N. meningitidis internalization fails to induce macrophage apoptosis. (A) MDM were infected with nonopsonized (Op−) or opsonized (Op+) N. meningitidis strain MC58, and at 4 or 20 h postinfection, the cell density was assessed and normalized to that of mock-infected (MI) cells (n = 6). (B) Percentage of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeled MDM after mock infection or infection with opsonized MC58 or S. pneumoniae (Spn), used as a control for MDM apoptosis. Data are means + SEM (n = 3). The positive control (DNase I treated) was set to 100%, and the negative control was set to 0% per the manufacturer's instructions. (C) Levels of apoptosis, as assessed by nuclear morphology after 4′,6′-diamidino-2-phenylindole (DAPI) staining, in MDM 4 or 20 h after mock infection or infection with an N. meningitidis strain lacking porB or the parental strain H44/76 (n = 4). The positive control (pneumococcal infection) value was 19.1 ± 0.9%. (D) Levels of apoptosis, as assessed by nuclear morphology after DAPI staining, in U937 cells, differentiated with 10 ng/ml phorbol 12-myristate 13-acetate (Sigma-Aldrich) for 3 days to produce adherent cells, 20 h after mock infection or infection with the porB mutant or H44/76 (n = 4). The positive control (pneumococcal infection) value was 18 ± 0.6%.

Enhanced nitric oxide production in macrophages infected with N. meningitidis mutants. Monocyte-derived macrophages were mock infected (MI) or infected with nonopsonized N. meningitidis parental MC58 cells or a strain lacking norB or cycP, and 20 h after infection, nitric oxide was detected by flow cytometry using 4-amino-5-methylamino-2′,7′-difluorescein diacetate (DAF-FM; Molecular Probes). Cells were unstained (U/S, upper panels) or stained with DAF-FM (lower panels). Representative dot plots from one experiment are shown (representative of three independent experiments). The percentages of positive cells are shown in the upper right corners of the graphs.

Macrophage resistance to apoptosis during N. meningitidis infection involves bacterial resistance to nitrosative stress. (A) Representative pictures of MDM demonstrating condensed or fragmented apoptotic nuclei (arrowheads) 20 h after mock infection (MI) or infection with the nonopsonized N. meningitidis parental MC58 strain or a strain lacking norB or cycP, as assessed by DAPI staining and imaged by a Nikon Eclipse TE300 microscope. (B) Levels of apoptosis in MDM, as assessed by nuclear morphology after DAPI staining, 4 or 20 h after mock infection or infection with nonopsonized (Op−) or opsonized (Op+) MC58 or the norB or cycP mutant (n = 12). (C and D) Levels of apoptosis in U937 cells, as assessed by nuclear morphology after DAPI staining, 20 h after mock infection or infection with MC58 or the norB or cycP mutant. **, P < 0.01; ***, P < 0.001 (analysis of variance [ANOVA] with Tukey's posttest).

Apoptosis induced by N. meningitidis mutants lacking genes encoding nitric oxide detoxification is caspase dependent. The graph shows levels of apoptosis, as assessed by nuclear morphology after DAPI staining, in monocyte-derived macrophages 20 h after mock infection (MI) or infection with the N. meningitidis parental MC58 strain or a strain lacking norB or cycP following treatment with 50 μM of the pan-caspase inhibitor zVAD [N-benzyloxycarbonyl-Val-Ala-Asp (O-methyl) fluoromethyl ketone; Enzyme Systems Products] or a 50 μM control, zFA (N-benzyloxycarbonyl-Phe-Ala fluoromethyl ketone; Enzyme Systems Products) (n = 5). Data are means + SEM. ***, P < 0.001 by ANOVA with Tukey's posttest.

Macrophages infected with N. meningitidis lacking porB undergo caspase-dependent apoptosis in the presence of exogenous nitric oxide. The graph shows levels of apoptosis, as assessed by nuclear morphology after DAPI staining, in monocyte-derived macrophages 20 h after mock infection (MI) or infection with the N. meningitidis parental strain H44/76 or a mutant lacking porB, treated with (+) or without (−) the nitric oxide donor SNAP (50 μM; Calbiochem), the pan-caspase inhibitor zVAD (50 μM), or control zFA (50 μM) (n = 5). Data are means + SEM. ***, P < 0.001 by ANOVA with Tukey's posttest.