Substitutions in the eyelet region disrupt cefepime diffusion through the Escherichia coli OmpF channel

The Escherichia coli OmpF porin is a nonspecific channel involved in the me mbrane translocation of small hydrophilic molecules and especially in the p assage of p-lactam antibiotics. In order to understand the dynamic of charg ed-compound uptake through bacterial porins, specific charges located in th e E, coli OmpF channel were mutated. Substitutions G119D and G119E, inserti ng a protruding acidic side chain into the pore, decreased cephalosporin an d colicin susceptibilities. Cefepime diffusion was drastically altered by t hese mutations. Conversely, substitutions R132A and R132D, changing a resid ue located in the positively charged cluster, increased the rate of cephalo sporin uptake without modifying colicin sensitivity. Modelling approaches s uggest that G119E generates a transverse hydrogen bond dividing the pore, w hile the two R132 substitutions stretch the channel size. These charge alte rations located in the constriction area have differential effects on cepha losporin diffusion and substantially modify the profile of antibiotic susce ptibility.