Serena Leone

The human milk oligosaccharide 2′-fucosyllactose modulates CD14 expression in human enterocytes, thereby attenuating LPS-induced inflammation

Background A major cause of enteric infection, Gram-negative pathogenic bacteria activate mucosal inflammation through lipopolysaccharide (LPS) binding to intestinal toll-like receptor 4 (TLR4). Breast feeding lowers risk of disease, and human milk modulates inflammation. Objective This study tested whether human milk oligosaccharides (HMOSs) influence pathogenic Escherichia coli-induced interleukin (IL)-8 release by intestinal epithelial cells (IECs), identified specific proinflammatory signalling molecules modulated by HMOSs, specified the active HMOS and determined its mechanism of action. Methods Models of inflammation were IECs invaded by type 1 pili enterotoxigenic E. coli (ETEC) in vitro: T84 modelled mature, and H4 modelled immature IECs. LPS-induced signalling molecules co-varying with IL-8 release in the presence or absence of HMOSs were identified. Knockdown and overexpression verified signalling mediators. The oligosaccharide responsible for altered signalling was identified. Results HMOSs attenuated LPS-dependent induction of IL-8 caused by ETEC, uropathogenic E. coli, and adherent-invasive E. coli (AIEC) infection, and suppressed CD14 transcription and translation. CD14 knockdown recapitulated HMOS-induced attenuation. Overexpression of CD14 increased the inflammatory response to ETEC and sensitivity to inhibition by HMOSs. 2′-fucosyllactose (2′-FL), at milk concentrations, displayed equivalent ability as total HMOSs to suppress CD14 expression, and protected AIEC-infected mice. Conclusions HMOSs and 2′-FL directly inhibit LPS-mediated inflammation during ETEC invasion of T84 and H4 IECs through attenuation of CD14 induction. CD14 expression mediates LPS-TLR4 stimulation of portions of the ‘macrophage migration inhibitory factors’ inflammatory pathway via suppressors of cytokine signalling 2/signal transducer and activator of transcription 3/NF-κB. HMOS direct inhibition of inflammation supports its functioning as an innate immune system whereby the mother protects her vulnerable neonate through her milk. 2′-FL, a principal HMOS, quenches inflammatory signalling.

Human colostrum oligosaccharides modulate major immunologic pathways of immature human intestine

The immature neonatal intestinal immune system hyperreacts to newly colonizing unfamiliar bacteria. The hypothesis that human milk oligosaccharides from colostrum (cHMOSs) can directly modulate the signaling pathways of the immature mucosa was tested. Modulation of cytokine immune signaling by HMOSs was measured ex vivo in intact immature (fetal) human intestinal mucosa. From the genes whose transcription was modulated by cHMOSs, Ingenuity Pathway Analysis identified networks controlling immune cell communication, intestinal mucosal immune system differentiation, and homeostasis. cHMOSs attenuate pathogen-associated molecular pattern-stimulated acute phase inflammatory cytokine protein levels (interleukin-8 (IL-8), IL-6, monocyte chemoattractant protein-1/2 and IL-1β), while elevating cytokines involved in tissue repair and homeostasis. In all, 3′-, 4-, and 6′-galactosyllactoses of cHMOSs account for specific immunomodulation of polyinosinic:polycytodylic acid-induced IL-8 levels. cHMOSs attenuate mucosal responses to surface inflammatory stimuli during early development, while enhancing signals that support maturation of the intestinal mucosal immune system.

NMR Spectroscopic Assignment of Backbone and Side-Chain Protons in Fully Protonated Proteins: Microcrystals, Sedimented Assemblies, and Amyloid Fibrils.

We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid-state NMR spectroscopy with 100-111 kHz magic-angle spinning (MAS). The excellent resolution in the Cα-Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα-Hα detection block was developed and applied for the sequence-specific backbone and aliphatic side-chain resonance assignment using only 500 μg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.

Detailed characterization of the lipid A fraction from the nonpathogen Acinetobacter radioresistens strain S13.

The genus Acinetobacter is composed of ubiquitous, generally nonpathogen environmental bacteria. Interest concerning these microorganisms has increased during the last 30 years, because some strains, belonging to the so-called A. baumannii-A. calcoaceticus complex, have been implicated in some severe pathological states in debilitated and hospitalized patients. The involvement of lipopolysaccharides (LPSs) as virulence factors in infections by Acinetobacter has been proven, and ongoing studies are aimed toward the complete serological characterization of the O-polysaccharides from LPSs isolated in clinical samples. Conversely, no characterization of the lipid A fraction from Acinetobacter strains has been performed. Here, the detailed structure of the lipid A fraction from A. radioresistens S13 is reported for the first time. A. radioresistens strains have never been isolated in cases of infectious disease. Nevertheless, it is known that the lipid A structure, with minor variations, is highly conserved across the genus; thus, structural details acquired from studies of this nonpathogen strain represent a useful basis for further studies of pathogen species.

Design of sweet protein based sweeteners: hints from structure-function relationships.

Sweet proteins represent a class of natural molecules, which are extremely interesting regarding their potential use as safe low-calories sweeteners for individuals who need to control sugar intake, such as obese or diabetic subjects. Punctual mutations of amino acid residues of MNEI, a single chain derivative of the natural sweet protein monellin, allow the modulation of its taste. In this study we present a structural and functional comparison between MNEI and a sweeter mutant Y65R, containing an extra positive charge on the protein surface, in conditions mimicking those of typical beverages. Y65R exhibits superior sweetness in all the experimental conditions tested, has a better solubility at mild acidic pH and preserves a significant thermal stability in a wide range of pH conditions, although slightly lower than MNEI. Our findings confirm the advantages of structure-guided protein engineering to design improved low-calorie sweeteners and excipients for food and pharmaceutical preparations.

Molecular Dynamics Driven Design of pH-Stabilized Mutants of MNEI, a Sweet Protein

MNEI is a single chain derivative of monellin, a plant protein that can interact with the human sweet taste receptor, being therefore perceived as sweet. This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry. Unfortunately, applications of MNEI have been so far limited by its intrinsic sensitivity to some pH and temperature conditions, which could occur in industrial processes. Changes in physical parameters can, in fact, lead to irreversible protein denaturation, as well as aggregation and precipitation. It has been previously shown that the correlation between pH and stability in MNEI derives from the presence of a single glutamic residue in a hydrophobic pocket of the protein. We have used molecular dynamics to study the consequences, at the atomic level, of the protonation state of such residue and have identified the network of intramolecular interactions responsible for MNEI stability at acidic pH. Based on this information, we have designed a pH-independent, stabilized mutant of MNEI and confirmed its increased stability by both molecular modeling and experimental techniques.

Sweeter and stronger: enhancing sweetness and stability of the single chain monellin MNEI through molecular design

Sweet proteins are a family of proteins with no structure or sequence homology, able to elicit a sweet sensation in humans through their interaction with the dimeric T1R2-T1R3 sweet receptor. In particular, monellin and its single chain derivative (MNEI) are among the sweetest proteins known to men. Starting from a careful analysis of the surface electrostatic potentials, we have designed new mutants of MNEI with enhanced sweetness. Then, we have included in the most promising variant the stabilising mutation E23Q, obtaining a construct with enhanced performances, which combines extreme sweetness to high, pH-independent, thermal stability. The resulting mutant, with a sweetness threshold of only 0.28 mg/L (25 nM) is the strongest sweetener known to date. All the new proteins have been produced and purified and the structures of the most powerful mutants have been solved by X-ray crystallography. Docking studies have then confirmed the rationale of their interaction with the human sweet receptor, hinting at a previously unpredicted role of plasticity in said interaction.

Influence of pH on the structure and stability of the sweet protein MNEI

MNEI is a single‐chain derivative of the sweet protein monellin that, in recent years, has become an accepted model for studying protein dynamic properties such as folding and aggregation. Although MNEI is very resistant at acidic pH, exposure to neutral or alkaline pH strongly affects its stability. We have performed a thorough NMR study of the dynamic properties of MNEI at different pHs. The results demonstrate that, at physiological temperature, exposure to higher pH increases MNEI flexibility. The changes, originating from a well‐defined region in the protein, are transmitted to the whole structure and are likely to be the key for triggering unfolding processes.

The structural elucidation of the Salmonella enterica subsp. enterica, reveals that it contains both O-factors 4 and 5 on the LPS antigen.

Spectroscopic investigation of the O-antigen from Salmonella enterica subsp. enterica revealed fine details on the acetylation pattern, the biological repeating unit and the polymerization degree. Acetylation at O-2 of the abequose residue, defined both O-factors 4 and 5 in the O-antigen chain of the lipopolysaccharide. NMR observation of the terminal non-reducing end of the polymer confirmed previous data regarding the biological repeating unit and showed an average polymerization degree of 5. The information about these structural elements might contribute to the understanding of key features of the biology of this pathogen, as phase variation and/or adaptation to the external environment.

The structure of the O-specific polysaccharide from the lipopolysaccharide of Pseudomonas sp. OX1 cultivated in the presence of the azo dye Orange II

The Gram-negative bacterium Pseudomonas sp. OX1, previously known as Pseudomonas stutzeri OX1, is endowed with a high metabolic versatility. In fact, it is able to utilize a wide range of toxic organic compounds as the only source of carbon and energy for growth. It has been recently observed that, while growing on a glucose-containing liquid medium, Pseudomonas sp. OX1 can reduce azo dyes, ubiquitous pollutants particularly resistant to chemical and physical degradation, with this azoreduction being a process able to generate enough energy to sustain bacterial survival. We have found that, under these conditions, modifications in the primary structure of the O-specific polysaccharide (OPS) within the lipopolysaccharides occur, leading to remarkable changes both in the monosaccharide composition and in the architecture of the repeating unit, with respect to the polysaccharide produced in the absence of azo dyes. In the present paper, we present the complete structure of this O-specific polysaccharide, whose repeating unit is the following: [Formula: see text] This structure is totally different from the one determined from Pseudomonas sp. OX1 grown on rich medium.