Role of putative loops 2 and 3 in imipenem passage through the specific porin OprD of Pseudomonas aeruginosa

Abstract

Mutant proteins with eight amino acid deletions in putative surface loops 2 and 3 of the imipenem-specific porin OprD of Pseudomonas aeruginosa failed to reconstitute imipenem susceptibility in an oprD-deficient background. The loop 3 deletion prevented the ability of imipenem to inhibit KCl conductance through the OprD channel, as previously shown for a loop 2 deletion. This suggests that both loops 2 and 3 have a role in imipenem binding to the OprD channel.

FIG. 1

Partial membrane topology model of P. aeruginosa outer membrane protein OprD showing the portion of the protein from β strands 3 to 8 and the surface loops 2, 3, and 4. The boundaries of the insertions made in this study are shown by arrows. Only that portion of the model that was predicted to be altered compared to the sequence of the previously published model (5) is presented. Numbering of amino acids refers to the mature OprD protein.

FIG. 2

Western immunoblot probed with antiserum specific for OprD (5) of P. aeruginosa strain H729 oprD::Km^r containing the following plasmids or standards: pHP23 (lane 1), pHP22 (lane 2), pHP21 (lane 3), pHP2 (lane 4), pXH2 (lane 5), vector control pUCP18 (lanes 6 and 8), chromogenic molecular weight standards (lane 7), pXH3 (lane 9), and pXH12 (lane 10).

FIG. 3

Inhibition by imipenem of macroscopic KCl conductance through native OprD and the loop 3 deletion mutant Δ156-163 expressed from pXH12. Macroscopic conductance inhibition experiments were performed exactly as described previously (6). Each point represents the average of four to seven measurements.