Infection of epithelial cells by pathogenic neisseriae reduces the levels of multiple lysosomal constituents

Abstract

Members of our group reported recently that neisseria infection of human epithelial cells results in accelerated degradation of the major lysosomal integral membrane protein LAMP1 and that this is due to hydrolysis of this glycoprotein at its immunoglobulin A1 (IgA1)-like hinge by the neisseria type 2 IgA1 protease (L. Lin et al., Mol. Microbiol. 24:1083-1094, 1997). We also reported that the IgA1 protease plays a major role in the ability of the pathogenic neisseriae to survive within epithelial cells and hypothesized that this is due to alteration of lysosomes as a result of protease-mediated LAMP1 degradation. In this study, we tested the hypothesis that neisseria infection leads to multiple changes in lysosomes. Here, we report that neisseria infection also reduces the levels of three other lysosomal markers: LAMP2, lysosomal acid phosphatase (LAP), and CD63. In contrast, neither the epidermal growth factor receptor level nor the beta-tubulin level is affected. A detailed examination of LAMP2 indicated that the reduced LAMP2 levels are not the result of an altered biosynthetic rate or of cleavage by the IgA1 protease. Nevertheless, the protease plays a role in reducing LAMP2 and LAP activity levels, as these are partially restored in cells infected with an iga mutant. We conclude that neisseria infection results in multiple changes to the lysosomes of infected epithelial cells and that these changes are likely an indirect result of IgA1 protease-mediated cleavage of LAMP1.

FIG. 1

LAMP2 and EGFR levels in neisseria-infected A431 human epithelial cells. (A) LAMP2 and β-tubulin signals in representative immunoblots of total cell proteins from uninfected cultures and cultures infected with piliated bacteria. U, uninfected cultures; GC, WT piliated GC (strain GCM740); GC iga, GCM740Δ4, a protease-deficient but otherwise isogenic derivative of GCM740; MC, WT piliated MC (strain 8013.6). (B) LAMP2 levels in infected A431 cultures relative to that in the uninfected control culture (means and standard deviations [vertical bar] from four independent experiments). (A′) LAMP2 and β-tubulin signals in representative immunoblots of total cell proteins from A431 cells infected with MS11A307, a nonpiliated, Opa⁻, low-adherence strain. (B′) LAMP2 levels in A431 cultures infected with piliated MS11A (GC P⁺) and nonpiliated MS11A307 (GC P⁻) relative to the uninfected control culture. (C) EGFR level in A431 cultures infected with strain GCM740 (GC) expressed relative to that in the uninfected (control) culture (U) (mean and standard deviation [vertical bar] from three independent experiments).

FIG. 2

Confocal laser scanning microscopy of GC-infected A431 cells double-stained for LAMP2 (green) and bacteria (red). Panels A and B show one horizontal 4-μm-thick optical section taken of the same field of cells. The arrow indicates the location of a cell demonstrated by phase-contrast microscopy (data not shown) and by its fluorescence properties to be infected.

FIG. 3

Biosynthesis rates of LAMP2 in uninfected A431 cell cultures (U) and cultures infected with WT GC (strain GCM740) (I). (A) A representative autoradiogram of LAMP2 immunoprecipitated from radiolabelled cultures. (B) Rates of incorporation of [³⁵S]Met-[³⁵S]Cys into LAMP2. Data are from three independent experiments.

FIG. 4

Purified human LAMP2 is not cleaved by neisseria type 2 IgA1 protease in vitro. Three nanograms of LAMP1 (A) or LAMP2 (B) was incubated with 300 ng of purified neisseria type 2 IgA1 protease in buffer at pH 7.5, 6.5, or 5.0, or with buffer alone, at 37°C for 4 h. The reaction products were resolved by SDS-PAGE, and the cleavage products were detected by immunoblotting with the anti-LAMP1 or anti-LAMP2 polyclonal antibody. The arrow indicates the position of the LAMP1 cleavage products.

FIG. 5

LAP activities in cultures of A431 cells infected with MC strain 8013.6 (MC) or GC strains GCM740 (GC) and GCM740Δ4 (GC iga) expressed relative to that in control uninfected cells (U). Results represent the means and standard deviations from three independent experiments.

FIG. 6

CD63 levels in uninfected A431 cells (A and B) and cells infected with N. meningitidis 8013.6 (C and D), visualized by double-label immunofluorescence microscopy. (A and C) Cells stained for CD63. (B and D) The same field stained for MC. Stained cells were examined and photographed with a Nikon Microphot FX at the same magnifications. Note that the uninfected cell within the center of this field of infected cells (panel D) had normal levels of CD63 (panel C).