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Acanthocheilonema viteae: characterization of a molt-associated excretory/secretory 18-kDa protein
T Pogonka1, U Oberländer, T Marti
1Department of Molecular Parasitology (Institute of Biology), Humboldt University Berlin, Philippstrasse 13, Berlin, 10115, USA.
Abstract:
Post-invasive third-stage larvae (pL3) of Acanthocheilonema viteae were labeled with [(35)S]-methionine in vivo, and proteins released into the culture supernatant before and during the third molt were analyzed. The molting supernatant (MSN) contained abundant proteins of 14, 18, 29, and 36 kDa. The 14- and 29-kDa proteins were exclusively found in the MSN, while the 18- and 36-kDa proteins were also produced by nonmolting pL3, albeit in much lower quantities. The cDNA for the most abundant protein in the MSN, an 18-kDa protein (Av18), was isolated by polymerase chain reaction (PCR) with reverse transcribed (RT) RNA of pL3, using information of the protein sequence. The Av18 full-length cDNA of 583 base pairs contained the 5' spliced leader sequence of nematodes, an open reading frame of 427 base pairs, and a poly(A) tail in typical distance to a polyadenylation signal. The deduced amino acid sequence encodes for a protein with a calculated size of 15.8 kDa. The N-terminus starts with a hydrophobic signal sequence and a predicted cleavage site after amino acid 20. The Av18 protein showed homologies to the deduced amino acid sequence of the larval transcripts Bm-alt-1 and alt-2 of Brugia malayi and to the Dirofilaria immitis proteins Di20/22 as well as to the Onchocerca volvulus proteins Ov-alt-1 and Ov-alt-2. Av18 is present in all parasite stages within the mammalian host, as determined by immunoblot with sera against the Escherichia coli-expressed protein and RT-PCR experiments. However, it was released into culture medium only by L3 and adult female worms. In female worms Av18 was localized in the cuticular region as demonstrated by immunofluorescent antibody tests using cryosections.
Insights
Researchers identified Av18, an abundant 18-kDa protein released by Acanthocheilonema viteae during molting. This protein is present in all parasite stages and localized to the cuticle in female worms.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Nematode molting involves the release of specific proteins into the surrounding environment.
- Understanding these secreted proteins is crucial for identifying potential drug targets and diagnostic markers for filarial infections.
Purpose of the Study:
- To identify and characterize proteins released by Acanthocheilonema viteae during the third larval molt.
- To isolate and analyze the cDNA and protein sequence of the most abundant secreted protein.
- To investigate the expression and localization of this protein in different parasite stages.
Main Methods:
- In vivo labeling of Acanthocheilonema viteae post-invasive third-stage larvae (pL3) with [(35)S]-methionine.
- Analysis of proteins in the molting supernatant (MSN) using SDS-PAGE.
- Isolation of Av18 cDNA using reverse transcription polymerase chain reaction (RT-PCR).
- Sequence analysis of Av18 cDNA and deduced amino acid sequence.
- Homology searches against known nematode proteins.
- Immunoblotting and RT-PCR to determine protein presence across parasite stages.
- Immunofluorescence antibody testing on cryosections to localize Av18 in female worms.
Main Results:
- The MSN contained abundant proteins of 14, 18, 29, and 36 kDa, with 14 and 29 kDa proteins being exclusive to MSN.
- The 18-kDa protein (Av18) was the most abundant protein in the MSN and its cDNA was successfully isolated.
- Av18 shares homology with larval proteins of Brugia malayi and Dirofilaria immitis, and Onchocerca volvulus.
- Av18 is expressed in all parasite stages in the mammalian host but is released into the medium only by L3 and adult female worms.
- Av18 was localized to the cuticular region of adult female worms.
Conclusions:
- Av18 is a significant component of the molting fluid of Acanthocheilonema viteae.
- The protein's conserved nature across different filarial species suggests a potentially important biological role.
- Av18's stage-specific release and localization indicate its potential as a target for diagnostics or therapeutics against filarial parasites.