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Self-promoted uptake hypothesis

This hypothesis states that cationic compounds, such as aminoglycosides and polymyxin B, interact at sites, on the outer membrane surface, at which divalent cations crossbridge adjacent lipopolysaccharide molecules. This causes a destabilization of the outer membrane that is proposed to permit uptake of the interacting molecule and/or of other molecules in the environment of the cell. The hypothesis derives its name from the observation that antibiotics are promoting their own uptake across the outer membrane.

Research Milestones.

1980-81. Formulation of the hypothesis based on studies with a polymyxin/gentamicin/EDTA resistant, outer membrane protein OprH (=H1) overproducing mutant of P. aeruginosa.

1981-84. Establishment of probes to demonstrate that molecules proposed to access this pathway lead to permeabilization of the outer membrane - probes utilized were lysozyme; the b-lactam nitrocefin and the hydrophobic fluorophor 1-N-phenyl napthylamine.

1985-86. Utilization of spin-label probes and the fluorescent probe dansyl polymyxin to demonstrate that antibiotics which utilize the self-promoted uptake pathway bind to divalent cation binding sites on Lipid A, LPS and intact cells.

1980-95. Demonstration that the following classes of antibiotics are permeabilizers that utilize the self-promoted uptake pathway, polymyxins (N.B. others had noted polymyxin was a permeabilizer but had not suggested that permeabilizing outer membranes was part of an uptake mechanism), aminoglycosides, gramicidin S, the new dibasic macrolide azithromycin, antimicrobial peptides from rabbit neutrophils and alveolar macrophages (now called defensins), and, novel dibasic deglucoteichoplainins and recombinant cationic antimicrobial peptides, and newly engineered gramicidin S variants.

1988. Correlation between enhanced affinity for aminoglycosides, enhanced permeabilization of outer membranes and supersusceptibility to aminoglycosides in a tolA-12 mutant of P. aeruginosa - these latter two observations and analogous ones with E. coli as well as kinetic studies indicated that self-promoted uptake was relevant to eventual cell killing.

1991. Demonstration that aminoglycosides, azithromycin, and deglucoteichoplainins are also taken up by self-promoted uptake in E. coli, and that cationic peptides also access this pathway in Enterobacteriaceae.

1986-present. Extending the self-promoted uptake hypothesis to include a large number of different types of cationic molecules and chelators from bacteria, insects, and various eukaryotic sources. Proof of concept with natural and synthetic cationic antimicrobial peptides including defensins, cecropin melittin hybrids, gramicidins and indolicidin.

1980-1991. Proposal that overproduction of an outer membrane protein OprH results in resistance to gentamicin and EDTA due to blocking of self-promoted uptake in P. aeruginosa and confirmation of this proposal using molecular genetic techniques.

1992. Structure of P. aeruginosa Lipid A.

1996. Construction of a membrane topology model for OprH as an 8-stranded ß-barrel, the smallest known b-barrel to date.

1999-2000. Demonstration that the two genes downstream of oprH, are phoP and phoQ. Cloning of these latter two genes and their mutation by interposons was employed to provide convincing evidence that they regulate self-promoted uptake and oprH (and consequently polymyxin resistance, resistance to some antimicrobial peptides, as well as virulence. Based on this mutational studies we demonstrtaed that OprH was co-regulated with this system but itself was not involved in resistance.