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Medical Microbiology Section Characterization of an Aspartate Aminotransferase Antigen of Wolbachia Endobacteria from Filarial Parasites Zusammenfassung Ein Gen, das <strong>für</strong> eine bakterielle Aspartat- Aminotransfease kodiert, wurde in den Filarien Onchocerca volvulus und Brugia malayi identifiziert. Eine Immunlokalisierung mit einem Antiserum gegen das rekombinante Protein, zeigte ein Vorkommen in den Wolbachia-Endobakterien der Filarien. Ein Teil der Personen mit Filarieninfektion weisen IgG1-Antikörper auf, die mit dem rekombinanten Antigen reagieren. Die <strong>für</strong> Filarien typischen IgG4- und IgE-Antikörper wurden nicht nachgewiesen. Eine sehr ähnliche Aminotransferase konnte in Wolbachien des Sandflohs Tunga penetrans nachgewiesen werden. Summary A gene coding for a bacterial aspartate aminotransferase was identified in the filarial parasites Onchocerca volvulus and Brugia malayi. Immunolocalization using an antibody raised against the recombinant protein showed strong granular staining of the Wolbachia endobacteria of the worms. A number of individuals with filarial infections showed IgG1 antibodies reactive with the recombinant antigen. IgG4 and IgE antibodies, which are typical for filarial infections, were not detected. A similar aminotransferase was detected in Wolbachia of the sand flea Tunga penetrans. Introduction Like many arthropods, most filarial parasites contain obligatory intracellular a-proteobacteria of the genus Wolbachia.The detailed characterization of their proteins may aid to elucidate the relationship between Wolbachia and their filarial host. This understanding can lead to novel strategies to control filarial infections. Wolbachia have a small genome and only very few genes of the 66 amino acid metabolism survived the reduction of the enzymatic machinery during evolution of the intracellular lifestyle. The aspartate aminotransferase is one of these genes. These aminotransferases are variously involved in amino acid metabolism, production of the neurotransmitter glutamate and in providing amino acids for energy winning processes. The aspartate aminotransferases of eukaryotes belong to the subclass 1a and show only a week similarity to the orthologous enzymes of most prokaryotes which are grouped in the subclass 1b. Project Description and Results A gene coding for an aspartate aminotransferase of subclass 1b was isolated from cDNA libraries and genomic DNA of the human pathogenic filarial parasites Onchocerca volvulus and Brugia malayi. A fragment of the gene was cloned and the recombinant protein was used to raise polyclonal antibodies. Immunolocalization in adult O. volvulus worms revealed that the protein is expressed in the Wolbachia endobacteria (Figure 1A, B). In addition, the antibody stained also tissue in the surrounding of the endobacteria. Since also the spermatozoa in the uterus and the male worms were labeled by the antibody, which are thought to be not infected by Wolbachia, a cross-reaction with the filarial ortholog was assumed. The recombinant aminotransferase was used to study whether individuals with filarial infections develop antibodies against Wolbachia antigens and to evaluate their specificity. The antigen was recognized exclusively by antibodies of the IgG1 isotype and not by IgG2, IgG3 or the for filarial infections characteristic IgG4 isotype or IgE antibodies. No cross-reactions of these sera with the filarial ortholog was detected. In 53% of individuals with O. volvulus infection and 70% of individuals with B. malayi infection reacted with the antigen, whereas none of 47 samples from healthy Figure 1: Localization of a bacterial aspartate aminotransferase in a female O. volvulus using an antiserum raised against the recombinant protein.A Pre-immune serum showing no distinct staining. B Immune serum showing granular staining of Wolbachia endobacteria (arrow) in the hypodermis of the worm (u, uterus).
Europeans, Europeans with intestinal helminths or with anti-rickettsial antibodies were positive. However, 28% of the sera from individuals from areas non-endemic for human filariasis in Africa recognized the antigen. Therefore, we searched for Wolbachia in non-filarial human parasites, which might induce also antibodies against Wolbachia antigens. Wolbachia were identified for the first time in large numbers in the ovary of the sand flea Tunga penetrans (Figure 2A). The aspartate aminotransferase gene of these Wolbachia was found to be very similar to the homolog in filarial Wolbachia and an immunological cross-reaction in humans is likely. The Wolbachia in T. penetrans can be also labeled by antibodies raised against a Wolbachia surface protein of filarial Wolbachia (Figure 2B). It can be concluded that specific IgG1 antibodies against Wolbachia antigens, such as aspartate aminotransferase, are induced by Wolbachia from filarial parasites, but a cross-reaction with Wolbachia from arthropods cannot be excluded. 67 Selected Publications • Büttner DW, Wanji S, Bazzocchi C, Bain O, Fischer P (2003). Obligatory symbiotic Wolbachia endobacteria are absent from Loa loa. Filaria J, 2:10. • Fischer P, Bonow I, Büttner DW, Kamal IH, Liebau E (2003). An aspartate aminotransferase of Wolbachia endobacteria of Onchocerca volvulus is recognized by IgG1 antibodies of residents from endemic areas. Parasitol Res, 90: 38-47. • Fischer P, Schmetz C, Bandi C, Bonow I, Mand S, Fischer K, Büttner DW (2002). Tunga penetrans: molecular identification of Wolbachia endobacteria and their recognition by antibodies against proteins of endobacteria from filarial parasites. Exp Parasitol, 102: 201-211. Funding • Vereinigung der Freunde des Tropeninstituts Hamburg e.V. Cooperating Partners • Claudio Bandi, University of Milano, Italy • Ibrahim H. Kamal, New England BioLabs, Beverly MA, USA Investigators • Peter Fischer • Insa Bonow • Michaela Krämling • Christel Schmetz • Dietrich W. Büttner Medical Microbiology Section Figure 2: Ultrastructural identification of Wolbachia in the ovary of the sand flea Tunga penetrans from Ghana. A Numerous Wolbachia endobacteria (inlay, arrows) in the cytoplasm of an egg follicle surrounded by follicular cells (fc) with large nuclei (nu). B Immunogold labeling of Wolbachia (arrow) using an antibody against a Wolbachia surface protein of Wolbachia from the filarial parasite Dirofilaria immitis.
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BERNHARD NOCHT INSTITUTE FOR TROPIC
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Human genetic variants influencing
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Research Funding / Haushaltsmittel
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Introduction The appearance of SARS
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tive project with the Faculty of Me
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since this group headed by Dr. Chri
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Vorwort Das Erscheinen der schweren
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Education and Teaching Lars Volkman
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Education and Teaching The objectiv
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Seminar Programme 2002 Prof. Aileen
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Seminar Programme Dr. Stefan Martin
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Symposia and Meetings 2002 Symposia
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Publications Book chapters and Mono
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Appendix November 2003 Inauguration
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Appendix Ansprache von Dr. Thomas F
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Diary 2004/2005 12.-14. November 20
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