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Humoral and cell-mediated autoimmune reactions to human acidic ribosomal P2 protein in individuals sensitized to Aspergillus fumigatus P2 protein.

ABSTRACT: A panel of cDNAs encoding allergenic proteins was isolated from an Aspergillus fumigatus cDNA library displayed on the surface of filamentous phage. Solid phase-immobilized serum immunoglobulin E (IgE) from A. fumigatus-allergic individuals was used to enrich phage displaying IgE-binding molecules. One of the cDNAs encoded a 11.1-kD protein that was identified as acidic ribosomal phosphoprotein type 2 (P2 protein). The allergen, formally termed rAsp f 8, shares >62% sequence identity and >84% sequence homology to corresponding eukaryotic P2 proteins, including human P2 protein. The sequences encoding human and fungal P2 protein were subcloned, expressed in Escherichia coli as His6-tagged fusion proteins, and purified by Ni2+-chelate affinity chromatography. Both recombinant P2 proteins were recognized by IgE antibodies from allergic individuals sensitized to the A. fumigatus P2 protein and elicited strong type 1-specific skin reactions in these individuals. Moreover, human and fungal P2 proteins induced proliferative responses in peripheral blood mononuclear cells of A. fumigatus- allergic subjects sensitized to the fungal P2 protein. These data provide strong evidence for in vitro and in vivo humoral and cell-mediated autoreactivity to human P2 protein in patients suffering from chronic A. fumigatus allergy.

SUBMITTER: Mayer C

PROVIDER: S-EPMC2193053 | biostudies-literature | 1999 May

REPOSITORIES: biostudies-literature

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Humoral and cell-mediated autoimmune reactions to human acidic ribosomal P2 protein in individuals sensitized to Aspergillus fumigatus P2 protein.

Mayer C C Appenzeller U U Seelbach H H Achatz G G Oberkofler H H Breitenbach M M Blaser K K Crameri R R

The Journal of experimental medicine 19990501 9

A panel of cDNAs encoding allergenic proteins was isolated from an Aspergillus fumigatus cDNA library displayed on the surface of filamentous phage. Solid phase-immobilized serum immunoglobulin E (IgE) from A. fumigatus-allergic individuals was used to enrich phage displaying IgE-binding molecules. One of the cDNAs encoded a 11.1-kD protein that was identified as acidic ribosomal phosphoprotein type 2 (P2 protein). The allergen, formally termed rAsp f 8, shares >62% sequence identity and >84% se ...[more]

PMID: 10224291

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Project description:The larval stage of Taenia multiceps, also known as coenurus, is the causative agent of coenurosis, which results in severe health problems in sheep, goats, cattle and other animals that negatively impact on animal husbandry. There is no reliable method to identify coenurus infected goats in the early period of infection.We identified a full-length cDNA that encodes acidic ribosomal protein P2 from the transcriptome of T. multiceps (TmP2). Following cloning, sequencing and structural analyses were performed using bioinformatics tools. Recombinant TmP2 (rTmP2) was prokaryotically expressed and then used to test immunoreactivity and immunogenicity in immunoblotting assays. The native proteins in adult stage and coenurus were located via immunofluorescence assays, while the potential of rTmP2 for indirect ELISA-based serodiagnostics was assessed using native goat sera. In addition, 20 goats were randomly divided into a drug treatment group and a control group. Each goat was orally given mature, viable T. multiceps eggs. The drug treatment group was given 10% praziquantel by intramuscular injection 45 days post-infection (p.i), and all goats were screened for anti-TmP2 antibodies with the indirect ELISA method established here, once a week for 17 weeks p.i.The open reading frame (366 bp) of the target gene encodes a 12.62 kDa protein, which showed high homology to that from Taenia solium (93% identity) and lacked a signal peptide. Immunofluorescence staining showed that TmP2 was highly localized to the parenchymatous zone of both the adult parasite and the coenurus; besides, it was widely distributed in cystic wall of coenurus. Building on good immunogenic properties, rTmP2-based ELISA exhibited a sensitivity of 95.0% (19/20) and a specificity of 96.3% (26/27) in detecting anti-P2 antibodies in the sera of naturally infected goats and sheep. In goats experimentally infected with T. multiceps, anti-TmP2 antibody was detectable in the control group from 3 to 10 weeks and 15 to 17 weeks p.i. In the drug-treated group, the anti-TmP2 antibody dropped below the cut-off value about 2 weeks after treatment with praziquantel and remained below this critical value until the end of the experiment.The indirect ELISA method developed in this study has the potential for detection of T. multiceps infections in hosts.

Project description:A human host exploits stresses such as acidic/alkaline pH, antifungal drugs, and reactive oxygen species to kill microbial pathogens such as the fungus Aspergillus fumigatus. However, A. fumigatus is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity of the human host? In this observation, we show that simultaneous exposure to acidic pH and oxidative stresses is much more potent than the individual stresses themselves and that this combinatorial stress kills A. fumigatus synergistically in vitro. Interestingly, A. fumigatus is resistant to the combination of alkaline pH and oxidative stress. Quantitative real-time PCR analyses showed that acidic/alkaline pH stress can mediate oxidative stress responses in A. fumigatus by regulating the expression of catalase-encoding genes. We further show that A. fumigatus is sensitive to the combination of acidic/alkaline stress and azole drug stress. Transcriptome analysis revealed that the sensitivity of A. fumigatus to azole drugs under acidic/alkaline conditions may be related to changes in genetic stability, sphingolipid metabolism, lipid metabolism, and amino acid metabolism. Collectively, our findings suggest that combinatorial stress represents a powerful fungicidal mechanism employed by hosts against pathogens, which suggests novel approaches to potentiate antifungal therapy. IMPORTANCE The human host combats fungal infections via phagocytic cells that recognize and kill fungal pathogens. Immune cells combat Aspergillus fumigatus infections with a potent mixture of chemicals, including reactive oxygen species, acidic/alkaline stress, and antifungal drugs. However, A. fumigatus is relatively resistant to these stresses in vitro. In this observation, we show that it is the combination of acidic/alkaline pH and oxidative or azole stress that kills A. fumigatus so effectively, and we define the molecular mechanisms that underlie this potency. Our findings suggest that combinatorial stress is a powerful fungicidal mechanism employed by hosts, which suggests novel approaches to potentiate antifungal therapy. This study provides a platform for future studies that will address the combinatorial impacts of various environmental stresses on A. fumigatus and other pathogenic microbes.

Dataset Information

Humoral and cell-mediated autoimmune reactions to human acidic ribosomal P2 protein in individuals sensitized to Aspergillus fumigatus P2 protein.

Publications

Humoral and cell-mediated autoimmune reactions to human acidic ribosomal P2 protein in individuals sensitized to Aspergillus fumigatus P2 protein.

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