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Updated: Jul 19, 2026
Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens
Published on: May 19, 2020
Inactivation of Bacillus anthracis spores in murine primary macrophages
Haijing Hu1, Qila Sa, Theresa M Koehler
1Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
Abstract:
The current model for pathogenesis of inhalation anthrax indicates that the uptake and fate of Bacillus anthracis spores in alveolar macrophages are critical to the infection process. We have employed primary macrophages, which are more efficient for spore uptake than the macrophage-like cell line RAW264.7, to investigate spore uptake and survival. We found that at a multiplicity of infection (moi) of 5, greater than 80% of the spores of the Sterne strain containing only the pXO1 plasmid were internalized within 1 h. Within 4 h post infection, viability of internalized Sterne spores decreased to approximately 40%. Intracellular vegetative bacteria represented less than 1% of the total spore inoculum throughout the course of infection suggesting effective killing of germinated spores and/or vegetative bacteria. The Sterne spores trafficked quickly to phagolysosomes as indicated by colocalization with lysosome-associated membrane protein 1 (LAMP1). Expression of a dominant-negative Rab7 that blocked lysosome fusion enhanced Sterne spore survival. Addition of d-alanine to the infection resulted in 75% inhibition of spore germination and increased survival of internalized spores of the Sterne strain and a pathogenic strain containing both the pXO1 and pXO2 plasmids. Inhibition was reversed by the addition of l-alanine, which resumed spore germination and subsequent spore killing. Our data indicate that B. anthracis spores germinate in and are subsequently killed by primary macrophages.
Insights
Primary macrophages efficiently kill Bacillus anthracis spores after uptake, but inhibiting germination with d-alanine enhances spore survival. L-alanine reverses this effect, confirming germination is key to macrophage killing of anthrax spores.
Area of Science:
- Microbiology
- Immunology
- Pathogenesis
Background:
- Inhalation anthrax pathogenesis hinges on Bacillus anthracis spore interaction with alveolar macrophages.
- Understanding spore uptake and intracellular fate is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the uptake, survival, and killing mechanisms of Bacillus anthracis spores within primary macrophages.
- To explore the role of spore germination and phagolysosomal trafficking in macrophage-mediated killing.
Main Methods:
- Primary macrophages were infected with Bacillus anthracis Sterne strain spores at a multiplicity of infection (moi) of 5.
- Spore internalization, viability, and intracellular trafficking (LAMP1 colocalization) were assessed.
- The effect of dominant-negative Rab7 on lysosome fusion and spore survival was evaluated.
- Spore germination was inhibited using d-alanine and its reversal by l-alanine was studied.
Main Results:
- Primary macrophages efficiently internalized >80% of Sterne spores within 1 hour.
- Internalized spore viability decreased to ~40% within 4 hours, with minimal intracellular vegetative growth.
- Spores trafficked to phagolysosomes, and blocking lysosome fusion enhanced spore survival.
- D-alanine significantly inhibited spore germination (~75%), increasing survival of both Sterne and pathogenic strains; L-alanine reversed this inhibition.
Conclusions:
- Bacillus anthracis spores are primarily killed by primary macrophages after germination.
- Phagolysosomal targeting is important for spore killing, but germination is the critical step targeted by macrophages.
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