Molecular Characteristics of the Fatty-Acid-Binding Protein (FABP) Family in Spirometra mansoni-A Neglected Medical Tapeworm

Abstract

The plerocercoid larva of the tapeworm Spirometra mansoni can parasitize humans and animals, causing serious parasitic zoonosis. The molecular characteristics and adaptive parasitism mechanism of Spirometra tapeworms are still unknown. In this study, 11 new members of the fatty-acid-binding protein (FABP) family were characterized in S. mansoni. A clustering analysis showed 11 SmFABPs arranged into two groups, and motif patterns within each group had similar organizations. RT-qPCR showed that SmFABPs were highly expressed in the adult stage, especially in gravid proglottid. A high genetic diversity of SmFABPs and relative conservation of FABPs in medical platyhelminthes were observed in the phylogenetic analysis. Immunolocalization revealed that natural SmFABP is mainly located in the tegument and parenchymal tissue of the plerocercoid and the uterus, genital pores, and cortex of adult worms. rSmFABP can build a more stable holo form when binding with palmitic acid to protect the hydrolytic sites of the protein. A fatty acid starvation induction test suggested that SmFABP might be involved in fatty acid absorption, transport, and metabolism in S. mansoni. The findings in this study will lay the foundation to better explore the underlying mechanisms of FABPs involved in Spirometra tapeworms as well as related taxa.

Keywords: Spirometra mansoni; fatty-acid-binding protein; gene expression; molecular characterization; tapeworm.

Figure 1

Fatty-acid-binding protein family members identified in Spirometra mansoni. (a) Multiple alignment of protein sequences of SmFABPs. Three areas are highlighted by black lines, representing the three elements that make up the PRINPS pattern PR00178 (fatty acid binding protein signature), which represents a fingerprint of the FABP family of fatty acids. Amino acids shaded in grey represent conserved positions, while those shaded in purple represent similar positions of amino acids in different proteins. Red bars indicate approximate positions of β-strands (βI-βX), while yellow bars indicate α-helices (αI and αII). They are all based on the EgFABP1 structure (PDB, 1o8v). Secondary structure predictions were performed using the PSIPRED tool. (b) Phylogenetic tree and conserved motifs of 11 SmFABPs. (c) Quantitative real-time PCR (qRT–PCR) was used to detect SmFABP gene expression in different tissues. GAPDH was used as an internal reference, and 2^−ΔΔCT values were used for calculation. All data are presented as the means and standard deviation (SD) with at least three repeats. * indicate significant differences (p < 0.01), ** indicate significant differences (p < 0.05). P., plerocercoid; IP, immature proglottide; MP, mature proglottide; GP, gravid proglottide.

Figure 2

Phylogenetic analysis of fatty-acid-binding protein sequences in medical helminths (cestodes + trematodes) based on the maximum-likelihood method. The FABP sequences from S. mansoni are represented by arrows. Values on branches represent bootstrap values, and only values with bootstrap values of >60 are displayed.

Figure 3

Molecular characterization of SmFABP. (a) Determination of the optimal antigen coating concentration. All data are presented as the means and standard deviation (SD) with at least three repeats. (b) Determination of anti-rSmFABP immune serum titer by indirect ELISA. The data were obtained from three repeated experiments. Red, orange, green, blue, purple, and rose red represent serum dilutions of 1:10², 1:10³, 1:10⁴, 1:10⁵, 1:10⁶, and 1:10⁷, respectively. (c) SDS–PAGE and Western blot analyses of the recombinant protein SmFABP. The loading amount of SDS was 5 μg per lane. M: protein marker; Lane 1: uninduced bacterial cultures; Lane 2: induced bacterial cultures; Lane 3: purified rSmFABP; Line 4: rSmFABP + anti-rSmFABP serum; Line 5: rSmFABP + preimmune serum. (d) Transcription pattern of the FABP gene in different developmental stages of Spirometra mansoni. M: DL2000 Marker; Egg: egg stage; Larva: plerocercoid stage; Adult: adult stage; H₂O: Milli-Q water control. * represents a statistically significant difference compared with the plerocercoid group (p < 0.05), ** represents a statistically significant difference compared with the plerocercoid group (p < 0.01). A housekeeping gene (Sm-GAPDH) was used as a positive control. H₂O was used as a negative control.

Figure 4

Immunofluorescence localization of FABP in different developmental stages of S. mansoni. (a) Immunofluorescence localization of SmFABP in the plerocercoid stage. Green fluorescence: SmFABP immune serum/infected mouse serum binding; blue fluorescence: nuclear DAPI staining. Head: the head of plerocercoid; body (cross): transverse section of plerocercoid; body: longitudinal section of plerocercoid; head and body (cross) scale: 200 μm; body scale: 100 μm. (b) Immunofluorescence localization of SmFABP in the adult stage. Green fluorescence: SmFABP immune serum/infected cat serum binding; blue fluorescence: DAPI staining of the nuclei. IMPR: immature proglottids; MPR: mature proglottids; GPR: gravid proglottids; IMPR scale: 500 μm; MPR, GPR and eggs scale: 200 μm.

Figure 5

Degradation pattern and stability of recombinant SmFABP and albumin after incubation with ArgC. (a): The degradation pattern and stability of rSmFABP with fatty acid. A1, A2: Apo form rSmFABP; Y1, Y2: rSmFABP is mixed with oleic acid; Z1, Z2: rSmFABP is mixed with palmitic acid; +ArgC: protein incubated with ArgC at 37 °C for 1 h and 18 h; −ArgC: protein without ArgC at 37 °C for 1 h and 18 h. (b): The degradation pattern and stability of albumin with fatty acid. AB: albumin detected in parallel with recombinant SmFABP; YB: albumin is mixed with oleic acid; ZB: albumin is mixed with palmitic acid. Arrows indicate SmFABP and albumin.

Figure 6

Expression of FABP in vitro culture of plerocercoids. The expression level of FABP in the plerocercoid at 0 h, 6 h, 12 h, 18 h, and 24 h after the fatty acid supply was cut off. The results were generated from the average of three independent replicates. Error bars represent SD (n = 3). The expression level of FABP at 0 h was used as a control. * represents a statistically significant difference compared with the expression level of FABP at 0 h (p < 0.05).