Functional inhibition of acid sphingomyelinase disrupts infection by intracellular bacterial pathogens

(A, B, D, E) Desipramine was added to RF/6A cells before infection with A. phagocytophilum and treatment was either maintained throughout the time course (A, B, D) or removed at 20 h (B, E). (C, F) Desipramine was added to A. phagocytophilum–infected RF/6A cells beginning at 20 h (C, F). DMSO served as vehicle control. At 20, 24, 28, and 32 h, the cells were fixed and examined by confocal microscopy for ApV maturation (A–C) and expansion (D–F). (A–C) Desipramine reversibly inhibits APH0032 expression and localization to the ApV. A. phagocytophilum–infected RF/6A cells were screened with antibodies targeting vimentin and APH0032 to demarcate and assess maturation of the ApV, respectively. DAPI was used to stain host cell nuclei and bacterial DNA. (A) Representative confocal micrographs of desipramine or DMSO-treated cells at the indicated postinfection time points. (B, C) Percentages of APH0032-positive ApVs determined for 100 cells for each of three biological replicates per condition. (D–F) Desipramine reversibly inhibits ApV expansion. The mean ApV pixel area was determined for 50 ApVs per time point per condition. Error bars indicate SD. t test was used to test for a significant difference among pairs. Statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) values are indicated. ns, not significant. Data shown are representative of three experiments conducted in triplicate with similar results. Scale bar = 10 μm.

(A) Desipramine inhibits aph1235 transcription. Desipramine-treated HL-60 cells were infected with A. phagocytophilum. Total RNA isolated at 24, 28, and 32 h was subjected to qRT-PCR. The 2^−ΔΔCT method was used to determine the relative aph1235 expression level normalized to that of A. phagocytophilum 16S rRNA. (B, C) Desipramine inhibits APH1235 protein expression. Desipramine-treated RF/6A cells were infected with A. phagocytophilum. At 24, 28, and 32 h, the cells were fixed, immunolabeled with APH1235 and vimentin antibodies, stained with DAPI, and visualized using confocal microscopy. (B) Representative confocal micrographs. Scale bar = 10 μM. (C) Percentage of APH1235-positive ApVs determined by counting 100 cells for each of triplicate samples per time point. (D) Desipramine inhibits A. phagocytophilum–infectious progeny production. RF/6A cells were treated with desipramine or DMSO followed by infection with A. phagocytophilum. At 48 h, the cells were mechanically disrupted followed by isolation and subsequent incubation of host cell–free bacteria with naïve untreated cells. At 24 h, the recipient cells were fixed and examined by immunofluorescence microscopy to determine the percentage that had become infected. Error bars indicate SD. t test was used to test for a significant difference among pairs. Statistically significant (**P < 0.01; ***P < 0.001; ****P < 0.0001) values are indicated. Data shown in (A–C) are representative of three experiments conducted in triplicate that yielded similar results. Data shown in (D) are representative of two experiments conducted in triplicate with similar results.

Desipramine treatment inhibits NPC1 localization to the ApV. Desipramine- or DMSO-treated RF/6A cells were infected A. phagocytophilum. (A–E) At 24 h, the cells were either fixed, immunolabeled with vimentin and NPC1 antibodies, stained with DAPI, and examined by confocal microscopy (A, B); or incubated with BODIPY ceramide (cer) or BODIPY cholesterol (chol), stained with Hoechst 33342, and visualized by live cell imaging (C–E). (A) Representative confocal micrographs of infected cells immunolabeled for vimentin and NPC1. Regions that are demarcated by hatched-line boxes are magnified in the inset panels. (B) Percentage of vimentin-positive ApVs to which NPC1 immunosignal localizes in DMSO- and desipramine-treated cells determined by counting 100 cells for each of triplicate samples per condition. (C) Representative live cell images of infected cells incubated with BODIPY-cer and BODIPY-chol. (D, E) Percentages of ApVs to which BODIPY-cer–positive (D) or BODIPY-chol–positive (E) vesicles localize. Error bars indicate SD. t test was used to test for a significant difference among pairs. Statistically significant (****P < 0.001) values are indicated. ns, not significant. Data shown are representative of three experiments conducted in triplicate that yielded similar results. Scale bar = 10 μm.

(A) A. phagocytophilum fails to productively infect ASM^−/− mice. (A) ASMase^−/− mice or WT mice were infected with A. phagocytophilum DC organisms. Peripheral blood drawn on days 4, 8, 12, 16, 21, and 28 d postinfection was analyzed by qPCR. Relative levels of the A. phagocytophilum 16S rRNA gene were normalized to those of β-actin using the 2^−ΔΔCT method. (B) Desipramine reduces the A. phagocytophilum DNA load in the peripheral blood when administered to infected mice. A. phagocytophilum–infected WT mice were administered desipramine or PBS on days 7 through 12 postinfection, and the bacterial DNA load in the peripheral blood was determined using qPCR. Error bars indicate SD. t test was used to test for a significant difference among pairs. Statistically significant (**P < 0.01) values are indicated. Data shown in (A) are representative of eight experiments each of which were conducted with 5–7 mice per group. Data shown in (B) are representative of three experiments each of which were conducted with 5–7 mice per group.

(A, B) mCherry-C. burnetii (Cb)–infected THP-1 macrophage-like cells (A) or mCherry-C. burnetii grown in axenic medium (B) were treated with desipramine or not treated with. The bacterial load was measured as relative fluorescent units. (C–E) C. burnetii was added to HeLa cells (C, E) or MH-S cells (D) that had been pretreated with desipramine or DMSO, or C. burnetii–infected cells were treated at the indicated days postinfection (E). Bacterial load was measured using a CFU assay. (F) CCV area was determined for desipramine and DMSO-treated C. burnetii–infected HeLa cells. (G) HeLa cells that had been treated with desipramine and infected with mCherry-C. burnetii were labeled with filipin and CD63 antibody. Error bars indicate SD. t test was used to test for a significant difference among pairs. Statistically significant (**P < 0.01; ***P < 0.001; ****P < 0.0001) values are indicated. ns, not significant. Data in panels A and B are representative of three experiments conducted in triplicate with similar results. Data in panels C through F are the means ± SD of three independent experiments. Scale bar = 50 μm.

(A–D) Desipramine or DMSO-treated HeLa cells infected with C. trachomatis (Ctr; A, B) or C. pneumoniae (Cpn; C, D) were either lysed to recover infectious progeny that were incubated with naïve HeLa cells to determine inclusion-forming units (A, C) or were fixed and assessed by immunofluorescence microscopy to determine inclusion area (B, D). (E–J) Desipramine-treated or control cells were infected with Ctr (E, G, I) or Cpn (F, H, J). The cells were either incubated with filipin (E, F) or screened with antibodies specific for LBPA (G, H) or CERT (I, J) together with antisera against C. trachomatis (E, G, I) or C. pneumoniae (F, H, J). DAPI or DRAQ5 was used to stain DNA. Regions that are demarcated by hatched-line boxes are magnified in the inset panels. Error bars indicate SD. t test was used to test for a significant difference among pairs. Statistically significant (*P < 0.05; ***P < 0.001) values are indicated. Data shown are representative of three experiments conducted in triplicate with similar results. Scale bar = 10 μm.