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Surface action of gentamicin on Pseudomonas aeruginosa
J L Kadurugamuwa1, A J Clarke, T J Beveridge
1Department of Microbiology, University of Guelph, Ontario.
Abstract:
The mode of action of gentamicin has traditionally been considered to be at the 30S ribosomal level. However, the inhibition of bacterial protein synthesis alone appears to be insufficient to entirely explain the bactericidal effects. Bacteriolysis is also mediated through perturbation of the cell surface by gentamicin (J.L. Kadurugamuwa, J.S. Lam, and T.J. Beveridge, Antimicrob. Agents Chemother. 37:715-721, 1993). In order to separate the surface effect from protein synthesis in Pseudomonas aeruginosa PAO1, we chemically conjugated bovine serum albumin (BSA) to gentamicin, making the antibiotic too large to penetrate through the cell envelope to interact with the ribosomes of the cytoplasm. Furthermore, this BSA-gentamicin conjugate was also used to coat colloidal gold particles as a probe for electron microscopy to study the surface effect during antibiotic exposure. High-performance liquid chromatography confirmed the conjugation of the protein to the antibiotic. The conjugated gentamicin and BSA retained bactericidal activity and inhibited protein synthesis on isolated ribosomes in vitro but not on intact cells in vivo because of its exclusion from the cytoplasm. When reacted against the bacteria, numerous gentamicin-BSA-gold particles were clearly seen on the cell surfaces of whole mounts and thin sections of cells, while the cytoplasm was devoid of such particles. Disruption of the cell envelope was also observed since gentamicin-BSA and gentamicin-BSA-gold destabilized the outer membrane, evolved outer membrane blebs and vesicles, and formed holes in the cell surface. The morphological evidence suggests that the initial binding of the antibiotic disrupts the packing order of lipopolysaccharide of the outer membrane, which ultimately forms holes in the cell envelope and can lead to cell lysis. It is apparent that gentamicin has two potentially lethal effects on gram-negative cells, that resulting from inhibition of protein synthesis and that resulting from surface perturbation; the two effects in concert make aminoglycoside drugs particularly effective antibiotics.
Insights
Gentamicin exhibits bactericidal effects through both protein synthesis inhibition and direct cell surface disruption. This study used a large gentamicin-bovine serum albumin conjugate to demonstrate gentamicin
Area of Science:
- Microbiology and Molecular Biology
- Antimicrobial Drug Action
Background:
- The established mechanism of gentamicin involves binding to the 30S ribosomal subunit, inhibiting bacterial protein synthesis.
- However, protein synthesis inhibition alone does not fully account for gentamicin's potent bactericidal activity against Gram-negative bacteria.
- Previous research suggests that gentamicin also perturbs the bacterial cell surface, contributing to its antimicrobial effects.
Purpose of the Study:
- To elucidate the distinct roles of protein synthesis inhibition versus cell surface perturbation in gentamicin's bactericidal action.
- To investigate the direct interaction of gentamicin with the bacterial cell envelope using a novel conjugate probe.
Main Methods:
- Chemical conjugation of bovine serum albumin (BSA) to gentamicin to create a large molecule unable to penetrate the bacterial cell envelope.
- Utilized BSA-gentamicin conjugate coated on colloidal gold particles as a probe for electron microscopy.
- High-performance liquid chromatography (HPLC) confirmed successful conjugation.
Main Results:
- The BSA-gentamicin conjugate retained bactericidal activity and inhibited protein synthesis in vitro but not in intact cells, confirming cytoplasmic exclusion.
- Electron microscopy revealed significant binding of BSA-gentamicin-gold particles to the bacterial cell surface, with no cytoplasmic localization.
- Observed destabilization of the outer membrane, formation of blebs and vesicles, and holes in the cell surface upon exposure to the conjugate.
Conclusions:
- Gentamicin exerts bactericidal effects through two primary mechanisms: inhibition of protein synthesis and disruption of the cell envelope.
- The surface perturbation mechanism involves disruption of lipopolysaccharide packing in the outer membrane, leading to cell envelope damage and potential lysis.
- The synergistic action of these two lethal effects contributes to the high efficacy of aminoglycoside antibiotics like gentamicin against Gram-negative bacteria.