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Characterization of macrophage sensitivity and resistance to anthrax lethal toxin
A M Friedlander1, R Bhatnagar, S H Leppla
1U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702-5011.
Abstract:
Anthrax lethal toxin, which consists of two proteins, protective antigen and lethal factor, is cytolytic for macrophages. Macrophages from different mouse strains were found to vary in their sensitivities to toxin. C3H mouse macrophages lysed by lethal factor concentrations of 0.001 micrograms/ml were 100,000 times more sensitive than those from resistant A/J mice. We analyzed various stages of the intoxication process to determine the basis for this resistance. Direct binding studies with radioiodinated protective antigen revealed that the affinity (Kd, approximately 0.5 nM) and number of receptors per cell (25,000 to 33,000) were the same in sensitive and resistant cells. Proteolytic activation of protective antigen by a cell surface protease and subsequent binding of lethal factor were also the same in both sensitive and resistant macrophages. Resistant A/J macrophages were not cross-resistant to other toxins and a virus which, like lethal toxin, require vesicular acidification for activity, implying that resistance is not due to a defect in vesicular acidification. When introduced into the cytosol by osmotic lysis of pinosomes, lethal factor in the absence of protective antigen was cytolytic for the sensitive macrophages while resistant cells were unaffected. Thus, lethal factor by itself possesses the toxic activity of lethal toxin. These results suggest that macrophage resistance is due to a defect at a stage occurring after toxin internalization. A/J macrophages may lack the putative lethal factor target in the cytosol or be defective in the further processing or activation of lethal factor in the cytosol or in endocytic vesicles.
Insights
Anthrax lethal toxin resistance in macrophages is not due to toxin binding or activation. A/J mouse macrophages resist lethal toxin due to a post-internalization defect, suggesting issues with lethal factor processing.
Area of Science:
- Immunology
- Cell Biology
- Toxicology
Background:
- Anthrax lethal toxin (LT) comprises protective antigen (PA) and lethal factor (LF).
- LT causes macrophage lysis, but sensitivity varies significantly across mouse strains.
- Understanding resistance mechanisms is crucial for developing countermeasures.
Purpose of the Study:
- To investigate the molecular basis of macrophage resistance to anthrax lethal toxin.
- To pinpoint the stage of intoxication affected by resistance in A/J mice.
Main Methods:
- Comparative analysis of toxin binding, PA activation, and LF-induced cytolysis in sensitive (C3H) and resistant (A/J) macrophages.
- Assessment of vesicular acidification function.
- Cytosolic delivery of LF to bypass initial uptake steps.
Main Results:
- Macrophage resistance to LT is not linked to PA receptor binding or proteolytic activation.
- A/J macrophages exhibit normal vesicular acidification, ruling out this as a resistance factor.
- Lethal factor alone is cytolytic to sensitive macrophages but not resistant ones when delivered to the cytosol.
Conclusions:
- Macrophage resistance to anthrax lethal toxin occurs after toxin internalization.
- The defect in resistant A/J macrophages likely involves intracellular processing or target interaction of lethal factor.
- Further research should focus on cytosolic LF targets or activation pathways.