Isolation and characterization of minicell-producing mutants of Shigella spp.

Minicells are small, anucleate cells resulting from aberrant cell divisions at the polar ends of bacilli. We have isolated minicell-producing mutant strains of Shigella flexneri 2a (MC-I) and Shigella dysenteriae 1 (MC-V) after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Microscopically, broth cultures of MC-I and MC-V were found to contain free minicells, normal cells, and filamentous cells with polar, attached minicells. Both strains retained their ability to provoke keratoconjunctivitis in guinea pigs and to invade HeLa cells. Purified suspensions of minicells containing less than one whole cell per 10(6) minicells were obtained by a combination of differential sedimentation and density gradient centrifugation (5 to 30% [wt/vol] linear sucrose gradients). Each MC-I minicell contained about 0.005 times the amount of deoxyribonucleic acid of one normal S. flexneri. The MC-V minicell had about 0.003 times the amount of deoxyribonucleic acid of one whole S. dysenteriae cell. Purified MC-V minicells were treated with polymyxin B to release Shiga toxin. Shiga toxin was readily detected in MC-V minicells by means of a microtiter HeLa cell cytotoxicity assay. Our findings indicate that such a minicell-producing alteration in the cell division cycle of shigellae has not significantly affected their virulence.