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Isolation and characterization of minicell-producing mutants of Shigella spp
Abstract:
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.
Insights
Researchers created minicell-producing mutants of Shigella flexneri and Shigella dysenteriae. These anucleate bacterial minicells retained virulence and could be used to isolate Shiga toxin.
Area of Science:
- Microbiology
- Bacterial Genetics
- Cell Biology
Background:
- Minicells are small, anucleate bacterial cells formed during aberrant cell division.
- Shigella species are known human pathogens causing dysentery and other infections.
- Understanding bacterial cell division is crucial for developing novel antimicrobial strategies.
Purpose of the Study:
- To isolate and characterize minicell-producing mutants of Shigella flexneri and Shigella dysenteriae.
- To assess the virulence and toxin production capabilities of these minicell mutants.
- To evaluate the potential of minicells as a platform for toxin isolation.
Main Methods:
- Mutagenesis of Shigella strains using N-methyl-N'-nitro-N-nitrosoguanidine.
- Microscopic examination of bacterial cultures to identify minicell production.
- Purification of minicells using differential sedimentation and density gradient centrifugation.
- Quantification of DNA content in minicells.
- Detection of Shiga toxin in minicells via HeLa cell cytotoxicity assay.
Main Results:
- Minicell-producing mutants (MC-I and MC-V) of Shigella flexneri and Shigella dysenteriae were successfully isolated.
- Both mutant strains maintained their ability to cause keratoconjunctivitis and invade HeLa cells, indicating retained virulence.
- Purified minicells contained minimal amounts of DNA (0.005% for MC-I, 0.003% for MC-V compared to normal cells).
- Shiga toxin was successfully detected in purified MC-V minicells, demonstrating their utility for toxin isolation.
Conclusions:
- Alterations in the bacterial cell division cycle leading to minicell formation do not significantly impair Shigella virulence.
- Minicell-producing Shigella strains can serve as a valuable tool for studying bacterial pathogenesis and for the purification of bacterial toxins.
- Further research into minicell technology could offer new avenues for diagnostics and therapeutics.