Variation in Shrimp Allergens: Place of Origin Effects on Food Safety Assessment.

Due to the widespread use of shellfish ingredients in food products, accurate food labelling is urgently needed for consumers with shellfish allergies. Most crustacean allergen detection systems target the immunorecognition of the allergenic protein tropomyosin. However, this mode of detection may be affected by an origin-dependent protein composition. This study determined if the geographic location of capture, or aquaculture, influenced the allergenic protein profiles of Black Tiger Shrimp (Penaeus monodon), one of the most farmed and consumed shrimp species worldwide. Protein composition was analysed in shrimp from nine different locations in the Asia-Pacific by SDS-PAGE, immunoblotting, and mass spectrometry. Ten of the twelve known shrimp allergens were detected, but with considerable differences between locations. Sarcoplasmic calcium-binding protein, myosin light chain, and tropomyosin were the most abundant allergens in all locations. Hemocyanin-specific antibodies could identify up to six different isoforms, depending on the location of origin. Similarly, tropomyosin abundance varied by up to 13 times between locations. These findings suggest that allergen abundance may be related to shrimp origin and, thus, shrimp origin might directly impact the readout of commercial crustacean allergen detection kits, most of which target tropomyosin, and this should be considered in food safety assessments.

Comparison of protein extraction protocols and allergen mapping from black soldier fly Hermetia illucens.

Exploration of important insect proteins - including allergens - and proteomes can be limited by protein extraction buffer selection and the complexity of the proteome. Herein, LC-MS/MS-based proteomics experiments were used to assess the protein extraction efficiencies for a suite of extraction buffers and the effect of ingredient processing on proteome and allergen detection. Discovery proteomics revealed that SDS-based buffer yields the maximum number of protein groups from three types of BSF samples. Bioinformatic analysis revealed that buffer composition and ingredient processing could influence allergen detection. Upon applying multi-level filtering criteria, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. A targeted LC-MRM-MS assay was developed for the pan-allergen tropomyosin and used to assess the influence of buffer composition and ingredient processing using peptide abundance measurements. SIGNIFICANCE: We demonstrated that the selection of protein extraction buffer and the processing method could influence protein yield and cross-reactive allergen detection from processed and un-processed black soldier fly (BSF) samples. In total, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. An LC-MRM-MS assay was developed for tropomyosin, indicating the importance of buffer selection and processing conditions to reduce BSF samples' allergenicity.

Recombinant Tropomyosin from the Pacific Oyster (Crassostrea gigas) for Better Diagnosis.

The Pacific oyster is a commercially important mollusc and, in contrast to most other shellfish species, frequently consumed without prior heat treatment. Oysters are rich in many nutrients but can also cause food allergy. Knowledge of their allergens and cross-reactivity remains very limited. These limitations make an optimal diagnosis of oyster allergy difficult, in particular to the Pacific oyster (Crassostrea gigas), the most cultivated and consumed oyster species worldwide. This study aimed to characterise IgE sensitisation profiles of 21 oyster-sensitised patients to raw and heated Pacific oyster extract using immunoblotting and advanced mass spectrometry, and to assess the relevance of recombinant oyster allergen for improved diagnosis. Tropomyosin was identified as the major allergen recognised by IgE from 18 of 21 oyster-sensitised patients and has been registered with the WHO/IUIS as the first oyster allergen (Cra g 1). The IgE-binding capacity of oyster-sensitised patients' IgE to purified natural and recombinant tropomyosin from oyster, prawn, and dust mite was compared using enzyme-linked immunosorbent assay. The degree of IgE binding varied between patients, indicating partial cross-sensitisation and/or co-sensitisation. Amino acid sequence alignment of tropomyosin from these three species revealed five regions that contain predicted IgE-binding epitopes, which are most likely responsible for this cross-reactivity. This study fully biochemically characterises the first and major oyster allergen Cra g 1 and demonstrates that the corresponding recombinant tropomyosin should be implemented in improved component-resolved diagnostics and guide future immunotherapy.

Effects of Extraction Buffer on the Solubility and Immunoreactivity of the Pacific Oyster Allergens.

Despite recent technological advances, novel allergenic protein discovery is limited by their low abundance, often due to specific physical characteristics restricting their recovery during the extraction process from various allergen sources. In this study, eight different extraction buffers were compared for their ability to recover proteins from Pacific oyster (Crassostrea gigas). The protein composition was investigated using high resolution mass spectrometry. The antibody IgE-reactivity of each extract was determined using a pool of serum from five shellfish-allergic patients. Most of the investigated buffers showed good capacity to extract proteins from the Pacific oyster. In general, a higher concentration of proteins was recovered using high salt buffers or high pH buffers, subsequently revealing more IgE-reactive bands on immunoblotting. In contrast, low pH buffers resulted in a poor protein recovery and reduced IgE-reactivity. Discovery of additional IgE-reactive proteins in high salt buffers or high pH buffers was associated with an increase in allergen abundance in the extracts. In conclusion, increasing the ionic strength and pH of the buffer improves the solubility of allergenic proteins during the extraction process for oyster tissue. This strategy could also be applied for other difficult-to-extract allergen sources, thereby yielding an improved allergen panel for increased diagnostic efficiency.

Protein extraction protocols for optimal proteome measurement and arginine kinase quantitation from cricket Acheta domesticus for food safety assessment.

Insects have been consumed by people for millennia and have recently been proposed as a complementary, sustainable source of protein to feed the world's growing population. Insects and crustaceans both belong to the arthropod family. Crustacean (shellfish) allergies are common and potentially severe; hence, the cross-reactivity of the immune system with insect proteins is a potential health concern. Herein, LC-MS/MS was used to explore the proteome of whole, roasted whole and roasted powdered cricket products. Eight protein extraction protocols were compared using the total number of protein and distinct peptide identifications. Within these data, 20 putative allergens were identified, of which three were arginine kinase (AK) proteoforms. Subsequently, a multiple reaction monitoring MS assay was developed for the AK proteoforms and applied to a subset of extracts. This targeted assay demonstrated that allergen abundance/detectability varies according to the extraction method as well as the food processing method.

Novel Allergen Discovery through Comprehensive De Novo Transcriptomic Analyses of Five Shrimp Species.

Shellfish allergy affects 2% of the world's population and persists for life in most patients. The diagnosis of shellfish allergy, in particular shrimp, is challenging due to the similarity of allergenic proteins from other invertebrates. Despite the clinical importance of immunological cross-reactivity among shellfish species and between allergenic invertebrates such as dust mites, the underlying molecular basis is not well understood. Here we mine the complete transcriptome of five frequently consumed shrimp species to identify and compare allergens with all known allergen sources. The transcriptomes were assembled de novo, using Trinity, from raw RNA-Seq data of the whiteleg shrimp (Litopenaeus vannamei), black tiger shrimp (Penaeus monodon), banana shrimp (Fenneropenaeus merguiensis), king shrimp (Melicertus latisulcatus), and endeavour shrimp (Metapenaeus endeavouri). BLAST searching using the two major allergen databases, WHO/IUIS Allergen Nomenclature and AllergenOnline, successfully identified all seven known crustacean allergens. The analyses revealed up to 39 unreported allergens in the different shrimp species, including heat shock protein (HSP), alpha-tubulin, chymotrypsin, cyclophilin, beta-enolase, aldolase A, and glyceraldehyde-3-phosphate dehydrogenase (G3PD). Multiple sequence alignment (Clustal Omega) demonstrated high homology with allergens from other invertebrates including mites and cockroaches. This first transcriptomic analyses of allergens in a major food source provides a valuable resource for investigating shellfish allergens, comparing invertebrate allergens and future development of improved diagnostics for food allergy.

A Comprehensive Review on Natural Bioactive Compounds and Probiotics as Potential Therapeutics in Food Allergy Treatment.

Food allergy is rising at an alarming rate and is a major public health concern. Globally, food allergy affects over 500 million people, often starting in early childhood and increasingly reported in adults. Commercially, only one approved oral immunotherapy-based treatment is currently available and other allergen-based immunotherapeutic are being investigated in clinical studies. As an alternative approach, a substantial amount of research has been conducted on natural compounds and probiotics, focusing on the immune modes of action, and therapeutic uses of such sources to tackle various immune-related diseases. Food allergy is primarily mediated by IgE antibodies and the suppression of allergic symptoms seems to be mostly modulated through a reduction of allergen-specific IgE antibodies, upregulation of blocking IgG, and downregulation of effector cell activation (e.g., mast cells) or expression of T-helper 2 (Th-2) cytokines. A wide variety of investigations conducted in small animal models or cell-based systems have reported on the efficacy of natural bioactive compounds and probiotics as potential anti-allergic therapeutics. However, very few lead compounds, unlike anti-cancer and anti-microbial applications, have been selected for clinical trials in the treatment of food allergies. Natural products or probiotic-based approaches appear to reduce the symptoms and/or target specific pathways independent of the implicated food allergen. This broad range therapeutic approach essentially provides a major advantage as several different types of food allergens can be targeted with one approach and potentially associated with a lower cost of development. This review provides a brief overview of the immune mechanisms underlying food allergy and allergen-specific immunotherapy, followed by a comprehensive collection of current studies conducted to investigate the therapeutic applications of natural compounds and probiotics, including discussions of their mode of action and immunological aspects of their disease-modifying capabilities.

Conservation Analysis of B-Cell Allergen Epitopes to Predict Clinical Cross-Reactivity Between Shellfish and Inhalant Invertebrate Allergens.

Understanding and predicting an individual's clinical cross-reactivity to related allergens is a key to better management, treatment and progression of novel therapeutics for food allergy. In food allergy, clinical cross-reactivity is observed in patients reacting to unexpected allergen sources containing the same allergenic protein or antibody binding patches (epitopes), often resulting in severe allergic reactions. Shellfish allergy affects up to 2% of the world population and persists for life in most patients. The diagnosis of shellfish allergy is however often challenging due to reported clinical cross-reactivity to other invertebrates including mites and cockroaches. Prediction of cross-reactivity can be achieved utilizing an in-depth analysis of a few selected IgE-antibody binding epitopes. We combined available experimentally proven IgE-binding epitopes with informatics-based cross-reactivity prediction modeling to assist in the identification of clinical cross-reactive biomarkers on shellfish allergens. This knowledge can be translated into prevention and treatment of allergic diseases. To overcome the problem of predicting IgE cross-reactivity of shellfish allergens we developed an epitope conservation model using IgE binding epitopes available in the Immune Epitope Database and Analysis Resource (http://www.iedb.org/). We applied this method to a set of four different shrimp allergens, and successfully identified several non-cross-reactive as well as cross-reactive epitopes, which have been experimentally established to cross-react. Based on these findings we suggest that this method can be used for advanced component-resolved-diagnosis to identify patients sensitized to a specific shellfish group and distinguish from patients with extensive cross-reactivity to ingested and inhaled allergens from invertebrate sources.

Defining specific allergens for improved component-resolved diagnosis of shrimp allergy in adults.

Shrimp is one of the predominant causes of food allergy among adults, often presenting with severe reactions. Current in vitro diagnostics are based on quantification of patient specific-IgE (sIgE) to shrimp extract. Tropomyosin is the known major shrimp allergen, but IgE sensitisation to other allergens is poorly characterised. In this study, the binding of IgE to various shrimp allergens, additional to tropomyosin, was investigated using sera from 21 subjects who had clinical reactions to one or more shellfish species. Total shrimp-sIgE was quantified using ImmunoCAP, while allergen-sIgEs were quantified using immunoblotting and mass spectrometry, and immuno-PCR to recombinant shrimp tropomyosin. Sixty-two percent of subjects (13/21) were positive to shrimp by ImmunoCAP. IgE from 43% of subjects (9/21) bound tropomyosin, while an additional 29% of subjects (6/21) demonstrated IgE-binding solely to other shrimp allergens, including sarcoplasmic calcium-binding protein, arginine kinase and hemocyanin. Furthermore, IgE sensitisation to other shrimp allergens was demonstrated in 50% of subjects (4/8) who were ImmunoCAP negative. The lack of standardised shrimp allergens and inadequacy of current extracts for shrimp allergy diagnosis is highlighted by this study. Comprehensive knowledge of less studied allergens and their inclusion in component-resolved diagnostics will improve diagnostic accuracy, benefitting the wider population suffering from shellfish allergy.

Seafood allergy: A comprehensive review of fish and shellfish allergens.

Seafood refers to several distinct groups of edible aquatic animals including fish, crustacean, and mollusc. The two invertebrate groups of crustacean and mollusc are, for culinary reasons, often combined as shellfish but belong to two very different phyla. The evolutionary and taxonomic diversity of the various consumed seafood species poses a challenge in the identification and characterisation of the major and minor allergens critical for reliable diagnostics and therapeutic treatments. Many allergenic proteins are very different between these groups; however, some pan-allergens, including parvalbumin, tropomyosin and arginine kinase, seem to induce immunological and clinical cross-reactivity. This extensive review details the advances in the bio-molecular characterisation of 20 allergenic proteins within the three distinct seafood groups; fish, crustacean and molluscs. Furthermore, the structural and biochemical properties of the major allergens are described to highlight the immunological and subsequent clinical cross-reactivities. A comprehensive list of purified and recombinant allergens is provided, and the applications of component-resolved diagnostics and current therapeutic developments are discussed.

Tus-Ter-lock immuno-PCR assays for the sensitive detection of tropomyosin-specific IgE antibodies.

BACKGROUND: The increasing prevalence of food allergies requires development of specific and sensitive tests capable of identifying the allergen responsible for the disease. The development of serologic tests that can detect specific IgE antibodies to allergenic proteins would, therefore, be highly received. RESULTS: Here we present two new quantitative immuno-PCR assays for the sensitive detection of antibodies specific to the shrimp allergen tropomyosin. Both assays are based on the self-assembling Tus-Ter-lock protein-DNA conjugation system. Significantly elevated levels of tropomyosin-specific IgE were detected in sera from patients allergic to shrimp. CONCLUSION: This is the first time an allergenic protein has been fused with Tus to enable specific IgE antibody detection in human sera by quantitative immuno-PCR.

Inhibitors of bacterial transcription initiation complex formation.

Antibiotic resistance is a growing global problem, with very few new compounds in development. Bacterial transcription is an underutilized target for antibiotics, which has been attributed to the similarity of the active site of RNA polymerases (RNAPs) across all domains of life and the ease with which resistance can arise through point mutation at multiple sites within this conserved region. In this study we have taken a rational approach to design a novel set of compounds that specifically target the formation of transcription initiation complexes by preventing the unique bacterial σ initiation factor from binding to RNAP. We have identified the region of RNAP to which these compounds bind and demonstrate that one compound, GKL003, has an inhibition constant in the low nanomolar range. This compound has activity against both Gram-positive and -negative organisms, including a community acquired methicillin-resistant strain of the major pathogen Staphylococcus aureus.

The interaction of Bacillus subtilis sigmaA with RNA polymerase.

RNA polymerase (RNAP) is an essential and highly conserved enzyme in all organisms. The process of transcription initiation is fundamentally different between prokaryotes and eukaryotes. In prokaryotes, initiation is regulated by sigma factors, making the essential interaction between sigma factors and RNAP an attractive target for antimicrobial agents. Our objective was to achieve the first step in the process of developing novel antimicrobial agents, namely to prove experimentally that the interaction between a bacterial RNAP and an essential sigma factor can be disrupted by introducing carefully designed mutations into sigma(A) of Bacillus subtilis. This disruption was demonstrated qualitatively by Far-Western blotting. Design of mutant sigmas was achieved by computer-aided visualization of the RNAP-sigma interface of the B. subtilis holoenzyme (RNAP + sigma) constructed using a homology modeling approach with published crystal structures of bacterial RNAPs. Models of the holoenzyme and the core RNAP were rigorously built, evaluated, and validated. To allow a high-quality RNAP-sigma interface model to be constructed for the design of mutations, a crucial error in the B. subtilis sigma(A) sequence in published databases at amino acid 165 had to be corrected first. The new model was validated through determination of RNAP-sigma interactions using targeted mutations.

The structure of bacterial RNA polymerase in complex with the essential transcription elongation factor NusA.

There are three stages of transcribing DNA into RNA. These stages are initiation, elongation and termination, and they are well-understood biochemically. However, despite the plethora of structural information made available on RNA polymerase in the last decade, little is available for RNA polymerase in complex with transcription elongation factors. To understand the mechanisms of transcriptional regulation, we describe the first structure, to our knowledge, for a bacterial RNA polymerase in complex with an essential transcription elongation factor. The resulting structure formed between the RNA polymerase and NusA from Bacillus subtilis provides important insights into the transition from an initiation complex to an elongation complex, and how NusA is able to modulate transcription elongation and termination.