Identification of the ligand of Pru p 3, a peach LTP.

KEY MESSAGE: Pru p 3, a peach LTP, is located in pollinated flower styles and secreting downy hairs, transporting a derivative of camptothecin bound to phytosphingosine. Pru p 3 may inhibit a second pollination and may keep away herbivores until seed maturation. The allergen Pru p 3, a peach lipid transfer protein, has been well studied. However, its physiological function remains to be elucidated. Our results showed that Pru p 3 usually carries a lipid ligand that play an essential role in its function in plants. Using ESI-qToF, we observed that the ligand was a derivative of camptothecin binding to phytosphingosine, wich that is inserted into the hydrophobic tunnel of the protein. In addition, the described ligand displayed topoisomerase I activity inhibition and self-fluorescence, both recognized as camptothecin properties. During flower development, the highest expression of Pru p 3 was detected in the styles of pollinated flowers, in contrast to its non-expression in unpollinated pistils, where expression decreased after anthesis. During ripening, the expression of Pru p 3 were observed mainly in peel but not in pulp. In this sense, Pru p 3 protein was also localized in trichomes covering the fruit epidermis.

LPS promotes Th2 dependent sensitisation leading to anaphylaxis in a Pru p 3 mouse model.

Pru p 3 is the major peach allergen in the Mediterranean area. It frequently elicits severe reactions, limiting its study in humans, raising the need for animal models to investigate the immunological mechanisms involved. However, no anaphylaxis model exists for Pru p 3. We aimed to develop a model of peach anaphylaxis by sensitising mice with Pru p 3 in combination with lipopolysaccharide (LPS) as an adjuvant. Four groups of mice were sensitised intranasally: untreated; treated with Pru p 3; treated with LPS; treated with Pru p 3 + LPS. After sensitisation mice were intraperitoneally challenged with Pru p 3 and in vivo and in vitro parameters were evaluated. Only mice in the Pru p 3 + LPS group showed anaphylaxis symptoms, including a decrease in temperature. Determination of in vitro parameters showed a Th2 response with an increase of Pru p 3-specific IgE and IgG1. Moreover, at the cellular level, we found increased levels of IgE and IgG1 secreting Pru p 3-specific cells and a proliferative CD4+ T-cell response. These results demonstrate that Pru p 3-specific anaphylaxis can be generated after nasal sensitisation to Pru p 3 in combination with LPS. This is a promising model for evaluating food allergy immunotherapies.

Pru p 3-Epitope-based sublingual immunotherapy in a murine model for the treatment of peach allergy.

SCOPE: Food-specific immunotherapy (SIT) is a promising treatment for lipid transfer protein (LTP)-syndrome. We propose a novel sublingual-SIT (SLIT) that combines a Pru p 3 T-cell peptide and an oligodeoxyribonucleotide (ODN) with CpG motifs (ODN-CpG) as adjuvants to induce a specific Th1/Treg response. METHODS AND RESULTS: LTP-peach allergic mice were treated sublingually with a combination of a CpG sequence and mono- or tetravalent systems including a Pru p 3 peptide, D1 (Prup3) or D4 (Prup3). Mice were challenged intraperitoneally with Pru p 3 one or three weeks after SLIT and tolerance was assessed. Mice treated with D1 (Prup3)+CpG were protected from anaphylaxis after Pru p 3 challenge. They showed no change in body temperature, lower levels of Pru p 3-specific IgE and IgG1 antibodies and higher levels of sIgG2a compared to the untreated group. They had fewer IgE and IgG1 secreting cells and more sIgG2a secreting cells. Moreover, a significantly lower number of Pru p 3-specific CD4+ T cells and a higher number of Treg cells were found, alongside a Th1 cytokine pattern. These changes were maintained for three weeks after stopping treatment. CONCLUSION: D1 Prup3+CpG represents a promising SIT for food allergy. It is easily synthesized and induces protection from anaphylaxis to Pru p 3 that is maintained for at least three weeks.

Characterisation of a flavonoid ligand of the fungal protein Alt a 1.

Spores of pathogenic fungi are virtually ubiquitous and cause human disease and severe losses in crops. The endophytic fungi Alternaria species produce host-selective phytotoxins. Alt a 1 is a strongly allergenic protein found in A. alternata that causes severe asthma. Despite the well-established pathogenicity of Alt a 1, the molecular mechanisms underlying its action and physiological function remain largely unknown. To gain insight into the role played by this protein in the pathogenicity of the fungus, we studied production of Alt a 1 and its activity in spores. We found that Alt a 1 accumulates inside spores and that its release with a ligand is pH-dependent, with optimum production in the 5.0-6.5 interval. The Alt a 1 ligand was identified as a methylated flavonoid that inhibits plant root growth and detoxifies reactive oxygen species. We also found that Alt a 1 changes its oligomerization state depending on the pH of the surrounding medium and that these changes facilitate the release of the ligand. Based on these results, we propose that release of Alt a 1 should be a pathogenic target in approaches used to block plant defenses and consequently to favor fungal entry into the plant.