Download CE James and JM. Pagès

Translocation of -lactams through Enterobacterial porins
C. E. James and J-M. Pagès
UMR-MD1, Université de la Méditerranée, Marseille, France.
Influx of antibiotics into the periplasm of Gram negative bacteria is facilitated by porins
that form channels in the outer membrane. In this study, transport through the
enterobacterial porins Omp36 of Enterobacter aerogenes and OmpF of E. coli was
explored.
The omp36 gene from E. aerogenes was cloned and expressed in the outer membrane of
porin-null E. coli BL21omp. Un-labeled porin was purified from outer membrane
extracts using anion-exchange chromatography. Single trimeric Omp36 channels were
reconstituted into planar lipid membranes and translocation characteristics of various lactams were investigated by analysing transient current blockages in their presence.
Concentration dependent ion current fluctuations were observed when ertapenem and
cefepime were added to the system, suggesting an interaction with the channel. MIC
assays were used to assess bacteriocidal activity of antibiotics on the porin-free E. coli
expressing Omp36 in the outer membrane. For optimal permeation of translocating
molecules, a balance between affinity and repulsion interactions is required inside the
channel. Strategically located residues create a strong electrostatic field within the
constriction zone of porin channels. Mutations at such key sites can dramatically alter the
efficiency of translocation through a channel. CFU assays were used here to assess the
effect of such mutations on the rate of -lactam influx into porin-free E. coli expressing
mutant OmpF porins in the outer membrane.
This study of antibiotic translocation at the single molecule and whole cell level provides
new insights for the design of novel drugs, highly effective at translocation through
bacterial porins for rapid accumulation of intracellular antibiotic concentrations.