UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Molecular studies of the structure and function of pseudomonas aeruginosa OprD: an imipenem specific porin Huang, Hongjin

Abstract

Pseudomonas aeruginosa OprD is a specific porin which facilitates the uptake of basic amino acids and imipenem, a carbapenem antibiotic with high potency against P. aeruginosa. To permit further studies of OprD, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1-kb BamHI/KpnI fragment. DNA sequencing predicted a 420 amino acid mature OprD protein with a 23 amino acid signal peptide. In addition, a putative oprD regulatory gene opdE was sequenced, which predicted a hydrophobic protein of 402 amino acids. A set of P. aeruginosa isogenic strains with genetically defined levels of OprD were constructed and utilized to characterize the in vivo function of OprD. The results clearly demonstrated that OprD could be utilized by imipenem and meropenem but, even when substantially overexpressed, could not be significantly utilized by other ß-lactams, quinolones or aminoglycosides. Regarding its function in uptake of nutrients, OprD selectively facilitated the diffusion of basic amino acids and gluconate under growth-rate limiting conditions. Competition experiments confirmed that imipenem shared common binding sites with basic amino acids in the OprD channel, but not with gluconate or glucose. In vitro functional studies using purified OprD provided direct evidence for the presence of a specific binding site(s) for imipenem in the OprD channel, with an I₅₀ value of 1.4 µM. An OprD topology model was proposed based on sequence alignment with E. coli porin OmpF and structure predictions. Sixteen ß-strands were predicted, connected by short turns at the periplasmic side, whereas the eight external loops were of variable length but tended to be much longer. In addition, multiple sequence alignments between OprD and seven representatives from the porin superfamily indicated that OprD was the first specific porin that could be aligned with members of the so-called porin superfamily. PCR-based site directed mutagenesis was performed to separately delete short stretches (4-8) of amino acid residues from each of the predicted external loops. Six out of eight mutants expressed in both E. coli and P. aeruginosa, maintained substantial resistance to trypsin treatment in the context of outer membranes, and formed functional channels, which supported the general accuracy of the model. The loop 2 deletion mutant only partially reconstituted sup ersusceptibility to imipenem in an OprD defective background, and showed much lower affinity to imipenem in the macroscopic conductance inhibition experiment, indicating its involvement in iniipenem binding. Deletions in loops 5, 7 or 8 resulted in a channel with enhanced permeability to antibiotics, but which retained the imipenem binding site(s). A model of the channel architecture of OprD was constructed based on these data, and the mechanism by which imipenem and basic amino acids pass through the OprD channel was discussed.

Item Media

Item Citations and Data

License

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

Usage Statistics