Article Figures & Data
Figures
- FIG. 1.
B. abortus rough mutants efficiently invade macrophages. Murine J774.A1 macrophages cultured in 24-well plate were infected with B. abortus S2308 and rough mutants (CA180 and BA582R) at an MOI of 200 as described in Materials and Methods. The cells were washed with Peptone saline and lysed with 0.5% Tween 20 following 1 h incubation in DMEM supplemented with 100 μg of gentamicin per ml. B. abortus uptake represents the percentage of added CFU protected from gentamicin killing. Data represent the means ± standard deviations from three independent experiments.
- FIG. 2.
B. abortus rough mutants replicate in macrophages efficiently. (A) Murine macrophages were infected with S2308 and rough mutants at an MOI of 200 as described in the legend to Fig. 1. After 1 h, the medium was removed and replenished with complete medium containing 20 μg of gentamicin per ml. At selected time points, the medium was removed and the cells were washed prior to lysis of the cell monolayer. Each point represents the mean ± standard deviation from three to five independent experiments. (B) Murine macrophages were infected with S2308 and rough mutant CA180 at an MOI of 200.The infected cells were cultured in complete medium without gentamicin. Results of a representative experiment are shown (C) Murine macrophages were infected with S2308 and rough mutant CA180 at an MOI of 20. The infected cells were cultured in complete medium with 20 μg of gentamicin per ml. In each experiment the cells were lysed with 0.5% Tween 20 at the indicated time points, and the CFU in each well were determined by plating serial dilutions on TSA or TSA containing kanamycin. Results of a representative experiment are shown.
- FIG. 3.
Intracellular replication of B. abortus in murine macrophages. J774.A1 cells were infected with S2308 and CA180 at an MOI of 20. The cells were fixed at 1 and 48 h p.i. and stained by indirect immunofluorescence assay. The intracellular bacteria (red) were observed by confocal microscopy. Individual smooth organisms fluoresce more intensely due to elevated levels of O-antigen antibody in the sera used.
- FIG. 4.
Morphology of B. abortus-infected macrophages. J774.A1 macrophages in 24-well tissue culture plates were infected with B. abortus S2308 or CA180 at an MOI of 200. Infected cells were observed at the times indicated by phase-contrast microscopy. Magnification, ×200. Arrowheads indicate dead cells.
- FIG. 5.
B. abortus rough mutants are cytopathic to J774.A1 macrophages. Macrophages cultured in 96-well plate were infected with S2308 and CA180 at an MOI of 200. The supernatants were collected at the indicated time points, and the LDH released by the infected cells was detected by CytoTox 96 nonradioactive cytotoxicity assay. Each point indicates the mean ± standard deviation from three independent experiments.
- FIG. 6.
B. abortus rough mutants are cytopathic to professional phagocytes but not epithelial cells. J774.A1, THP-1, bovine macrophage, Vero, BHK, and primary bovine epithelial cells were infected with S2308 and CA180 at an MOI of 200. The supernatants were collected at 24 h p.i., and the LDH was detected by CytoTox 96 nonradioactive cytotoxicity assay. Each point indicates the mean ± standard deviation from three independent experiments.
- FIG. 7.
Necrotic cell death in macrophages associated with B. abortus rough mutant infection. J774.A1 macrophages cultured in 96-well plate were uninfected (A and B), infected with S2308 (C and D) or CA180 (E and F) at an MOI of 200 for 24 h, or treated with 5 μM gliotoxin for 5 h (G and H). The cells were stained with annexin V (green) and PI (red) and observed by phase-contrast (A, C, E, and G) or fluorescence (B, D, F, and H) microscopy. Magnification, ×200.
- FIG. 8.
CPE of CA180 infection correlate with the number of necrotic cells. J774.A1 macrophages cultured in 96-well plates were infected with either S2308, CA180, or BA582 at an MOI of 200. The cells were stained with annexin V and PI at the indicated time points. The necrotic cells (annexin V and PI positive) and apoptotic cells (annexin V positive and PI negative) were observed and counted by fluorescence microscopy. The data represent the percentages of necrotic and apoptotic cells in representative fields having at least 600 cells.
- FIG. 9.
Morphological characteristics of CA180-infected cells. (A and B) J774.A1 macrophages treated with gliotoxin for 6 h (A) and infected with CA180 for 24 h (B). The morphology of the cells was observed by phase-contrast microscopy. Magnification, ×600. (C and D) The nuclei of the cells treated with gliotoxin (C) or infected with CA180 (D) were visualized after annexin V and PI staining.
- FIG. 10.
Effect of glycine treatment on the CPE induced by CA180 infection. J774.A1 cells were infected with CA180 for 24 h at an MOI of 200 or treated with 200 μM TBH or 5 μM gliotoxin for 20 h in the presence or absence of 5 μM glycine. CPE was determined by LDH release. Each point indicates the mean ± standard deviation from two or three independent experiments.
Tables
- TABLE 1.
CPE induced by B. abortus rough mutant infection in J774.A1 cells
MOI h p.i. CPEa with strains S2308 CA180 CA353 CA533 CA613 2,000 8 − ++++ ++ + +++ 12 − ++++ ++++ ++ ++++ 24 − ++++ ++++ ++++ ++++ 48 − ++++ ++++ ++++ ++++ 200 8 − + + − + 12 − ++ + + ++ 24 − ++++ ++ + ++ 48 − ++++ +++ +++ +++ 20 8 − − − − − 12 − + − − − 24 − + − − − 48 − ++ + + + ↵ a ++++, 75 to 100% CPE; +++, 50 to 75% CPE; ++, 25 to 50% CPE; +, <25% CPE; −, no CPE.
