Statistical Study of Low-Intensity Single-Molecule Recognition Events Using DeepTipTM Probes: Application to the Pru p 3-Phytosphingosine System.

The interaction between the plant lipid transfer protein Pru p 3 and phytosphingosine was assessed using an atomic force microscope. Phytosphingosine was covalently immobilized on DeepTipTM probes and Pru p 3 on MicroDeckTM functionalized substrates. Single-molecular interaction events between both molecules were retrieved and classified and the distribution for each one of the identified types was calculated. A success rate of over 70% was found by comparing the number of specific Pru p 3-phytosphingosine interaction events with the total number of recorded curves. The analysis of the distribution established among the various types of curves was further pursued to distinguish between those curves that can mainly be used for assessing the recognition between phytosphingosine (sensor molecule) and Pru p 3 (target molecule) in the context of affinity atomic force microscopy, and those that entail details of the interaction and might be employed in the context of force spectroscopy. The successful application of these functionalized probes and substrates to the characterization of the low-intensity hydrophobic interaction characteristic of this system is a clear indication of the potential of exploiting this approach with an extremely wide range of different biological molecules of interest. The possibility of characterizing molecular assembly events with single-molecule resolution offers an advantageous procedure to plough into the field of molecular biomimetics.

Corrigendum: Suitability of potyviral recombinant virus-like particles bearing a complete food allergen for immunotherapy vaccines.

[This corrects the article DOI: 10.3389/fimmu.2022.986823.].

Natural variation in Avr3D1 from Zymoseptoria sp. contributes to quantitative gene-for-gene resistance and to host specificity.

Successful host colonization by plant pathogens requires the circumvention of host defense responses, frequently through sequence modifications in secreted pathogen proteins known as avirulence factors (Avrs). Although Avr sequences are often polymorphic, the contribution of these polymorphisms to virulence diversity in natural pathogen populations remains largely unexplored. We used molecular genetic tools to determine how natural sequence polymorphisms of the avirulence factor Avr3D1 in the wheat pathogen Zymoseptoria tritici contributed to adaptive changes in virulence. We showed that there is a continuous distribution in the magnitude of resistance triggered by different Avr3D1 isoforms and demonstrated that natural variation in an Avr gene can lead to a quantitative resistance phenotype. We further showed that homologues of Avr3D1 in two nonpathogenic sister species of Z. tritici are recognized by some wheat cultivars, suggesting that Avr-R gene-for-gene interactions can contribute to nonhost resistance. We suggest that the mechanisms underlying host range, qualitative resistance, and quantitative resistance are not exclusive.

Critical features of an in vitro intestinal absorption model to study the first key aspects underlying food allergen sensitization.

New types of protein sources will enter our diet in a near future, reinforcing the need for a straightforward in vitro (cell-based) screening model to test and predict the safety of these novel proteins, in particular their potential risk for de novo allergic sensitization. The Adverse Outcome Pathway (AOP) for allergen sensitization describes the current knowledge of key events underlying the complex cellular interactions that proceed allergic food sensitization. Currently, there is no consensus on the in vitro model to study the intestinal translocation of proteins as well as the epithelial activation, which comprise the first molecular initiation events (ME1-3) and the first key event of the AOP, respectively. As members of INFOGEST, we have highlighted several critical features that should be considered for any proposed in vitro model to study epithelial protein transport in the context of allergic sensitization. In addition, we defined which intestinal cell types are indispensable in a consensus model of the first steps of the AOP, and which cell types are optional or desired when there is the possibility to create a more complex cell model. A model of these first key aspects of the AOP can be used to study the gut epithelial translocation behavior of known hypo- and hyperallergens, juxtaposed to the transport behavior of novel proteins as a first screen for risk management of dietary proteins. Indeed, this disquisition forms a basis for the development of a future consensus model of the allergic sensitization cascade, comprising also the other key events (KE2-5).

Arabidopsis immune responses triggered by cellulose- and mixed-linked glucan-derived oligosaccharides require a group of leucine-rich repeat malectin receptor kinases.

The plant immune system perceives a diversity of carbohydrate ligands from plant and microbial cell walls through the extracellular ectodomains (ECDs) of pattern recognition receptors (PRRs), which activate pattern-triggered immunity (PTI). Among these ligands are oligosaccharides derived from mixed-linked ß-1,3/ß-1,4-glucans (MLGs; e.g. ß-1,4-D-(Glc)2 -ß-1,3-D-Glc, MLG43) and cellulose (e.g. ß-1,4-D-(Glc)3 , CEL3). The mechanisms behind carbohydrate perception in plants are poorly characterized except for fungal chitin oligosaccharides (e.g. ß-1,4-d-(GlcNAc)6 , CHI6), which involve several receptor kinase proteins (RKs) with LysM-ECDs. Here, we describe the isolation and characterization of Arabidopsis thaliana mutants impaired in glycan perception (igp) that are defective in PTI activation mediated by MLG43 and CEL3, but not by CHI6. igp1-igp4 are altered in three RKs - AT1G56145 (IGP1), AT1G56130 (IGP2/IGP3) and AT1G56140 (IGP4) - with leucine-rich-repeat (LRR) and malectin (MAL) domains in their ECDs. igp1 harbors point mutation E906K and igp2 and igp3 harbor point mutation G773E in their kinase domains, whereas igp4 is a T-DNA insertional loss-of-function mutant. Notably, isothermal titration calorimetry (ITC) assays with purified ECD-RKs of IGP1 and IGP3 showed that IGP1 binds with high affinity to CEL3 (with dissociation constant KD  = 1.19 ± 0.03 µm) and cellopentaose (KD  = 1.40 ± 0.01 µM), but not to MLG43, supporting its function as a plant PRR for cellulose-derived oligosaccharides. Our data suggest that these LRR-MAL RKs are components of a recognition mechanism for both cellulose- and MLG-derived oligosaccharide perception and downstream PTI activation in Arabidopsis.

Integration of Multiple Interferometers in Highly Multiplexed Diagnostic KITs to Evaluate Several Biomarkers of COVID-19 in Serum.

In the present work, highly multiplexed diagnostic KITs based on an Interferometric Optical Detection Method (IODM) were developed to evaluate six Coronavirus Disease 2019 (COVID-19)-related biomarkers. These biomarkers of COVID-19 were evaluated in 74 serum samples from severe, moderate, and mild patients with positive polymerase chain reaction (PCR), collected at the end of March 2020 in the Hospital Clínico San Carlos, in Madrid (Spain). The developed multiplexed diagnostic KITs were biofunctionalized to simultaneously measure different types of specific biomarkers involved in COVID-19. Thus, the serum samples were investigated by measuring the total specific Immunoglobulins (sIgT), specific Immunoglobulins G (sIgG), specific Immunoglobulins M (sIgM), specific Immunoglobulins A (sIgA), all of them against SARS-CoV-2, together with two biomarkers involved in inflammatory disorders, Ferritin (FER) and C Reactive Protein (CRP). To assess the results, a Multiple Linear Regression Model (MLRM) was carried out to study the influence of IgGs, IgMs, IgAs, FER, and CRP against the total sIgTs in these serum samples with a goodness of fit of 73.01% (Adjusted R-Squared).

Suitability of potyviral recombinant virus-like particles bearing a complete food allergen for immunotherapy vaccines.

Virus-like particles (VLPs) have been gaining attention as potential platforms for delivery of cargos in nanomedicine. Although animal viruses are largely selected due to their immunostimulatory capacities, VLPs from plant viruses constitute a promising alternative to be considered. VLPs derived from Turnip mosaic virus (TuMV) have proven to present a tridimensional structure suited to display molecules of interest on their surface, making them interesting tools to be studied in theragnostic strategies. Here, we study their potential in the treatment of food allergy by genetically coupling TuMV-derived VLPs to Pru p 3, one of the most dominant allergens in Mediterranean climates. VLPs-Pru p 3 were generated by cloning a synthetic gene encoding the TuMV coat protein and Pru p 3, separated by a linker, into a transient high-expression vector, followed by agroinfiltration in Nicotiana benthamiana plants. The generated fusion protein self-assembled in planta to form the VLPs, which were purified by exclusion chromatography. Their elongated morphology was confirmed by electron microscopy and their size (~400 nm), and monodispersity was confirmed by dynamic light scattering. Initial in vitro characterization confirmed that they were able to induce proliferation of human immune cells. This proliferative capability was enhanced when coupled with the natural lipid ligand of Pru p 3. The resultant formulation, called VLP-Complex, was also able to be transported by intestinal epithelial cells, without affecting the monolayer integrity. In light of all these results, VLP-Complex was furtherly tested in a mouse model of food allergy. Sublingual administration of VLP-Complex could effectively reduce some serological markers associated with allergic responses in mice, such as anti-Pru p 3 sIgE and sIgG2a. Noteworthy, no associated macroscopic, nephritic, or hepatic toxicity was detected, as assessed by weight, blood urea nitrogen (BUN) and galectin-3 analyses, respectively. Our results highlight the standardized production of allergen-coated TuMV-VLPs in N. benthamiana plants. The resulting formula exerts notable immunomodulatory properties without the need for potentially hazardous adjuvants. Accordingly, no detectable toxicity associated to their administration was detected. As a result, we propose them as good candidates to be furtherly studied in the treatment of immune-based pathologies.

Alt a 1 Promotes Allergic Asthma In Vivo Through TLR4-Alveolar Macrophages.

The mold Alternaria alternata is one of the main sources of asthma exacerbation, being its major allergen, Alt a 1, indispensable for its development. The main objective of this work was to answer two main questions: 1) can Alt a 1 by itself (without any other context) induce an asthmatic profile in vivo?; and 2) Which molecular mechanisms take place during this phenomenon? To answer both questions, we have developed a mouse model of allergic asthma using only Alt a 1 for mice sensitization. We also made use of in-vitro cellular models and computational studies to support some aspects of our hypothesis. Our results showed that Alt a 1 can induce an asthmatic phenotype, promoting tissue remodeling and infiltration of CD45+ cells, especially eosinophils and macrophages (Siglec F+ and F4/80+). Also, we have found that Alt a 1 sensitization is mediated by the TLR4-macrophage axis.

Lipid Ligands and Allergenic LTPs: Redefining the Paradigm of the Protein-Centered Vision in Allergy.

Lipid Transfer Proteins (LTPs) have been described as one of the most prevalent and cross-reactive allergen families in the general population. They are widely distributed among the plant kingdom, as well as in different plant organs ranging from pollen to fruits. Thus, they can initiate allergic reactions with very different outcomes, such as asthma and food allergy. Several mouse models have been developed to unravel the mechanisms that lead LTPs to promote such strong sensitization patterns. Interestingly, the union of certain ligands can strengthen the allergenic capacity of LTPs, suggesting that not only is the protein relevant in the sensitization process, but also the ligands that LTPs carry in their cavity. In fact, different LTPs with pro-allergenic capacity have been shown to transport similar ligands, thus positioning lipids in a central role during the first stages of the allergic response. Here, we offer the latest advances in the use of experimental animals to study the topic, remarking differences among them and providing future researchers a tool to choose the most suitable model to achieve their goals. Also, recent results derived from metabolomic studies in humans are included, highlighting how allergic diseases alter the lipidic metabolism toward a pathogenic state in the individual. Altogether, this review offers a comprehensive body of work that sums up the background evidence supporting the role of lipids as modulators of allergic diseases. Studying the role of lipids during allergic sensitization might broaden our understanding of the molecular events leading to tolerance breakdown in the epithelium, thus helping us to understand how allergy is initiated and established in the individuals.

Identification and molecular characterization of a novel non-specific lipid transfer protein (TdLTP2) from durum wheat.

Non-specific lipid transfer proteins (nsLTPs) are small, cysteine-rich proteins, a part of the pathogenesis-related protein family, and numerous of them act as positive regulators during plant disease resistance, growth, and reproduction. These proteins are involved also in the intracellular transfer of lipids, as well as in plant immune responses. Besides their differences in sequences, they show similar features in their structure. However, they show distinct lipid-binding specificities signifying their various biological roles that dictate further structural study. This study reports the identification, in silico characterization and purification of a novel member of the nsLTP2 protein family from durum wheat, TdLTP2. It was generated and purified using the combination of gel filtration chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its identity was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MALDI-TOF). TdLTP2 had been expressed in different stress to detect its localization; therefore, fluor-immunolocalization studies accomplished this data. In this approach, to assess the allergenicity of TdLTP2, thirty patients with baker's asthma were enrolled and ELISA to detect the presence of specific IgE antibodies tested their sera. Moreover, the lipid-binding properties of TdLTP2 were examined in vitro and validated using a molecular docking study. In summary, our results demonstrate a new addition of member in plant nsLTPs family, TdLTP2, which can develop a better understanding about its biological functions and shed light on future applications.

NLRP3 priming due to skin damage precedes LTP allergic sensitization in a mouse model.

Allergic sensitization is initiated by protein and epithelia interaction, although the molecular mechanisms leading this encounter toward an allergic phenotype remain unknown. Here, we apply the two-hit hypothesis of inflammatory diseases to the study of food allergy sensitization. First, we studied the effects of long-term depilation in mice by analyzing samples at different time points. Several weeks of depilation were needed until clear immunological changes were evidenced, starting with upregulation of NLRP3 protein levels, which was followed by overexpression of Il1b and Il18 transcripts. Secondly, we assessed the effects of allergen addition (in this case, Pru p 3 in complex with its natural lipid ligand) over depilated skin. Systemic sensitization was evaluated by intraperitoneal provocation with Pru p 3 and measure of body temperature. Anaphylaxis was achieved, but only in mice sensitized with Prup3_complex and not treated with the NLRP3 inhibitor MCC950, thus demonstrating the importance of both hits (depilation + allergen addition) in the consecution of the allergic phenotype. In addition, allergen encounter (but not depilation) promoted skin remodeling, as well as CD45+ infiltration not only in the sensitized area (the skin), but across several mucosal tissues (skin, lungs, and gut), furtherly validating the systemization of the response. Finally, a low-scale study with human ILC2s is reported, where we demonstrate that Prup3_complex can induce their phenotype switch (↑CD86, ↑S1P1) when cultured in vitro, although more data is needed to understand the implications of these changes in food allergy development.

Plant non-specific lipid transfer proteins: An overview.

Plant non-specific lipid transfer proteins (nsLTPs) are usually defined as small, basic proteins, with a wide distribution in all orders of higher plants. Structurally, nsLTPs contain a conserved motif of eight cysteines, linked by four disulphide bonds, and a hydrophobic cavity in which the ligand is housed. This structure confers stability and enhances the ability to bind and transport a variety of hydrophobic molecules. Their highly conserved structural resemblance but low sequence identity reflects the wide variety of ligands they can carry, as well as the broad biological functions to which they are linked to, such as membrane stabilization, cell wall organization and signal transduction. In addition, they have also been described as essential in resistance to biotic and abiotic stresses, plant growth and development, seed development, and germination. Hence, there is growing interest in this family of proteins for their critical roles in plant development and for the many unresolved questions that need to be clarified, regarding their subcellular localization, transfer capacity, expression profile, biological function, and evolution.

New applications of advanced instrumental techniques for the characterization of food allergenic proteins.

Current approaches based on electrophoretic, chromatographic or immunochemical principles have allowed characterizing multiple allergens, mapping their epitopes, studying their mechanisms of action, developing detection and diagnostic methods and therapeutic strategies for the food and pharmaceutical industry. However, some of the common structural features related to the allergenic potential of food proteins remain unknown, or the pathological mechanism of food allergy is not yet fully understood. In addition, it is also necessary to evaluate new allergens from novel protein sources that may pose a new risk for consumers. Technological development has allowed the expansion of advanced technologies for which their whole potential has not been entirely exploited and could provide novel contributions to still unexplored molecular traits underlying both the structure of food allergens and the mechanisms through which they sensitize or elicit adverse responses in human subjects, as well as improving analytical techniques for their detection. This review presents cutting-edge instrumental techniques recently applied when studying structural and functional aspects of proteins, mechanism of action and interaction between biomolecules. We also exemplify their role in the food allergy research and discuss their new possible applications in several areas of the food allergy field.

Alternaria as an Inducer of Allergic Sensitization.

Alternaria alternata is a saprophytic mold whose spores are disseminated in warm dry air, the typical weather of the Mediterranean climate region (from 30° to 45°), with a peak during the late summer and early autumn. Alternaria spores are known to be biological contaminants and a potent source of aeroallergens. One consequence of human exposure to Alternaria is an increased risk of developing asthma, with Alt a 1 as its main elicitor and a marker of primary sensitization. Although the action mechanism needs further investigation, a key role of the epithelium in cytokine production, TLR-activated alveolar macrophages and innate lymphoid cells in the adaptive response was demonstrated. Furthermore, sensitization to A. alternata seems to be a trigger for the development of co-sensitization to other allergen sources and may act as an exacerbator of symptoms and an elicitor of food allergies. The prevalence of A. alternata allergy is increasing and has led to expanding research on the role of this fungal species in the induction of IgE-mediated respiratory diseases. Indeed, recent research has allowed new perspectives to be considered in the assessment of exposure and diagnosis of fungi-induced allergies, although more studies are needed for the standardization of immunotherapy formulations.

The key to the allergenicity of lipid transfer protein (LTP) ligands: A structural characterization.

Plant lipid transfer proteins are a large family that can be found in all land plants. They have a hydrophobic cavity that allows them to harbor lipids and facilitates their traffic between membranes. However, in humans, this plant protein family is responsible for the main food allergies in the Mediterranean area. Nevertheless, not only the protein itself but also its ligand is relevant for allergic sensitization. The main aim of the present work is to analyse the natural ligands carried by four allergenic LTPs (Tri a 14, Art v 3, Par j 2, and Ole e 7), compared with the previously identified ligand of Pru p 3 (CPT-PHS ligand), and clarify their role within the immunological reactions. Results showed that the ligands of the LTPs studied shared a chemical identity, in which the presence of a polar head was essential to the protein-ligand binding. This ligand was transported through a skin cellular model, and phosphorylated phytosphingosine could be detected as result of cell metabolism. Since sphingosine kinase 1 was overexpressed in keratinocytes incubated with the LTP-ligand complex, this enzyme might be responsible for the phosphorylation of the phytosphingosine fraction of the CPT-PHS ligand. This way, phytosphingosine-1-phosphate could be mimicking the role of the human inflammatory mediator sphingosine-1-phosphate, explaining why LTPs are associated with more severe allergic responses. In conclusion, this work contributes to the understanding of the chemical nature and behavior of lipid ligands carried by allergens, which would help to gain insight into their role during allergic sensitization.

The Role of Sphingolipids in Allergic Disorders.

Allergy is defined as a complex chronic inflammatory condition in which genetic and environmental factors are implicated. Sphingolipids are involved in multiple biological functions, from cell membrane components to critical signaling molecules. To date, sphingolipids have been studied in different human pathologies such as neurological disorders, cancer, autoimmunity, and infections. Sphingolipid metabolites, in particular, ceramide and sphingosine-1-phosphate (S1P), regulate a diverse range of cellular processes that are important in immunity and inflammation. Moreover, variations in the sphingolipid concentrations have been strongly associated with allergic diseases. This review will focus on the role of sphingolipids in the development of allergic sensitization and allergic inflammation through the activation of immune cells resident in tissues, as well as their role in barrier remodeling and anaphylaxis. The knowledge gained in this emerging field will help to develop new therapeutic options for allergic disorders.

A computational structural study on the DNA-protecting role of the tardigrade-unique Dsup protein.

The remarkable ability of tardigrades to withstand a wide range of physical and chemical extremes has attracted a considerable interest in these small invertebrates, with a particular focus on the protective roles of proteins expressed during such conditions. The discovery that a tardigrade-unique protein named Dsup (damage suppressor) protects DNA from damage produced by radiation and radicals, has raised expectations concerning its potential applications in biotechnology and medicine. We present in this paper what might be dubbed a "computational experiment" on the Dsup-DNA system. By means of molecular modelling, calculations of electrostatic potentials and electric fields, and all-atom molecular dynamics simulations, we obtained a dynamic picture of the Dsup-DNA interaction. Our results suggest that the protein is intrinsically disordered, which enables Dsup to adjust its structure to fit DNA shape. Strong electrostatic attractions and high protein flexibility drive the formation of a molecular aggregate in which Dsup shields DNA. While the precise mechanism of DNA protection conferred by Dsup remains to be elucidated, our study provides some molecular clues of their association that could be of interest for further investigation in this line.

Correction: Pru p 9, a new allergen eliciting respiratory symptoms in subjects sensitized to peach tree pollen.

[This corrects the article DOI: 10.1371/journal.pone.0230010.].

Structural Dynamics of the Lipid Antigen-Binding Site of CD1d Protein.

CD1 molecules present lipid antigens to T-cells in early stages of immune responses. Whereas CD1‒lipid‒T-cell receptors interactions are reasonably understood, molecular details on initial trafficking and loading of lipids onto CD1 proteins are less complete. We present a molecular dynamics (MD) study of human CD1d, the isotype that activates iNKT cells. MD simulations and calculations of properties and Poisson-Boltzmann electrostatic potentials were used to explore the dynamics of the antigen-binding domain of the apo-form, CD1d complexes with three lipid-antigens that activate iNKT cells and CD1d complex with GM2AP, a protein that assists lipid loading onto CD1 molecules in endosomes/lysosomes. The study was done at pH 7 and 4.5, values representative of strongly acidic environments in endosomal compartments. Our findings revealed dynamic features of the entrance to the hydrophobic channels of CD1d modulated by two α helices with sensitivity to the type of lipid. We also found lipid- and pH-dependent dynamic changes in three exposed tryptophans unique to CD1d among the five human CD1 isotypes. On the basis of modelled structures, our data also revealed external effects produced by the helper protein GM2AP only when it interacts in its open form, thus suggesting that the own assistant protein also adapts conformation to association with CD1d.