C-terminal region of Pseudomonas aeruginosa outer membrane porin OprD modulates susceptibility to meropenem

Abstract

We investigated the unusual susceptibility to meropenem observed for seven imipenem-resistant clinical isolates of Pseudomonas aeruginosa. These strains were genetically closely related, expressed OprD, as determined by Western blot analyses, and were resistant to imipenem (>5 microg/ml) but susceptible to meropenem (<1 microg/ml). The oprD genes from two isolates were entirely sequenced, and their deduced protein sequences showed 93% identity with that of OprD of strain PAO1. The major alteration consisted of the replacement of a stretch of 12 amino acids, located in putative external loop L7 of OprD, by a divergent sequence of 10 amino acid residues. The oprD gene variants and the wild-type oprD gene were cloned and expressed in a defined oprD mutant. The meropenem MICs for strains carrying the oprD genes from clinical isolates were four times lower than that for the strain carrying the wild-type oprD gene. Imipenem activities, however, were comparable for all strains. Furthermore, meropenem hypersusceptibility was obtained with a hybrid OprD porin that consisted of the PAO1 oprD gene containing loop L7 from a clinical isolate. These results show that the C-terminal portion of OprD, in particular, loop L7, was responsible for the unusual meropenem hypersusceptibility. Competition experiments suggested that the observed OprD modifications in the clinical isolates did not affect antagonism between imipenem and the basic amino acid L-lysine. We further propose that shortening of putative loop L7 of the OprD porin by 2 amino acid residues sufficiently opens the porin channel to allow optimal penetration of meropenem and increase its activity. In contrast, this alteration would not affect susceptibility to a smaller carbapenem molecule, such as imipenem.

FIG. 1

Western blot analysis of strains used in this study. (A) Outer membrane proteins (OMPs) of clinical strains were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with OprM polyclonal antiserum. Lanes: 1, PAO1; 2, PP2; 3, PP63; 4, PP18; 5, PP148. (B) OMP preparations were probed with OprD polyclonal antiserum. Lanes: 1, PAO1; 2, PP2; 3, PP63; 4, PP73; 5, PP108; 6, PP118; 7, PP133; 8, PP148; 9, PAO1; 10, PASE1. (C) Crude cell extracts were probed with OprD polyclonal antiserum and OprF monoclonal antiserum MA5–8. Lanes: 1, PAO1; 2, PASE1(pSE43); 3, PASE1(pCS2); 4, PASE1(pCS148); 5, PASE1(pαΩM1Wt); 6, PASE1(pαΩM2Wt); 7, PASE1; 8, PASE1(pSE43); 9, PAO1. For each lane in panels A and B, 10 μg of OMPs was loaded; in panel C, 10 μl of crude extract was loaded per lane.

FIG. 2

Multiple sequence alignment of OprD protein variants from PAO1 and seven clinical strains. Black boxes indicate a change in amino acid charge or replacement of a stretch of residues; grey boxes indicate other amino acid substitutions. The small open box shows the single amino acid change observed in strain PP148. Amino acids in italics represent the N-terminal signal sequence. Locations of putative loops L1 to L8 are shown by bold lines; amino acids in grey boxes in loop L7 indicate the deletion (OprDΔL7) described by Huang et al. (11). The fusion junction in the OprD hybrid proteins is boxed and indicated by an arrow. Numbering includes the signal sequence.