Fine-tuning carbapenem resistance by reducing porin permeability of bacteria activated in the selection process of conjugation

Abstract

Antibiotic resistance is an emerging public health issue. Plasmids are one of the popular carriers to disseminate resistance genes among pathogens. However, the response of plasmid-carrying bacteria to antibiotic treatment and how these bacteria evolve to increase their resistance remain elusive. In this study, we conjugated plasmid pNDM-HK to E. coli J53 recipient cells and selected survivors using different concentrations of the broad spectrum antibiotic meropenem. After selection, transconjugants conferred varying minimum inhibitory concentrations with respect to carbapenems. We sequenced and compared the transcriptomes of transconjugants that exhibited distinct carbapenem susceptibilities, and found that the loss of outer membrane proteins led to antibiotic resistance. Moreover, we identified a novel mutation, G63S, in transcription factor OmpR which moderates the expression of outer membrane proteins. The loss of porins was due to incapability of phosphorylation, which is essential for porin transcription and carbapenem resistance. We also characterized other genes that are regulated by ompR in this mutant, which contributed to bacterial antibiotic resistance. Overall, our studies suggest antibiotic pressure after conjugation might be an alternative pathway to promote antimicrobial resistance.

Emerging Transcriptional and Genomic Mechanisms Mediating Carbapenem and Polymyxin Resistance in Enterobacteriaceae : a Systematic Review of Current Reports

“…Kong et al (51) revealed that the Gly63Ser amino acid substitution within the N-terminal phosphorylation domain of OmpR affects the phosphorylation of OmpR by EnvZ. The OmpR mutant, after that, failed to initiate porin transcription, resulting in a change in membrane permeability and, subsequently, carbapenem resistance (51). The study further went on to show the synergistic effect of OmpR mutants and carbapenemase activity, with the transformation of OmpR mutants with a NDM-harboring plasmid resulting in a 100-fold increase in the carbapenem MIC (Table 1) (51). SDS-PAGE analysis of the outer membrane porins of OmpR mutants revealed a sharp decrease in the expression of both major porin groups (49).…”

“…The loss of major outer membrane proteins, OmpK36 and OmpK35, is frequently observed in CRE, resulting in reduced permeability of the outer membrane due to structural changes in porin channels restricting the uptake of charged molecules through the bacterial cell wall ( Fig. 1 ) ( 3 , 49 , 51 ). These structural changes were due to mutations that either reduced the channel size of porins or modified its electrostatics ( 52 ).…”

“…This is accomplished through phosphorylation or dephosphorylation of OmpR, the transcriptional factor responsible for porin gene activation ( 54 ). The phosphorylation of OmpR results in a structural change in the protein, increasing its binding affinity to the major porin transcriptional factor binding site ( 51 ). Kong et al ( 51 ) revealed that the Gly63Ser amino acid substitution within the N-terminal phosphorylation domain of OmpR affects the phosphorylation of OmpR by EnvZ.…”

“…The phosphorylation of OmpR results in a structural change in the protein, increasing its binding affinity to the major porin transcriptional factor binding site ( 51 ). Kong et al ( 51 ) revealed that the Gly63Ser amino acid substitution within the N-terminal phosphorylation domain of OmpR affects the phosphorylation of OmpR by EnvZ. The OmpR mutant, after that, failed to initiate porin transcription, resulting in a change in membrane permeability and, subsequently, carbapenem resistance ( 51 ).…”

Mechanism and challenges associated with adaptation and evolution of drug-resistant bacteria: an overview

“…In the case of Escherichia coli certain general porins such as OmpF and PhoE act as a gateway for the diffusion of hydrophilic molecules (Achouak et al, 2001;Fernández & Hancock, 2012). In some cases the regular dosage of antibiotic pressure initiate certain mutational changes on transcription factor leading to the loss of porins expression as indicated by the development of carbapenem resistance in Escherichia coli trans conjugant (Kong et al, 2018). Carbapenem belongs to a class of β-lactam antibiotics which is also effective against disease caused by Pseudomonas aeruginosa (Hong et al, 2015).…”