Figure 1.
Representative fluorescence micrographs of internalization of C. difficile spores by Raw 264.7 cells.
C. difficile spores were labeled with biotoin and Alexa Fluor 488 (green) prior to infection of monolayers of Raw 264.7 cells (red). Infected Raw 264.7 cells were washed; fixed and extracellular spores were stained with Streptavidin-Alexa Fluor 350 conjugate (blue), stained for F-actin and analyzed by fluorescence microscopy as described in Methods section. Representative micrographs of phagocytosis of C. difficile spores are shown: A) Extracellular C. difficile spores (blue); B) Total C. difficile spores (green); C) Merged images. Bars represent 5 µm. White arrows highlight internalized spores.
Figure 2.
Adherence and internalization of C. difficile spores by Raw 264.7 cells.
Monolayers of Raw 264.7 cells were infected at an MOI of 10 with Alexa- and biotoin-labeled C. difficile spores of strains 630 and Pitt177, unbound spores washed, and samples prepared for fluorescence microscopy. Percentage of raw macrophages with at least one spore (A), number of spores per macrophage complex (B), and percentage of intracellular spores (C) were quantified as described in Methods section. D, E, F) The effect of cytochalasin D on the relative binding of spores to Raw 264.7 cells (D), relative number of spores per Raw-spore complex (E), and relative percentage of internalization (F) was evaluated without (white bars) and with 1 µM cytochalasin D (grey bars). Relative values refer to the relative percentage of Raw 264.7 cells with at least one spore (D), relative number of spores per Raw-spore complex (E), and relative percentage of internalization (F) in presence of cytochalasin D normalized to the culture medium control. Results are combined from at least three independent experiments and error bars are standard error of the mean. Asterisks (*) denote statistical difference at p<0.05, and double asterisks (**) denote statistical difference at p<0.01 compared to culture medium control.
Figure 3.
Complement decreases internalization of C. difficile spores by Raw 264.7 cells.
Alexa- biotin-labeled C. difficile spores of strains 630 and Pitt177 were incubated for 30 min with culture medium (white bars), fetal bovine serum (FBS) (light grey bars), heat inactivated FBS (dark grey bars), and heat inactivated FBS supplemented with rabbit complement (black bars) prior to infection of monolayers of Raw 264.7 cells as described figure legend of Figure 2. The relative percentage of Raw 264.7 cells with at least one spore (A), relative number of spores per Raw-spore complex (B), and relative percentage of internalization (C) were quantified and calculated as described in Methods section and legend of Figure 2. Results are combined from at least three independent experiments and error bars are standard error of the mean. Asterisks (*) denote statistical difference at p<0.05, and double asterisks (**) denote statistical difference at p<0.01 compared to culture medium control.
Figure 4.
Sonication does not affect binding and internalization of C. difficile spores by Raw 264.7 cells.
Monolayers of Raw 264.7 cells were infected with untreated (white bars) and sonicated (grey bars) C. difficile spores of strains 630 and Pitt177 at an MOI of 10 for 30 min, and analyzed by fluorescence microscopy for: relative percentage of Raw 264.7 cells with at least one spore (A), relative number of spores per Raw-spore complex (B), and relative percentage of internalization (C) as described in Methods section and in the legend of figure 2. Results are the average of at least three independent experiments and error bars are standard error of the mean.
Figure 5.
Raw 264.7 cells bind and phagocytose C. difficile spores.
A,B,C,D,E,F) SEM of Raw 264.7 cells infected with C. difficile spores under aerobic conditions. Note the active phagocytosis of the spores by Raw 264.7 cells in both panels. White arrows denote coiling phagocytosis. F) Transmission electron microscopy (TEM) of Raw 264.7 cells infected with C. difficile 630 spores at an MOI of 10 for 30 min under aerobic conditions. The area of adherence of C. difficile spores occurred at patchy regions at the end of protrusions from the surface of Raw 264.7 cells. Bar: 1 µm.
Figure 6.
Survival of C. difficile spores and vegetative cells during infection of Raw 264.7 macrophages.
Monolayers of Raw 264.7 cells were infected at an MOI of 10 with C. difficile strain 630: A) spores; B) vegetative cells, and unbound spores and vegetative cells rinsed off and further incubated under aerobic conditions for various periods of time and spore or vegetative cell viability was determined as described in Methods section. Results are the average of at least three independent experiments and error bars are standard error of the mean. Asterisks (*) denote statistical difference at p<0.05, and double asterisks (**) denote statistical difference at p<0.01 compared to time 0 h.
Figure 7.
C. difficile spores remain intact inside the phagosome of Raw 264.7 cells.
TEM images of Raw 264.7 cells infected with C. difficile spores under aerobic conditions (A–C, E, F) and with B. subtilis spores (D). TEM micrographs were taken after 30 min (A,B and D–F) and 24 h of infection. A) TEM shows that C. difficile spores are efficiently phagosytosed by Raw 264.7 cells. B), Phagosome containing C. difficile spores fuses with lysosomes, white arrows denotes fusion of lysosomes with the phagosome. C), C. difficile spores remain intact after 24 h of infection with Raw 264.7 macrophages. D) TEM micrograph of phagosytosed B. subtilis spores by Raw 264.7 cells. Phagosome’s membrane remains intact. E) TEM image showing phagocytosed C. difficile spore in a phagosome with membrane damage, white arrows denote disrupted phagosome membrane. F) TEM image rendering direct interactions between the surface of C. difficile spores and the phagosome’s membrane. White scale bar is 500 nm for panels A–C, and 100 m, for panels D-F.
Figure 8.
Factors affecting survival of C. difficile spores inside Raw 264.7 cells.
A) Monolayers of Raw 264.7 cells were infected at an MOI of 10 with dormant C. difficile spores; and with spores incubated for 30 min with 1% sodium taurocholate (ST) in DMEM and 1% sodium taurocholate-5 mM L-glycine (STG) in DMEM. After 30 min of infection, unbound spores were washed, and Raw 264.7 cells were incubated in DMEM with no FBS under: Aer, aerobic conditions-5% CO2 for 24 h; Ana, anaerobic conditions for 24 h. Loss of spore viability was determined as described in Methods section. B) Monolayers of Raw 264.7 cells were infected with non-heat activated C. difficile spores treated for 30 min with: C, DMEM; 0.1-STG, 0.1% sodium taurocholate-5 mM L-glycine in DMEM; HS, 50% human serum in DMEM; HS 0.1-STG, 0.1% sodium taurocholate-5 mM L-glycine-50% human serum in DMEM; HA HS 0.1-STG, with heat activated C. difficile spores treated for 30 min with 0.1% sodium taurocholate-5 mM L-glycine-50% human serum in DMEM. Infected monolayers of Raw 264.7 cells were incubated for 24 h and viable spores were determined by plating aliquots of lysed Raw 264.7 cells onto BHIS agar plates as described in Methods section. Treatment C corresponds to data from Fig. 7A and is shown for comparative purposes. C,D) Fluorescence micrographs of Raw 264.7 cells infected with 1.0%-STG C. difficile spores. Infected Raw 264.7 cells were incubated for 24 h under aerobic (panel C) and anaerobic (panel D) conditions, fixed and DNA material of either Raw 264.7 cells and C. difficile vegetative cells were stained with DAPI as described in Methods section. White arrows denote growing C. difficile vegetative cells. Results are the average of at least three independent experiments and error bars are standard error of the mean.
Figure 9.
C. difficile spores affect viability of Raw 264.7 cells.
Monolayers of Raw 264.7 cells were infected for 24 (grey bars) and 48 h (black bars) with C. difficile spores at MOIs of 1 and 10 and effects on Raw 264.7 cells was measured by quantification of: A) dead Raw 264.7 cells with ethidium homodimer-1; or B) live Raw 264.7 cells with calcein AM. Results were also confirmed with: C) cell viability assay with the membrane impermeable trypan blue as described in Methods section. Dark cells indicate disruption of the plasma membrane. Double asterisk indicate statistical significant difference (p<0.05) between treatments highlighted by brackets. Results are the average of at least three independent experiments and error bars are standard error of the mean.