Functional Characterization of <i>E. coli</i> LptC: Interaction with LPS and a Synthetic Ligand

Sestito, Stefania E.; Sperandeo, Paola; Santambrogio, Carlo; Ciaramelli, Carlotta; Calabrese, Valentina; Rovati, G. Enrico; Zambelloni, Luca; Grandori, Rita; Polissi, Alessandra; Peri, Francesco

doi:10.1002/cbic.201300805

Cited by 18 publications

(11 citation statements)

References 41 publications

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“…In vitro studies on the soluble domain of LptC (amino acids 24–191) show that LPS co‐elutes with 6xHis‐tagged LptC from affinity resin and suggest that the transfer of LPS from LptC to LptA is unidirectional . In addition, a dissociation constant was determined for LptC:LOS (lipo‐oligosaccharide, LPS lacking the O‐antigen) . In vivo studies on 23 mutants of LptC substituted with the unnatural amino acid p PBA indicated that LPS could cross‐link with four of the sites tested, the N‐terminal site T47 p PBA and interior‐facing sites F78 p PBA, A172 p PBA, and Y182 p PBA upon irradiation with UV light, supporting the hypothesis that LPS binds to the interior fold of LptC .…”

Section: Introductionmentioning

confidence: 99%

Characterization of and lipopolysaccharide binding to the E. coliLptC protein dimer

Schultz

Klug

2017

Protein Science

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Lipopolysaccharide (LPS, endotoxin) is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium. LPS is a large lipid containing several acyl chains as its hydrophobic base and numerous sugars as its hydrophilic core and O-antigen domains, and is an essential element of the organisms' natural defenses in adverse environmental conditions. LptC is one of seven members of the lipopolysaccharide transport (Lpt) protein family that functions to transport LPS from the inner membrane (IM) to the outer leaflet of the outer membrane of the bacterium. LptC is anchored to the IM and associated with the IM LptFGB complex. It is hypothesized that LPS binds to LptC at the IM, transfers to LptA to cross the periplasm, and is inserted by LptDE into the outer leaflet of the outer membrane. The studies described here comprehensively characterize and quantitate the binding of LPS to LptC. Site-directed spin labeling electron paramagnetic resonance spectroscopy was utilized to characterize the LptC dimer in solution and monitor spin label mobility changes at 10 sites across the protein upon addition of exogenous LPS. The results indicate that soluble LptC forms concentration-independent N-terminal dimers in solution, LptA binding does not change the conformation of the LptC dimer nor appreciably disrupt the LptC dimer in vitro, and LPS binding affects the entire LptC protein, with the center and C-terminal regions showing a greater affinity for LPS than the N-terminal domain, which has similar dissociation constants to LptA.

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Section: Introductionmentioning

confidence: 99%

Characterization of and lipopolysaccharide binding to the E. coliLptC protein dimer

Schultz

Klug

2017

Protein Science

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“…The in vitro LPS binding assay was based on a previous protocol with a few modifications (Sestito et al 2014). Briefly, purified ArnT protein (200 µg) was incubated in 0.5 mL of Phosphate/NaCl buffer (50 mM phosphate buffer, pH 8.0, 150 mM NaCl) with 0.06 mL of HisLink protein purification resin (Promega) for 90 min on a rotary shaker at 4°C.…”

Section: Lps Binding By Resin-capture Methodsmentioning

confidence: 99%

“…This region in the predicted ArnT model is the only one that has a pocket of a diameter such that could accommodate the lipid A molecule. To explore this hypothesis, we performed an in vitro binding assay using purified ArnT proteins and a resin-capture procedure previously described (Sestito et al 2014). ArnT was immobilized on the Ni 2+ -NTA resin and then Alexa-conjugated LPS from S. enterica Serovar Minnesota was added.…”

Section: The C-terminal Domain Of Arnt Is Required For Lps Bindingmentioning

confidence: 99%

ArnT proteins that catalyze the glycosylation of lipopolysaccharide share common features with bacterialN-oligosaccharyltransferases

Tavares-Carreón¹,

Mohamed²,

Andrade³

et al. 2015

Glycobiology

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ArnT is a glycosyltransferase that catalyzes the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A moiety of the lipopolysaccharide. This is a critical modification enabling bacteria to resist killing by antimicrobial peptides. ArnT is an integral inner membrane protein consisting of 13 predicted transmembrane helices and a large periplasmic C-terminal domain. We report here the identification of a functional motif with a canonical consensus sequence DEXRYAX (5) MX (3) GXWX (9) YFEKPX (4) W spanning the first periplasmic loop, which is highly conserved in all ArnT proteins examined. Site-directed mutagenesis demonstrated the contribution of this motif in ArnT function, suggesting that these proteins have a common mechanism. We also demonstrate that the Burkholderia cenocepacia and Salmonella enterica serovar Typhimurium ArnT C-terminal domain is required for polymyxin B resistance in vivo. Deletion of the C-terminal domain in B. cenocepacia ArnT resulted in a protein with significantly reduced in vitro binding to a lipid A fluorescent substrate and unable to catalyze lipid A modification with L-Ara4N. An in silico predicted structural model of ArnT strongly resembled the tertiary structure of Campylobacter lari PglB, a bacterial oligosaccharyltransferase involved in protein N-glycosylation. Therefore, distantly related oligosaccharyltransferases from ArnT and PglB families operating on lipid and polypeptide substrates, respectively, share unexpected structural similarity that could not be predicted from direct amino acid sequence comparisons. We propose that lipid A and protein glycosylation enzymes share a conserved catalytic mechanism despite their evolutionary divergence.

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“…Based on Lpt architecture and function, either the assembly of the machinery or the binding of individual proteins to LPS can be targeted and inhibited. Inhibitors of the LptB ATPase and of the LptC IM bitopic proteins have been identified. Still, none of these compounds is able to kill bacteria possessing an intact OM, highlighting once more the importance of the OM barrier properties for bacterial survival.…”

Section: Targeting Lps Biogenesis Pathway For the Discovery Of Novel mentioning

confidence: 99%

Fat Matters for Bugs: How Lipids and Lipid Modifications Make the Difference in Bacterial Life

Sperandeo

Polissi

Fabiani

2019

Euro J Lipid Sci & Tech

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Lipids are building blocks of biological membranes in the three domains of life and are endowed with several important biological functions. Lipopolysaccharide (LPS) is a peculiar glycolipid that represents the hallmark of the cell envelope of Gram‐negative bacteria, a group comprising several important human pathogens. The presence of LPS in the outer membrane (OM) of Gram‐negative bacteria correlates not only with the intrinsic resistance of this group of bacteria toward several antibiotics currently in use, but also with the worrisome increase in antibiotic resistance that is depleting the arsenal of efficacious molecules to cure bacterial infections. LPS consists of a conserved internal moiety decorated by a more variable distal unit. Several modifications occur at the canonical LPS structure during bacterial infection which are exploited by the microorganism to co‐evolve with the host thus evading its immune system. This viewpoint article discusses the current knowledge on LPS biogenesis and modification systems and their consequences on recognition by immune cells and antimicrobial resistance. It also discusses how knowledge on LPS biogenesis can be exploited to develop molecules able to dismantle the Gram‐negative protective barrier and to interfere with the interaction with the mammalian host. Practical Applications: Antibiotic resistance is a major threat for human health leading to inefficacy of many antibiotics to treat infectious diseases. Resistance to antibiotics causes around 25 000 deaths per year in the European Union alone and 700 000 deaths per year globally. This results in an increase of 1.5 billion euros per year in healthcare costs and productivity losses for illness. Gram‐negative bacteria are intrinsically resistant to many antibiotics, due to their LPS‐coated cell surface that protects them from the environment. LPS is sensed by the host immune system as a signal of bacterial infection, moreover bacteria exploit the complexity of LPS modifications to modulate the host immune response and eventually escape it. A deep understanding of the molecular mechanisms underlying LPS biogenesis, including the physico‐chemical and biological consequences of its lipid modifications, will be instrumental to develop novel antibiotic treatments aimed at dismantling the Gram‐negative bacteria barrier and restoring drug sensitivity. The hallmark of Gram‐negative bacteria is the presence of an asymmetric outer membrane (OM) surrounding the cytoplasmic membrane (inner membrane, IM), whose outer leaflet is made by lipopolysaccharide (LPS). A layer of peptidoglycan (PG) is sandwiched in between. LPS endows the bacteria with increased resistance to many antibiotics and is the first line of interaction with the host cell immune system. Chemical modifications of the canonical LPS structure determine different stimulatory effects of the immune response and allow the bacteria to escape from the killing action of effector molecules such as antimicrobial peptides.

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Functional Characterization of E. coli LptC: Interaction with LPS and a Synthetic Ligand

Cited by 18 publications

References 41 publications

Characterization of and lipopolysaccharide binding to the E. coliLptC protein dimer

Characterization of and lipopolysaccharide binding to the E. coliLptC protein dimer

ArnT proteins that catalyze the glycosylation of lipopolysaccharide share common features with bacterialN-oligosaccharyltransferases

Fat Matters for Bugs: How Lipids and Lipid Modifications Make the Difference in Bacterial Life

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