Dissecting Escherichia coli Outer Membrane Biogenesis Using Differential Proteomics

Martorana, Alessandra M.; Motta, Sara; Silvestre, Dario Di; Falchi, Federica A.; Dehò, Gianni; Mauri, Pierluigi; Sperandeo, Paola; Polissi, Alessandra

doi:10.1371/journal.pone.0100941

Cited by 32 publications

(28 citation statements)

References 83 publications

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“…2A). In keeping with this finding, our previous transcriptomics results showed that colistin treatment up-regulated the expression of the Mla system ( M aintenance of OM l ipid a symmetry) in A. baumannii ATCC 19606, which is responsible for transporting excess phospholipids in the outer leaflet back to the inner membrane to maintain the OM asymmetry415152. Significant changes to the OM lipids, as observed at both the transcriptomics and metabolomics levels, are highly consistent with the proposed bactericidal mechanism of colistin via lipid exchange between the inner and outer membrane11.…”

Section: Discussionsupporting

confidence: 63%

Untargeted metabolomics analysis reveals key pathways responsible for the synergistic killing of colistin and doripenem combination against Acinetobacter baumannii

Maifiah

Creek

Nation

et al. 2017

Sci Rep

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Combination therapy is deployed for the treatment of multidrug-resistant Acinetobacter baumannii, as it can rapidly develop resistance to current antibiotics. This is the first study to investigate the synergistic effect of colistin/doripenem combination on the metabolome of A. baumannii. The metabolite levels were measured using LC-MS following treatment with colistin (2 mg/L) or doripenem (25 mg/L) alone, and their combination at 15 min, 1 hr and 4 hr (n = 4). Colistin caused early (15 min and 1 hr) disruption of the bacterial outer membrane and cell wall, as demonstrated by perturbation of glycerophospholipids and fatty acids. Concentrations of peptidoglycan biosynthesis metabolites decreased at 4 hr by doripenem alone, reflecting its mechanism of action. The combination induced significant changes to more key metabolic pathways relative to either monotherapy. Down-regulation of cell wall biosynthesis (via D-sedoheptulose 7-phosphate) and nucleotide metabolism (via D-ribose 5-phosphate) was associated with perturbations in the pentose phosphate pathway induced initially by colistin (15 min and 1 hr) and later by doripenem (4 hr). We discovered that the combination synergistically killed A. baumannii via time-dependent inhibition of different key metabolic pathways. Our study highlights the significant potential of systems pharmacology in elucidating the mechanism of synergy and optimizing antibiotic pharmacokinetics/pharmacodynamics.

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Section: Discussionsupporting

confidence: 63%

Untargeted metabolomics analysis reveals key pathways responsible for the synergistic killing of colistin and doripenem combination against Acinetobacter baumannii

Maifiah

Creek

Nation

et al. 2017

Sci Rep

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“…Notably, the same mlaA in-frame deletion (named mlaA*) has been recently isolated as responsible for suppressing the bacitracin sensitivity of an lptD mutant defective in LPS assembly at the OM (44). Moreover, the IM MlaD protein increases in abundance in cells where LPS transport is severely impaired (63). These findings further strengthen a functional link between the Mla system and the Lpt machinery.…”

Section: Discussionmentioning

confidence: 75%

Mutation and Suppressor Analysis of the Essential Lipopolysaccharide Transport Protein LptA Reveals Strategies To Overcome Severe Outer Membrane Permeability Defects in Escherichia coli

et al. 2018

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In Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane (OM), preventing the entry of toxic molecules, such as detergents and antibiotics. LPS is transported from the inner membrane (IM) to the OM by the Lpt multiprotein machinery. Defects in LPS transport compromise LPS assembly at the OM and result in increased antibiotic sensitivity. LptA is a key component of the Lpt machine that interacts with the IM protein LptC and chaperones LPS through the periplasm. We report here the construction of , a quadruple mutant in four conserved amino acids potentially involved in LPS or LptC binding. Although viable, the mutant displays increased sensitivity to several antibiotics (bacitracin, rifampin, and novobiocin) and the detergent SDS, suggesting that affects LPS transport. Indeed, is defective in Lpt complex assembly, and its lipid A carries modifications diagnostic of LPS transport defects. We also selected and characterized two phenotypic bacitracin-resistant suppressors of One mutant, in which only bacitracin sensitivity is suppressed, harbors a small in-frame deletion in , which codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other mutant, in which bacitracin, rifampin, and SDS sensitivity is suppressed, harbors an additional amino acid substitution in LptA41 and a nonsense mutation in, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharide synthesis. Characterization of the suppressor mutants highlights different strategies adopted by the cell to overcome OM defects caused by impaired LPS transport. Lipopolysaccharide (LPS) is the major constituent of the outer membrane (OM) of most Gram-negative bacteria, forming a barrier against antibiotics. LPS is synthesized at the inner membrane (IM), transported across the periplasm, and assembled at the OM by the multiprotein Lpt complex. LptA is the periplasmic component of the Lpt complex, which bridges IM and OM and ferries LPS across the periplasm. How the cell coordinates the processes involved in OM biogenesis is not completely understood. We generated a mutant partially defective in that exhibited increased sensitivity to antibiotics and selected for suppressors of the mutant. The analysis of two independent suppressors revealed different strategies adopted by the cell to overcome defects in LPS biogenesis.

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“…For this we induced severe OM assembly stress by depleting LptC, an essential component of the lipopolysaccharide (LPS) export machinery. It is known that E. coli arrests growth but is capable to survive the depletion of lptC (25). We grew cells of BW25113araBplptC in the presence of arabinose and then inoculated them into growth media with or without arabinose, and in the presence or absence of copper chloride, and followed cell growth (optical density, OD) and viability (colony forming units, CFU) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Copper inhibits peptidoglycan LD-transpeptidases suppressing β-lactam resistance due to bypass of penicillin-binding proteins

Peters

Pazos

Edoo

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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The peptidoglycan (PG) layer stabilizes the bacterial cell envelope to maintain the integrity and shape of the cell. Penicillin-binding proteins (PBPs) synthesize essential 4–3 cross-links in PG and are inhibited by β-lactam antibiotics. Some clinical isolates and laboratory strains of Enterococcus faecium and Escherichia coli achieve high-level β-lactam resistance by utilizing β-lactam–insensitive LD-transpeptidases (LDTs) to produce exclusively 3–3 cross-links in PG, bypassing the PBPs. In E. coli, other LDTs covalently attach the lipoprotein Lpp to PG to stabilize the envelope and maintain the permeability barrier function of the outermembrane. Here we show that subminimal inhibitory concentration of copper chloride sensitizes E. coli cells to sodium dodecyl sulfate and impair survival upon LPS transport stress, indicating reduced cell envelope robustness. Cells grown in the presence of copper chloride lacked 3–3 cross-links in PG and displayed reduced covalent attachment of Braun’s lipoprotein and reduced incorporation of a fluorescent d-amino acid, suggesting inhibition of LDTs. Copper dramatically decreased the minimal inhibitory concentration of ampicillin in E. coli and E. faecium strains with a resistance mechanism relying on LDTs and inhibited purified LDTs at submillimolar concentrations. Hence, our work reveals how copper affects bacterial cell envelope stability and counteracts LDT-mediated β-lactam resistance.

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Dissecting Escherichia coli Outer Membrane Biogenesis Using Differential Proteomics

Cited by 32 publications

References 83 publications

Untargeted metabolomics analysis reveals key pathways responsible for the synergistic killing of colistin and doripenem combination against Acinetobacter baumannii

Untargeted metabolomics analysis reveals key pathways responsible for the synergistic killing of colistin and doripenem combination against Acinetobacter baumannii

Mutation and Suppressor Analysis of the Essential Lipopolysaccharide Transport Protein LptA Reveals Strategies To Overcome Severe Outer Membrane Permeability Defects in Escherichia coli

Copper inhibits peptidoglycan LD-transpeptidases suppressing β-lactam resistance due to bypass of penicillin-binding proteins

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