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Delivery of the non-membrane-permeative antibiotic gentamicin into mammalian cells by using Shigella flexneri
J L Kadurugamuwa1, T J Beveridge
1Canadian Bacterial Diseases Network, Department of Microbiology, College of Biological Science, University of Guelph, Ontario, Canada. Jag.Kaduru@roche.com
Abstract:
We developed a model to test whether non-membrane-permeative therapeutic agents such as gentamicin could be delivered into mammalian cells by means of bacterial membrane vesicles. Many gram-negative bacteria bleb off membrane vesicles (MVs) during normal growth, and the quantity of these vesicles can be increased by brief exposure to gentamicin (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995), which can be entrapped within the MVs. Gentamicin-induced MVs (g-MVs) were isolated from Shigella flexneri and contained 85 +/- 2 ng of gentamicin per microgram of MV protein. Immunogold electron microscopic labeling of thin sections with antibodies specific to S. flexneri lipopolysaccharide (LPS) demonstrated the adherence and subsequent engulfment of MVs by the human Henle 407 intestinal epithelial cell line. Further incubation of g-MVs with S. flexneri-infected Henle cells revealed that the g-MVs penetrated throughout the infected cells and reduced the intracellular pathogen by approximately 1.5 log10 CFU in the first hour of incubation. Antibiotic was detected in the cytoplasms of host cells, indicating the intracellular placement of the drug following the penetration of g-MVs. Soluble antibiotic, added as a fluid to the tissue culture growth medium, had no effect on intracellular bacterial growth, confirming the impermeability of the cell membranes of the tissue to gentamicin. Western blot analysis of MVs with S. flexneri Ipa-specific antibodies demonstrated that the invasion protein antigens IpaB, IpaC, and IpaD were present in MVs. Being bilayered, with outer faces composed of LPS and Ipa proteins, these MVs were readily engulfed by the otherwise impermeable membranes and eventually liberated their contents into the cytoplasmic substance of the host tissue.
Insights
Bacterial membrane vesicles (MVs) can deliver non-membrane-permeative drugs like gentamicin into mammalian cells. Gentamicin-induced MVs (g-MVs) successfully delivered the antibiotic intracellularly, reducing bacterial load in infected cells.
Area of Science:
- Microbiology
- Cell Biology
- Drug Delivery
Background:
- Many therapeutic agents cannot penetrate mammalian cell membranes.
- Gram-negative bacteria naturally produce membrane vesicles (MVs).
- Gentamicin exposure can increase MV production and entrapment of the antibiotic within MVs.
Purpose of the Study:
- To investigate the potential of bacterial membrane vesicles (MVs) for delivering non-membrane-permeative therapeutic agents into mammalian cells.
- To evaluate the efficacy of gentamicin-loaded MVs (g-MVs) in reducing intracellular bacterial infections.
Main Methods:
- Isolation of gentamicin-induced MVs (g-MVs) from Shigella flexneri.
- Characterization of gentamicin content in g-MVs.
- Incubation of g-MVs with human Henle 407 intestinal epithelial cells.
- Assessment of intracellular bacterial reduction and antibiotic localization using microscopy and CFU counts.
- Western blot analysis to detect bacterial invasion proteins (IpaB, IpaC, IpaD) in MVs.
Main Results:
- Isolated g-MVs contained significant amounts of gentamicin.
- Immunogold electron microscopy confirmed MV adherence and engulfment by host cells.
- g-MVs penetrated infected cells and reduced intracellular Shigella flexneri by approximately 1.5 log10 CFU within one hour.
- Antibiotic was detected within host cell cytoplasm, confirming intracellular drug delivery.
- Western blot confirmed the presence of invasion proteins (IpaB, IpaC, IpaD) on MVs, suggesting a mechanism for host cell entry.
Conclusions:
- Bacterial membrane vesicles (MVs) can serve as effective carriers for intracellular delivery of non-membrane-permeative drugs like gentamicin.
- Gentamicin-loaded MVs (g-MVs) demonstrate potential for treating intracellular bacterial infections by delivering antibiotics directly into host cells.
- The presence of invasion proteins on MVs may facilitate their entry into host cells, enhancing therapeutic delivery.