Phagocytosis, germination and killing of Bacillus subtilis spores presenting heterologous antigens in human macrophages

Bacillus subtilis is a Gram-positive spore-bearing bacterium long used as a probiotic product and more recently regarded as an attractive vehicle for delivering heterologous antigens to be used for mucosal vaccination. This report describes the in vitro interaction between human macrophages and B. subtilis spores displaying the tetanus toxin fragment C or the B subunit of the heat-labile toxin of Escherichia coli on their surface in comparison to spores of the parental strain. Recombinant and parental B. subtilis spores were similarly internalized by human macrophages, at a frequency lower than 2.5%. Inside macrophages, nearly all spores germinated and were killed within 6 h. Using germination-defective spores and inhibiting spore germination inside macrophages, evidence was produced that only germinated spores were killed by human macrophages and that intracellular spore germination was mediated by an alanine-dependent pathway. The germinated spores were killed by macrophages before any round of cell duplication, as estimated by fluorescence microscopy analysis of macrophages infected with spores carrying the gfp gene fused to abrB, a B. subtilis gene shown here to be expressed at the transition between outgrowth and vegetative growth. Monitoring of macrophage infection never revealed cytotoxic effects being exerted by B. subtilis spores. These in vitro data support the hypothesis that B. subtilis spores may potentially be used as a suitable and safe vehicle for administering heterologous antigens to humans.