Comparative analysis of two fatty acid binding proteins (FABPS) from fasciola Gigantica

• Main Author: Supatra Chunchob
• Other Authors: Suksiri Vichasri-Grams
• Language: English
• Published: Mahidol University. Mahidol University Library and Knowledge Center 2023
• Subjects: Electrophoresis, Gel, Two-Dimensional; Fasciola gigantica; Fatty acid-binding proteins
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/89319
• Institution: Mahidol University

Fasciola gigantica, an infective parasite, is incapable of de novo synthesizing its own lipids and cholesterol. Fatty acid binding proteins (FABPs) are acquired to facilitate the uptake and transport of host-derived fatty acids. Nevertheless, knowledge about the functional differences of the various FABP isoforms in this parasite is still limited. The major objective of this study was to clone and comparatively characterize two different FABP isoforms of F. gigantica (FgFABP1 and FgFABP3) at the nucleic acid and protein levels. Consequently, a full-length FgFABP3 cDNA was isolated from the metacercarial stage of the cDNA library, and further characterized by Southern and Northern analyses. The stage specific expression of FgFABP1 and FgFABP3 were investigated by using an RT-PCR procedure. The recombinant proteins were produced in bacterial and yeast expression systems, then purified by Ni-NTA affinity chromatography and used for production of specific polyclonal antibodies. The immunogenic potential of recombinant proteins was determined by Western analyses. Distribution of native proteins in parasite tissues was demonstrated by immunohistochemistry. The presence of various FgFABP isoforms in parasite extracts was determined by 2D gel electrophoresis (2DE) and mass spectrometry. Moreover, the humoral immune responses were preliminarily evaluated in experimental mice using an enzyme linked immunosorbent assay (ELISA). The FgFABP3 cDNA had a size of 544 base pairs and the FgFABP3 gene existed in a single copy in the parasite genome. The observed transcript size was 600 nucleotides. The deduced amino acid sequences of both FgFABPs shared a 67% identity and had different isoelectric points (pI 4.9 and pI 9.6). Reverse transcription PCR analysis demonstrated that both FgFABPs were present in juvenile and adult parasites whereas only FgFABP3 was found in their eggs. 14 kDa of FgFABPs were detected in an immunoblotted crude worm (CW), excretion/secretion (ES) product and soluble egg (SE) extract. Sera of an experimentally infected rabbit reacted earlier with FgFABP3 (week 6 after infection) than with FgFABP1 (week 12 after infection). Immunohistochemistry showed the presence of both FgFABPs in the parenchyma. However, differential tissue-specific distribution was found in digestive and reproductive organs. By using 2DE and mass spectrometry analysis, at least five FgFABP isoforms were detected in the CW extract. FgFABP1 and FgFABP3 were identified in the ES product whereas only FgFABP3 was identified in the SE extract. In addition, dominant IgG1 and significant IgG2a antibody responses in the immunized mice indicated that both FgFABP isoforms induced a mixed Th1/Th2 cell response. The results suggest different biological roles of the two isoforms during the parasite's life cycle and might be helpful to find a more effective vaccine candidate, diagnostic tool, and/or drug to target against fasciolosis.