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