Establishing outer membrane Integrity in Klebsiella pneumoniae

Abstract

The Outer Membrane (OM) of Gram-negative bacteria is the primary barrier for the cell, with its asymmetric composition of lipopolysaccharides (LPS) at the outer leaflet and glycerophospholipids (GPL) at the inner leaflet. The stability of this membrane is important not only as a barrier but also for viability of the cell as it provides an anchor for all outer membrane components necessary for bacterial survival. Thus, defects in the outer membrane, exacerbated by the packing and flipping of GPLs from the inner leaflet to the outer leaflet, allow for the diffusion of compounds that are toxic to the cell, and critically membrane integrity. Despite the apparent importance of the outer membrane, the implications of OM defects on drug- or host-pathogen interactions is not well studied.

Antibiotic resistance is a major health priority, which is exacerbated by the increased antibiotic use, rising rates of bacterial antibiotic resistance and a reducing pool of effective drugs. Whilst horizontal gene transfer remains a major route of AMR gene acquisition, chromosomally encoded intrinsic systems play a significant role in conferring protection against both antibiotics and the host immune response. In Klebsiella pneumoniae, the intrinsic transcriptional regulator, RamA, has been shown to regulate membrane permeability via the activation of increased efflux (acrAB) and decreased influx (ompF). Furthermore, RamA has been shown to alter host-pathogen interactions via changes to lipid A, which has been implicated in immune evasion and increased dissemination. As such, RamA remains a key regulator of membrane permeability and integrity in Klebsiella pneumoniae.

Transcriptome analyses to define the RamA regulon and proteomic analyses to establish RamA-dependent membrane changes also demonstrate that RamA functions as an activator of the mla (maintaining lipid membrane asymmetry) operon. The recently described mla operon has been attributed with trafficking of GPLs to and from the OM in addition to promoting outer membrane vesicle biogenesis. The mla operon is also involved in host-/ drug-pathogen interactions, as it is responsive to iron limitation via the Fur repressor in vivo and contributes to antibiotic susceptibility. Taken together, the overriding hypothesis of this work is the RamA-mediated activation of the mla operon results in altered OM composition and stability thus contributing to changes in host-pathogen interactions. To address this hypothesis, we aimed to investigate the mla operon, and its contribution to the RamA phenotype in K. pneumoniae, by specifically characterising the changes in outer membrane stability and integrity. Therefore, the specific aims were, to determine the level of changes in membrane stability caused by mla overexpression in a ΔramR background, the effect of these outer membrane changes on drug- and host-pathogen interactions.

The RamA overexpressing strain (Ecl8ΔramR) showed significant increase in membrane stability compared to the wildtype Ecl8, when plated on SDS-EDTA plates, a common OM integrity assay. This marked increase was in part due to the mla operon, as deletion of mlaCDEF in the Ecl8ΔramR showed a decrease in survival. The same decrease was also noted in the Ecl8 background in mlaCDEF deletions. Further to this, membrane hydrophobicity, an indicator of GPL accumulation at the OM, as measured via xylene and crystal violet assays, showed a marked decrease in the Ecl8ΔramR compared to Ecl8, whilst mla deletions in the Ecl8ΔramR and Ecl8 showing an increase in hydrophobicity.

Changes to the GPL content at the OM and IM in the Ecl8, Ecl8ΔramR and mlaE deletions were investigated via Thin layer Chromatography and Gas Chromatography, to examine changes in head group and fatty acid tails respectively. A significant increase of phosphatidylethanolamine (PE), one of the most abundant GPLs, was seen at the OM in Ecl8ΔramR compared to Ecl8. A PagP assay, that cleaves palmitic acid tails from OM outer leaflet exposed GPLs, demonstrated that this increase of PE was confined to the inner leaflet of the OM, confirming that RamA overexpression leads to alterations of the OM to maintain stability. The mlaE deletion in an Ecl8 background demonstrated an increase in PE content at the OM, however the same increase was not noted in an mlaE deletion in the RamA overexpressing background Ecl8ΔramR. Via a PagP assay the mlaE deletion in both Ecl8 and Ecl8ΔramR backgrounds showed a significant increase in surface exposed GPLs, confirming the phenotypic data that mla expression is responsible for the reduction of GPL accumulation at the outer leaflet of the OM, maintaining membrane stability. Gas chromatography (GC) of the IM and OM fatty acid tails additionally demonstrated that Ecl8ΔramR showed a significant increase in palmitic acid levels at the OM compared to Ecl8, with a slight decrease noted in the Ecl8ΔramR mlaE deletion. The mlaE deletions in both backgrounds showed imbalance in the palmitic acid and vaccenic acid tails in the OM and IM. The lipidomic work indicates that overexpression of RamA led to changes in GPL and fatty acid content at the OM, some dependant of the mla operon and others via potential alterations of other pathways, specifically GPL and Fatty acid (FA) synthesis, leading to the reduced susceptibility phenotype.

More importantly when a ramR deletion in Kp52145 was created, the same trends as Ecl8ΔramR were seen, demonstrating the changes were not strain dependant.

Due to RamA dependant but mla independent changes being noted at the OM, it was important to investigate further the role that RamA could be having on GPL synthesis. This was carried out via metabolomics and proteomics, specifically focussing at FA and GPL synthesis proteins.

Importantly, Ecl8ΔramR showed a significant increase in several FAS II proteins, with expression variation known to affect FA tail length, along with PssA the first committed step to PE synthesis. In addition, Glucose-6-phosphate dehydrogenase (zwf), the main producer of NADPH, also showed a significant increase in Ecl8ΔramR (8.1-fold), whilst in a gene reporter fusion assay it was significantly upregulated. With a 5.4-fold NADPH increase in Ecl8ΔramR compared to Ecl8. NADPH is a key cofactor for the FA elongation pathway and turnover. In conjunction with changes seen at the OM via TLC and GC, this demonstrated that RamA overexpression influenced GPL composition as well as having a tight control of the OM/IM via mla trafficking. As the OM is the primary point of contact it is given that any changes would immediately influence host-pathogen interactions. Using pooled human serum, it had already been established that Ecl8ΔramR was more resistant than Ecl8 to complement. With an mlaE deletion, an increase in sensitivity was observed relative to the wild type and RamA overexpressor. In vivo infections of G. mellonella, showed an increase in killing, melanisation and dissemination in Ecl8ΔramR compared to Ecl8, demonstrating an increase in virulence.

Interestingly, the mlaE deletion in Ecl8 showed a significant increase in killing and melanisation, suggesting a role for the mla operon directly in virulence.

However, the mlaE deletion in Ecl8ΔramR did not show a significant change post 24hrs for any of the assays. In conjunction with the complement assay, this would suggest that the RamA regulation of the mla operon plays an important role in immune evasion.

Therefore, this study shows that the changes at the OM mediated by RamA have a key role in altering antibiotic susceptibility and host-pathogen interactions; where these perturbations are not limited to is the active efflux and influx systems but affect the actual outer membrane barrier which has significant implications to the host- and drug- pathogen interactions.