OmpK26, a novel porin associated with carbapenem resistance in Klebsiella pneumoniae

Abstract

Clinical isolates of Klebsiella pneumoniae resistant to carbapenems are being isolated with increasing frequency. Loss of the expression of the major nonspecific porins OmpK35/36 is a frequent feature in these isolates. In this study, we looked for porins that could compensate for the loss of the major porins in carbapenem-resistant organisms. Comparison of the outer membrane proteins from two K. pneumoniae clinical isogenic isolates that are susceptible (KpCS-1) and resistant (KpCR-1) to carbapenems revealed the absence of OmpK35/36 and the presence of a new 26-kDa protein in the resistant isolate. An identical result was obtained when another pair of isogenic isolates that are homoresistant (Kpn-3) and heteroresistant (Kpn-17) to carbapenems were compared. Mass spectrometry and DNA sequencing analysis demonstrated that this new protein, designated OmpK26, is a small monomeric oligogalacturonate-specific porin that belongs to the KdgM family of porins. Insertion-duplication mutagenesis of the OmpK26 coding gene, yjhA, in the carbapenem-resistant, porin-deficient isolate KpCR-1 caused the expression of OmpK36 and the reversion to the carbapenem-susceptible phenotype, suggesting that OmpK26 is indispensable for KpCR-1 to lose OmpK36 and become resistant to these antibiotics. Moreover, loss of the major porin and expression of OmpK26 reduced in vitro fitness and attenuated virulence in a murine model of acute systemic infection. Altogether, these results indicate that expression of the oligogalacturonate-specific porin OmpK26 compensates for the absence of OmpK35/36 and allows carbapenem resistance in K. pneumoniae but cannot restore the fitness of the microorganism.

Fig. 1.

SDS-PAGE analysis (A) and Western blotting with anti-OmpK36 serum (B) of the outer membrane proteins of K. pneumoniae carbapenem-susceptible clinical strain KpCS-1 and the carbapenem-heteroresistant strain Kpn-3 and their derivative carbapenem-resistant and -homoresistant isolates KpCR-1 and Kpn-17. Molecular size markers are indicated in kDa on the left. White arrows indicate the position of OmpK36, and the black arrow indicates the position of the novel protein present in the resistant isolates.

Fig. 2.

Alignment of the deduced OmpK26 sequence from K. pneumoniae with the sequences of NanC from E. coli and KdgM from D. dadantii. Secondary structure motifs are described on the basis of the crystal structure of NanC. The numbering is based on the structure of the mature NanC. Conserved amino acids (highlighted in black), the two facing strings of arginines and lysines inside the OmpK26 pore (black circles) and gaps introduced to maximize alignment (hyphens) are indicated.

Fig. 3.

(A) Schematic representation of the insertion-duplication mutagenesis of the OmpK26 encoding gene, yjhA, in K. pneumoniae KpCR-1. The K. pneumoniae chromosome in strain KpCR-1 is shown on the upper part, and the chromosome of its isogenic yjhA mutant is on the lower part. DNA fragment size in the schematic is not to scale. The line between the black boxes in the mutant genome represents the DNA plasmid integrated into the chromosome. The black boxes indicate the probe used in the Southern blot analysis. The expected sizes of the HindIII fragments that hybridize with the probe described above are indicated in kilobases. (B) Southern blot analysis of K. pneumoniae KpCR-1 and the isogenic OmpK26-deficient mutant KpCR-1ΔOmpK26 chromosomes digested with HindIII. Molecular size markers (in kilobases) are shown to the left of the blots. (C and D) SDS-PAGE analysis (C) and Western blot analysis with anti-OmpK36 serum (D) of the outer membrane proteins from K. pneumoniae KpCS-1, KpCR-1, and the isogenic OmpK26-deficient mutant KpCR-1ΔOmpK26. Molecular size markers are indicated in kDa on the left. Arrows indicate the position of OmpK36 and OmpK26.

Fig. 4.

Results of in vitro competition experiments. In vitro competitions were performed in LB broth tubes in which bacteria were grown at 37°C and 180 rpm for 9 h as described in Materials and Methods. The CI values obtained for each of the independent experiments are plotted. The median CI values are shown in parentheses.

Fig. 5.

Results of in vivo competition experiments. In vivo competitions were conducted in a murine model of systemic infection in which bacteria were isolated from the spleen and quantified 24 h after infection. The CI values obtained for each of the at least eight independent experiments are plotted. The median CI values are shown in parentheses.

Fig. 6.

Percentages of survival over time for groups of 18 mice inoculated intraperitoneally with KpCS-1, KpCR-1, or KpCR-1ΔOmpK26. Significant differences are indicated with an asterisk (P < 0.05).