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

Falchi, Federica A.; Maccagni, Elisa A.; Puccio, Simone; Peano, Clelia; Castro, Cristina De; Palmigiano, Angelo; Garozzo, Domenico; Martorana, Alessandra M.; Polissi, Alessandra; Dehò, Gianni; Sperandeo, Paola

doi:10.1128/jb.00487-17

Cited by 35 publications

(29 citation statements)

References 94 publications

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“…Cell disruption releases the naturally occurring lipopolysaccharides (LPS), also known as endotoxins or pyrogens, from the external cover layer, contaminating the released target biomolecules [2,3]. LPS can act as an immunostimulant that activates the complement system causing pyrogenic reactions in humans if present in parenteral drug products.…”

Section: Introductionmentioning

confidence: 99%

Effect of electrolytes as adjuvants in GFP and LPS partitioning on aqueous two-phase systems: 1. Polymer-polymer systems

Lopes

Molino

Santos-Ebinuma

et al. 2018

Separation and Purification Technology

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The production of recombinant biopharmaceuticals is highly dependent of a proper choice of the downstream processing stages. Particularly, the purification that must ensure that all the endotoxins (lipopolysaccharides-LPS) are efficiently removed from the final product. The efficient removal of LPS has a direct impact on the manufacturing of therapeutic biopharmaceuticals, since LPS is naturally presented in Gram-negative bacterial expression systems. In order to provide a more simple and faster technique for the purification of green fluorescent protein (GFP) and LPS removal, aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and poly(acrylic acid) (NaPA) and electrolytes were studied. Firstly, the binodal curves of PEG/ NaPA + salt systems were established using NaCl, Li 2 SO 4 , KI, and KNO 3 as additives. It was demonstrated that the formation of ATPS is enhanced following the anion tendency: SO 4 2− > Cl − ≫ NO 3 − > I −. The stability of GFP in the presence of all different phase forming agents (polymers and salts) was evaluated, and the high biocompatibility of these ATPS demonstrated by the maintenance of GFP fluorescence in all conditions under study. Then, the GFP extraction and LPS removal aptitude of each ATPS was investigated. GFP and LPS were preferentially partitioned into the top (PEG-rich) phase (K GFP > 20), but with a removal of 35% of LPS was attained. Hydrophobic and electrostatic interactions were found to be the major driving forces for GFP partitioning and LPS removal. Moreover, a new effect was found, the presence of high loads of LPS can affect (decrease) the K GFP values (K GFP without LPS > K GFP with 10 4 EU/mL > K GFP with 10 6 EU/mL). The ATPS with best GFP extraction performance was selected for the recovery directly from the cell lysates of Escherichia coli. In this experiment, the system composed of 12 wt% PEG 1000 g/mol, 12 wt% NaPA 8000 g/mol, and 0.25 M Li 2 SO 4 lead to a LPS removal (REM LPS) of 13%, K GFP of 20.2, and high selectivity relatively to the total proteins (S = 20), since the majority of contaminants proteins were preferentially partitioned into the bottom (NaPArich) phase (purification factor of 4-fold). It is here demonstrated that the ATPS composed of PEG/NaPA + salts as additives can be used as a first step for the recovery of GFP and removal of contaminants (LPS in part, and most contaminant proteins) from cell lysates by applying a system with low polymer content and using mild conditions.

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Section: Introductionmentioning

confidence: 99%

Effect of electrolytes as adjuvants in GFP and LPS partitioning on aqueous two-phase systems: 1. Polymer-polymer systems

Lopes

Molino

Santos-Ebinuma

et al. 2018

Separation and Purification Technology

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“…However, less is known about how modification of cell membrane biochemistry affects resistance. Changes in membrane phospholipid composition was recently shown to increase Staphylococcus aureus resistance to daptomycin (41), and changes in LPS biosynthesis in E. coli increased sensitivity to bacitracin and rifampicin (30). Our work adds to the evidence that resistance can repeatedly evolve through diverse mechanisms.…”

Section: Discussionmentioning

confidence: 55%

“…Mutations in mdoG and mdoH were only observed in monocultures of E. coli and S. enterica under rifampicin selection. Both mdoG and mdoH likely influence cell membrane permeability (29,30). The mutations were not associated with any changes in monoculture or co-culture growth rates (Supplementary figure 2).…”

Section: Resultsmentioning

confidence: 97%

Cross-feeding modulates the rate and mechanism of antibiotic resistance evolution in a model microbial community of Escherichia coli and Salmonella enterica

Adamowicz

Muza

Chacón

et al. 2019

Preprint

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Author contributions: E.M.A, M.A.M., J.M.C. and W.R.H. designed research; E.M.A, M.A.M., and J.M.C. performed research; E.M.A., J.M.C., and W.R.H. analyzed data; and E.M.A., J.M.C., and W.R.H. wrote the paper. AbstractWith antibiotic resistance rates on the rise, it is critical to understand how microbial species interactions influence the evolution of resistance. We have previously shown that in obligate mutualisms the survival of any one species (regardless of its intrinsic resistance) is contingent on the resistance of its cross-feeding partners, setting the community antibiotic tolerance at that of the 'weakest link' species. In this study, we extended that hypothesis to test whether obligate cross-feeding would limit the extent and mechanisms of antibiotic resistance evolution. In both rifampicin and ampicillin treatments, we observed that resistance evolved more slowly in obligate co-cultures of E. coli and S. enterica than in monocultures. While we observed similar mechanisms of resistance arising under rifampicin selection, under ampicillin selection different resistance mechanisms arose in co-cultures and monocultures. In particular, mutations in an essential cell division protein, ftsI, arose in S. enterica only in co-culture. A simple mathematical model demonstrated that reliance on a partner is sufficient to slow the rate of adaptation, and can change the distribution of adaptive mutations that are acquired. Our results demonstrate that cooperative metabolic interactions can be an important modulator of resistance evolution in microbial communities. Significance statementLittle is known about how ecological interactions between bacteria influence the evolution of antibiotic resistance. We tested the impact of metabolic interactions on resistance evolution in an engineered two-species bacterial community. Through experimental and modeling work, we found that obligate metabolic interdependency slows the rate of resistance acquisition, and can change the type and magnitude of resistance mutations that evolve. This work suggests that resistance evolution may be slowed by targeting both a pathogen and its metabolic partners with antibiotics. Additionally, we showed that community context can generate novel trajectories through which antibiotic resistance evolves.

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“…All but one of these mutations were insertions or deletions of 1-6bp causing frameshift mutations (S1 Table). Both mdoG and mdoH likely influence cell membrane permeability [31,32]. The mutations were not associated with any changes in monoculture or co-culture growth rates (S3 Fig).…”

Section: S2 Fig)mentioning

confidence: 98%

Cross-feeding modulates the rate and mechanism of antibiotic resistance evolution in a model microbial community of Escherichia coli and Salmonella enterica

et al. 2020

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With antibiotic resistance rates on the rise, it is critical to understand how microbial species interactions influence the evolution of resistance. In obligate mutualisms, the survival of any one species (regardless of its intrinsic resistance) is contingent on the resistance of its cross-feeding partners. This sets the community antibiotic sensitivity at that of the 'weakest link' species. In this study, we tested the hypothesis that weakest link dynamics in an obligate cross-feeding relationship would limit the extent and mechanisms of antibiotic resistance evolution. We experimentally evolved an obligate co-culture and monoculture controls along gradients of two different antibiotics. We measured the rate at which each treatment increased antibiotic resistance, and sequenced terminal populations to question whether mutations differed between mono-and co-cultures. In both rifampicin and ampicillin treatments, we observed that resistance evolved more slowly in obligate co-cultures of E. coli and S. enterica than in monocultures. While we observed similar mechanisms of resistance arising under rifampicin selection, under ampicillin selection different resistance mechanisms arose in co-cultures and monocultures. In particular, mutations in an essential cell division protein, ftsI, arose in S. enterica only in co-culture. A simple mathematical model demonstrated that reliance on a partner is sufficient to slow the rate of adaptation, and can change the distribution of adaptive mutations that are acquired. Our results demonstrate that cooperative metabolic interactions can be an important modulator of resistance evolution in microbial communities.

show abstract

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

Cited by 35 publications

References 94 publications

Effect of electrolytes as adjuvants in GFP and LPS partitioning on aqueous two-phase systems: 1. Polymer-polymer systems

Effect of electrolytes as adjuvants in GFP and LPS partitioning on aqueous two-phase systems: 1. Polymer-polymer systems

Cross-feeding modulates the rate and mechanism of antibiotic resistance evolution in a model microbial community of Escherichia coli and Salmonella enterica

Cross-feeding modulates the rate and mechanism of antibiotic resistance evolution in a model microbial community of Escherichia coli and Salmonella enterica

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