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Published on: May 2, 2018
OmpK36 GD insertion induces RpoS-dependent blaKPC overexpression in carbapenem-resistant Klebsiella pneumoniae
Jinnuo Zhu1, Yizhen Wang1, Changqing Mei2
1Department of Clinical Laboratory, Graduate Joint Training Base of Zhejiang Chinese Medical University (Lishui Joint Training Base, Lishui Central Hospital), Lishui, China.
Abstract:
Gly115-Asp116 (GD) insertion in the outer membrane porin OmpK36 is prevalent in carbapenem-resistant, KPC-producing Klebsiella pneumoniae, but the molecular mechanisms linking this mutation to enhanced resistance cannot be explained simply by reduced drug permeability. Given that porin mutations compromise bacterial fitness, we investigated the potential role of the stress response regulator RpoS in OmpK36-mediated carbapenemase regulation. Our results demonstrated that among 127 clinical isolates, OmpK36 GD insertion occurred in 50.4% of isolates and was associated with a significantly elevated carbapenem minimum inhibitory concentration and enhanced high-level resistance. In genetically modified strains, the OmpK36 GD insertion impaired bacterial growth and led to a 3.7-fold upregulated rpoS expression and a 2.7-fold increase in blaKPC-2 expression. Bioinformatics analysis identified a putative RpoS binding site in the blaKPC promoter region, which was validated by electrophoretic mobility shift assays. Moreover, RpoS overexpression enhanced blaKPC-2 expression 1.7-fold and increased carbapenem resistance, while disruption of the RpoS binding site reduced blaKPC-2 expression fourfold and decreased resistance levels. Subinhibitory antibiotic treatment upregulated both rpoS and blaKPC-2 expression across multiple strains, further validating a RpoS-mediated regulatory mechanism. These results suggest that OmpK36 GD insertion contributes to carbapenem resistance through RpoS-mediated transcriptional upregulation of blaKPC, revealing a stress-activated regulatory mechanism linking porin structural alterations to modulated carbapenemase expression in K. pneumoniae.
Insights
The OmpK36 GD insertion in Klebsiella pneumoniae upregulates the stress regulator RpoS, which in turn boosts carbapenemase expression, increasing resistance. This reveals a stress-activated mechanism linking porin changes to carbapenem resistance in K. pneumoniae.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Carbapenem resistance in Klebsiella pneumoniae is a major public health threat.
- The OmpK36 porin mutation, specifically the Gly115-Asp116 (GD) insertion, is common in carbapenem-resistant strains.
- Reduced drug permeability alone does not fully explain the enhanced resistance.
Purpose of the Study:
- To investigate the role of the stress response regulator RpoS in OmpK36-mediated carbapenemase regulation.
- To elucidate the molecular mechanisms linking OmpK36 GD insertion to carbapenem resistance in K. pneumoniae.
Main Methods:
- Analysis of 127 clinical isolates for OmpK36 GD insertion prevalence and carbapenem resistance.
- Genetic modification of K. pneumoniae strains to study the effects of OmpK36 GD insertion on rpoS and blaKPC expression.
- Bioinformatics analysis to identify RpoS binding sites in the blaKPC promoter.
- Electrophoretic mobility shift assays (EMSAs) to validate RpoS binding.
- Overexpression and disruption studies of RpoS and its binding site.
Main Results:
- OmpK36 GD insertion was found in 50.4% of isolates, associated with higher carbapenem minimum inhibitory concentrations.
- OmpK36 GD insertion impaired bacterial growth, upregulated rpoS expression (3.7-fold), and increased blaKPC-2 expression (2.7-fold).
- RpoS binding to the blaKPC promoter was confirmed; RpoS overexpression increased resistance, while binding site disruption reduced blaKPC-2 expression (fourfold) and resistance.
Conclusions:
- OmpK36 GD insertion contributes to carbapenem resistance in K. pneumoniae.
- This occurs via RpoS-mediated transcriptional upregulation of blaKPC.
- A stress-activated regulatory mechanism links porin alterations to carbapenemase expression.
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