Proteomic analysis and molecular characterization of Anisakis pegreffii allergenic and immunogenic proteins

Aibinu, I 2018, Proteomic analysis and molecular characterization of Anisakis pegreffii allergenic and immunogenic proteins, Doctor of Philosophy (PhD), Science, RMIT University.

Document type: Thesis
Collection: Theses

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Title Proteomic analysis and molecular characterization of Anisakis pegreffii allergenic and immunogenic proteins
Author(s) Aibinu, I
Year 2018
Abstract In the nematode genus Anisakis, nine species are currently genetically recognized among which Anisakis simplex (s. s.) and Anisakis pegreffii have been recognised to be relevant for humans as a result of their zoonotic role in causing the disease, Anisakiasis. In addition to infection with this parasite, Anisakis can also cause allergic sensitisation. To date, A. simplex allergens have been described to represent the largest number for any parasite nematode accepted by the WHO/IUIS nomenclature committee. However, few data exist on the existence of such proteins in the sibling species, A. pegreffii. A. pegreffii has been reported as the causative agent of invasive anisakiasis in Europe, Japan and South Korea. It is reported as the most widespread anisakid species known to affect commercial fish from Mediterranean waters. Studies on A. pegreffii and identification of molecules released at the interface of host-parasite relationship are of crucial importance and may provide a basis for designing better novel diagnostic and therapeutic strategies.

A detailed review on the current knowledge regarding Anisakis spp, particularly A. simplex, and its immunogenic proteins is presented in Chapter 1. This chapter presents an understanding of the current status on the increasing number of Anisakis simplex molecules identified to attack key pathways in the mammalian immune system. Through phylogenetic analysis, relationships of these proteins with homologs in other nematodes and invertebrates are presented and major A. simplex allergenic protein structures were modelled. This provides the foundation for further investigation of these proteins and their presence in A. pegreffii, and further guides their biological and genomic explorations.

Chapter 2 describes the materials and methods generally used in this study while Chapter 3 follows up on the information provided in Chapter 1 on A. simplex immunogenic proteins. These proteins were investigated in A. pegreffii using high throughput mass spectrometry (LC/MS-MS). This method analysed and identified proteins present in the crude extract (CE) as well as excretory/secretory (ES) products of A. pegreffii. The results obtained showed that over 90 % of allergenic and immunogenic proteins identified in A. pegreffii proteome have also been described in A. simplex. Furthermore, most of the proteins identified in A. pegreffii ES (~80%) were found to be homologs of proteins in the ES of other helminths. The results of this chapter therefore emphasizes the cryptic speciation of the two sibling species, A. simplex and A. pegreffii, as well as affirming the notion that parasites employ a conserved set of proteins for parasite–host interaction mechanisms and host immune response evasion. Furthermore, the result from this chapter also suggests the probable absence of allergy-reducing molecules in A. pegreffii, which may be a contributing factor as to why Anisakis nematodes are able to elicit overt hypersensitivity reactions (allergy); in addition to inducing a Th-2 biased immune response.

One of the main discoveries in the proteomic analysis of A. pegreffii CE and ES in chapter 3 was the observation that a number of proteins identified as part of A. pegreffii ES molecules in this study were not predicted to be secreted molecules. This raised the thought that such proteins must have reached the exterior or released by novel or alternative mechanisms, Hence, Chapter 4 was initiated to investigate and identify by LC-MS/MS, the exosomes of A. pegreffii and their cargo content. Abundant round-shaped materials with the expected size of exosomes were obtained after ultracentrifugation and they were visualized by transmission electron microscopy (TEM). Among the proteins identified were key exosome markers which include Heat Shock protein (HSP)-70, enolase and elongation factor 1-alpha. The result from this chapter constitutes the first report of the existence and composition of exosome-like vesicles in the L3 larvae of the parasite, A. pegreffii. The identified structures appear to play critical role in transportation of immunomodulatory and allergenic proteins such as leucine aminopeptidase (LAP) and tropomyosin (TM), respectively. In addition, high portions of proteins enriched in A. pegreffii exosomes were implicated in carbohydrate metabolism, indicative that the parasite's main energy source is probably derived from carbohydrate metabolism. Exosomes might be involved in transporting proteins needed for this function within the parasites and to the host for parasite survival. These proteins are stabilized against degradation by encapsulation within vesicles. It is demonstrated in this study for the first time, that parasite exosomes contain high concentrations of allergens, including the pan-allergen tropomyosin, providing evidence for the route of allergic sensitisation to live parasites. The result of this chapter, suggests that the secretion of certain proteins in this parasite, follow non-conventional pathways.

Chapter 5 investigates, through immunoproteomic analysis, proteins from the CE and ES of A. pegreffii that are cross-reactive with serum IgE antibody of confirmed shellfish allergic patients. In the ES, we identified 2 different reactive proteins that satisfied the criteria for putative cross-reactive allergens as defined for this study and these were fructose bisphosphate aldolase 1 and enolase. In the CE, these proteins were also identified- tropomyosin as fructose bisphosphate aldolase 1 and enolase. Tropomyosin, one of the three proteins identified, had been previously described as a cross-reactive allergen in both shellfish and Anisakis parasite. The two other novel putative cross- reactive allergens described in this chapter are proteins with close homologues in fish.

Finally, in Chapter 6, a protease, leucine aminopeptidase (LAP) reported to be implicated in immunomodulation in other helminths, was characterized. LAP of Anisakis was cloned, expressed and purified by IMAC in a bacterial host. The activity of the enzyme was investigated and its location in A. pegreffii determined using histochemical methods. This protease was found predominantly in the gut lumen of A. pegreffii and in addition was shown to interact with cathepsin proteases by cleaving in particular, the inactivated cathepsin L5 of Fasciola hepatica and releasing the activated form. The result of this study depicts Anisakis LAP as a protein of interest in immunomodulatory activities and further investigation of this enzyme as a potential therapeutic candidate could be explored.

In summary, the results of this study highlights the proteins that are enriched in the proteome of A. pegreffii and the mechanisms employed by this parasite to release secreted molecules to sites of activity. It also demonstrates that A. pegreffii secretes specific sets of proteins that are preserved against degradation by being enclosed within vesicles. In addition, putative cross-reactive allergens were defined for A. pegreffii and an immunogenic protein (LAP) was characterized. Opportunities for further exploitation of the proteins identified in A. pegreffii, in a therapeutic context, are provided by the results of this study.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Science
Subjects Bioinformatics
Medical Biochemistry: Proteins and Peptides (incl. Medical Proteomics)
Keyword(s) Anisakiasis
Anisakis pegreffii
Proteomic analysis
Cross-reactivity, Allergens
Fructose bisphosphate Aldolase 1
Recombinant Protein
Leucine Aminopeptidase (LAP)
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Created: Fri, 22 Jun 2018, 10:45:45 EST by Denise Paciocco
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