Global Trends in Proteome Remodeling of the Outer Membrane Modulate Antimicrobial Permeability in Klebsiella pneumoniae

Abstract

In Gram-negative bacteria, the permeability of the outer membrane governs rates of antibiotic uptake and thus the efficacy of antimicrobial treatment. Hydrophilic drugs like β-lactam antibiotics depend on diffusion through pore-forming outer membrane proteins to reach their intracellular targets. In this study, we investigated the distribution of porin genes in more than 2,700 Klebsiella isolates and found a widespread loss of OmpK35 functionality, particularly in those strains isolated from clinical environments. Using a defined set of outer-membrane-remodeled mutants, the major porin OmpK35 was shown to be largely responsible for β-lactam permeation. Sequence similarity network analysis characterized the porin protein subfamilies and led to discovery of a new porin family member, OmpK38. Structure-based comparisons of OmpK35, OmpK36, OmpK37, OmpK38, and PhoE showed near-identical pore frameworks but defining differences in the sequence characteristics of the extracellular loops. Antibiotic sensitivity profiles of isogenic Klebsiella pneumoniae strains, each expressing a different porin as its dominant pore, revealed striking differences in the antibiotic permeability characteristics of each channel in a physiological context. Since K. pneumoniae is a nosocomial pathogen with high rates of antimicrobial resistance and concurrent mortality, these experiments elucidate the role of porins in conferring specific drug-resistant phenotypes in a global context, informing future research to combat antimicrobial resistance in K. pneumoniae. IMPORTANCE Klebsiella pneumoniae is a pathogen of humans with high rates of mortality and a recognized global rise in incidence of carbapenem-resistant K. pneumoniae (CRKP). The outer membrane of K. pneumoniae forms a permeability barrier that modulates the ability of antibiotics to reach their intracellular target. OmpK35, OmpK36, OmpK37, OmpK38, PhoE, and OmpK26 are porins in the outer membrane of K. pneumoniae, demonstrated here to have a causative relationship to drug resistance phenotypes in a physiological context. The data highlight that currently trialed combination treatments with a carbapenem and β-lactamase inhibitors could be effective on porin-deficient K. pneumoniae. Together with structural data, the results reveal the role of outer membrane proteome remodeling in antimicrobial resistance of K. pneumoniae and point to the role of extracellular loops, not channel parameters, in drug permeation. This significant finding warrants care in the development of phage therapies for K. pneumoniae infections, given the way porin expression will be modulated to confer phage-resistant—and collateral drug-resistant—phenotypes in K. pneumoniae.

Stepwise evolution of carbapenem-resistance, captured in patient samples and evident in global genomics of Klebsiella pneumoniae

“…3, Supplementary Table 4). Consistent with patterns observed globally, the most detected defective porin was OmpK35 (7).…”

“…Porins are beta-barrel proteins in the outer membrane of Gram-negative bacteria and defects in genes encoding porins have been associated with carbapenem resistance (7,18,26).…”

“…There is a current acceptance for a set of 289 genes that are known to contribute to AMR (6,7,9,19), and these include the various isoforms of beta-lactamases, carbapenemases, porins and efflux pumps of interest in our study. We identified 45 of these AMR genes across the 43 genomes (Supplementary Table 4).…”

“…These drugs enter the periplasm via porins in the bacterial outer membrane, and can be secreted by efflux pumps along the outer membrane (5). As a result, a key non-CP CRE mechanism in strains that express an extended spectrum beta-lactamase (ESBL), is either loss-of-function mutations in outer membrane porins to diminish beta-lactam entry (6,7), and/or overproduction of efflux pumps to increase drug export from the periplasm (8,9). Together, genotypes such as this result in a decreased concentration of carbapenem in the periplasm, which if sufficiently low, can be cleared by the action of ESBLs to generate a non-CP CRE phenotype (6).…”

The component parts of bacteriophage virions accurately defined by a machine-learning approach built on evolutionary features

“…B5055 is a multidrug-resistant K. pneumoniae (40, 41) strain with a K2-type capsule considered indicative of hypervirulent K. pneumoniae (hvKp) (42). To avoid isolating phages that use the major porin for entry into K. pneumoniae (33) and, thus circumvent the prospect of phage-resistance acquired by decreased expression of porins (43) and collateral increases in drug-resistant phenotype in the infection (44), we constructed as bait a strain that has no OmpK36. This Δ ompK36 mutant strain of K. pneumoniae B5055 was constructed by “gene gorging” as previously described (45, 46) utilizing the donor and helper plasmids described in Supplementary Table 10.…”