processed for immunofluorescence analyses using anti-Met antibodies. The colocalization of Met, CD9, PI4KIIα, and Listeria and of Met, CD63, PI4KIIα, and Listeria is observed (arrows). Boxes in the merge panels have been enlarged as insets. Bar, 3 μm.

FIG. 4.

Inactivation of CD81 inhibits the entry of Listeria into HeLa cells. Cells were treated with specific siRNA against CD9, CD63, or CD81 for 72 h, and then cells were infected with L. monocytogenes strain BUG 1641 for 1 h. Bacteria were washed, and cells were incubated in the presence of gentamicin for 1 h; afterwards, cells were lysed, and bacteria that survived the gentamicin treatment were distributed on brain heart infusion plates for counting of the CFU 24 h later. Results are expressed as the percentage of the inoculum that survived the gentamicin treatment (the data shown are representative of results from four independent experiments). Analysis for statistical significance was performed using Student's t test.

FIG. 5.

Distribution of PI4KIIα in Listeria-infected cells in which CD9, CD63, or CD81 has been inactivated. Cells were treated with specific siRNA against CD9, CD63, or CD81 for 72 h, and then cells were infected with L. monocytogenes strain BUG 1641 for 10 min; bacteria were washed, and cells were fixed, permeabilized with Triton X-100, and treated for immunofluorescence analyses using anti-CD9, anti-CD63, anti-CD81, and anti-PI4KIIα antibodies. Recruitment of PI4KIIα is still detected in cells in which CD9 or CD63 is inactivated but not in cells in which CD81 is inactivated. Arrows indicate bacteria that colocalize with PI4KIIα in cells with CD9 or CD63 inactivated; arrowheads indicate bacteria that do not colocalize with PI4KIIα in cells with CD81 inactivated. Boxes labeled a to f in the merge panels have been enlarged at the top of the figure. Bar, 3 μm.

FIG. 6.

Distribution of Met and PI4KIIα in Listeria-infected cells in which CD9, CD63, or CD81 has been inactivated. Cells were treated with specific siRNA against CD9, CD63, or CD81 for 72 h, and then cells were infected with L. monocytogenes strain BUG 1641 for 10 min; bacteria were washed, and cells were fixed, permeabilized with Triton X-100, and treated for immunofluorescence analyses using anti-Met and anti-PI4KIIα antibodies. Recruitment of Met by Listeria is detected in all cases. As in Fig. 5, bacterial colocalization with PI4KIIα is still detected in cells with CD9 or CD63 inactivated but not in cells with CD81 inactivated. Arrows indicate bacteria that colocalize with Met and PI4KIIα in cells with CD9 or CD63 inactivated; arrowheads indicate bacteria that colocalize with Met but do not colocalize with PI4KIIα in cells with CD81 inactivated. Boxes labeled a to f in the merge panels have been enlarged at the top of the figure. Bar, 3 μm.

FIG. 7.

Inactivation of CD81 reduces PI4KIIα recruitment to invading Listeria bacteria. Cells were treated with specific siRNA against CD9 or CD81 for 72 h, and then cells were infected with L. monocytogenes strain BUG 1641 and processed for immunofluorescence analyses as described in the legend to Fig. 6. Samples were analyzed by fluorescence microscopy, and bacteria which showed colocalization with the receptor Met were scored for the recruitment of PI4KIIα. As shown, inactivation of CD81 reduces the recruitment of PI4KIIα to invading bacteria interacting with Met. Results shown are the averages of results from three different infection experiments, and in each experiment, counts of more than 500 bacteria positive for Met were performed. Analysis for statistical significance was performed using Student's t test.