Effects of Clostridium perfringens alpha-toxin (PLC) and perfringolysin O (PFO) on cytotoxicity to macrophages, on escape from the phagosomes of macrophages, and on persistence of C. perfringens in host tissues

Abstract

Clostridium perfringens is the most common cause of clostridial myonecrosis (gas gangrene). Polymorphonuclear cells (PMNs) appear to play only a minor role in preventing the onset of myonecrosis in a mouse animal model of the disease (unpublished results). However, the importance of macrophages in the host defense against C. perfringens infections is still unknown. Two membrane-active toxins produced by the anaerobic C. perfringens, alpha-toxin (PLC) and perfringolysin O (PFO), are thought to be important in the pathogenesis of gas gangrene and the lack of phagocytic cells at the site of infection. Therefore, C. perfringens mutants lacking PFO and PLC were examined for their relative cytotoxic effects on macrophages, their ability to escape the phagosome of macrophages, and their persistence in mouse tissues. C. perfringens survival in the presence of mouse peritoneal macrophages was dependent on both PFO and PLC. PFO was shown to be the primary mediator of C. perfringens-dependent cytotoxicity to macrophages. Escape of C. perfringens cells from phagosomes of macrophage-like J774-33 cells and mouse peritoneal macrophages was mediated by either PFO or PLC, although PFO seemed to play a more important role in escape from the phagosome in peritoneal macrophages. At lethal doses (10(9)) of bacteria only PLC was necessary for the onset of myonecrosis, while at sublethal doses (10(6)) both PFO and PLC were necessary for survival of C. perfringens in mouse muscle tissue. These results suggest PFO-mediated cytotoxicity toward macrophages and the ability to escape macrophage phagosomes may be important factors in the ability of C. perfringens to survive in host tissues when bacterial numbers are low relative to those of phagocytic cells, e.g., early in an infection.

FIG. 1.

Schematic diagram illustrating the integration event of pSM250 into the chromosome of the pfoA gene of C. perfringens strains 13 and PLC-. Southern blot analysis showed pSM250 integrated into the chromosome in the doublet form, illustrated in the figure. EcoRI and XhoI fragments used for the Southern hybridization analysis are shown at the bottom of the figure.

FIG. 2.

(A) Survival of C. perfringens strains incubated with fluid thioglycolate-elicited mouse peritoneal macrophages, with an MOI of 1:1. (B) Survival of C. perfringens strains incubated under aerobic conditions in DMEM cell culture medium in the absence of macrophages, with an inoculum of ∼1.5 × 10⁶ bacteria/well. Squares, strain 13; triangles, DOB3; circles, PLC-; diamonds, DOB4. A representative experiment from one of at least three independent experiments is shown. The values are shown as percent survival, beginning with 100% of the inoculum at 0 h.

FIG. 3.

(Top) Representative images of macrophages incubated for 24 h with supernatants from the cultures listed below each image. Magnification, ×250. (Bottom) Cytotoxicity of cell-free supernatants from cultures grown with the C. perfringens strain indicated. J774 macrophages were incubated with 30 μl of culture supernatant for 24 h. Cytotoxicity was determined using the Cytotox 96 assay kit. The values are shown as percent lysis, in which the amount of LDH released in wells with treated macrophages was compared to that in control wells in which all of the macrophages had been deliberately lysed. The values shown are the means and standard deviations of quadruplicate samples from three independent experiments.

FIG. 4.

C. perfringens whole-cell-dependent cytotoxicity towards macrophages after 5 h of incubation under aerobic conditions. (A) C. perfringens-dependent cytotoxicity towards J774-33 cells incubated with the indicated strains at an MOI of 1:1. (B and C) C. perfringens-dependent cytotoxicity towards mouse peritoneal macrophages incubated with the indicated strains at an MOI of 1:1 (B) or 10:1 (C). Cytotoxicity was determined using the Cytotox 96 assay kit. The values are shown as percent cytotoxicity, in which the amount of lysis that occurred in wells with treated macrophages was compared to that in control wells, in which all of the macrophages had been deliberately lysed. The means and standard deviations of at least three independent experiments performed on quadruplicate samples are shown.

FIG. 5.

Transmission electron microscopy images of J774-33 cells and mouse peritoneal macrophages infected with C. perfringens strain 13. Macrophages were infected for 60 min, fixed, and processed for electron microscopy. (A and B) C. perfringens in the cytoplasm (A) and in the phagosome (B) of J774-33 cells. (C and D) C. perfringens in the cytoplasm (C) and in the phagosome (D) of mouse peritoneal macrophages. Note the presence of a phagosomal membrane around the bacteria in panels B and D. The clear zones around the intracellular bacteria in panels A and C are not associated with a phagosomal membrane. These clear areas have been seen previously with intracellular C. perfringens (25) and may represent the presence of a polysaccharide capsule on strain 13 (26). Magnification: (A and C) ×10,000; (B) ×15,000; (D) ×7,500.

FIG. 6.

(A) Percentage of intracellular C. perfringens bacteria that were determined to be in the cytoplasm of J774-33 macrophages by using electron microscopy (see Materials and Methods). Bacteria clearly lacking a phagosomal membrane around them were scored as being in the cytoplasm of the cells. See Fig. 5A for an example. (B and C) Percentage of intracellular C. perfringens bacteria that were determined to be in the cytoplasm of mouse peritoneal macrophages. Bacteria clearly lacking a phagosomal membrane around them were scored as being in the cytoplasm of the cells. See Fig. 5C for an example. The macrophages in panels A and B were incubated with C. perfringens bacteria for 1 h, while panel C shows results from peritoneal macrophages incubated with the bacteria for 2 h before the cells were fixed and processed for electron microscopy.

FIG. 7.

In vivo survival of C. perfringens injected into the hind leg muscles of mice. The strains injected were strain 13 (A), DOB3 (B), PLC- (C), and DOB4 (D). Thirty-six mice were injected in the left femoral muscle with 10⁶ bacteria/mouse, and six mice were sacrificed at each time point shown. Each triangle represents the number of CFU obtained from one mouse. Dashes represent the mean value of the number of CFU recovered from the six mice analyzed at each time point indicated. The horizontal line represents the lowest level of detection, and values shown below the line indicate no CFU were detected in that mouse. Asterisks above each group represent the statistical difference between the means of mice injected with wild-type C. perfringens (A) at each time indicated and the corresponding mutant strain at the same time interval using the two-tailed t test. *, P ≤ 0.0001; **, P ≤ 0.0025.