Research Group Heussler (Malaria I) - Bernhard-Nocht-Institut für ...

Medical Microbiology Section
Characterization of an Aspartate Aminotransferase Antigen of
Wolbachia Endobacteria from Filarial Parasites
Zusammenfassung
Ein Gen, das für 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 für 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
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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).