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Clonorchis sinensis acetoacetyl-CoA thiolase: identification and characterization of its potential role in surviving in the bile duct.

Lin J, Qu H, Chen G, He L, Xu Y, Xie Z, Ren M, Sun J, Li S, Chen W, Chen X, Wang X, Li X, Liang C, Huang Y, Yu X - Parasit Vectors (2015)

Bottom Line: CsACAT was confirmed to be a member of the thiolase family and present in the excretory/secretory proteins of C. sinensis.Our results implied that C. sinensis might sense lipid levels and survive better in the bile environment with higher lipid levels.C. sinensis might modulate the expression and enzymatic activity of CsACAT, an enzyme involved in fatty acid metabolism, for energy or physical requirements to adapt to the host.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, People's Republic of China. linjinsi1989@gmail.com.

ABSTRACT

Background: Clonorchis sinensis (C. sinensis) inhabits in bile duct of the host. However, the mechanisms involved in why C. sinensis can survive in the bile environment containing lipids have not yet been explored. In this study, C. sinensis acetoacetyl-CoA thiolase (CsACAT), a member of the thiolase family which has a key role in the beta oxidation pathway of fatty acid production, was identified and characterized to understand its potential role in adapting to the bile environment.

Methods: The encoding sequence, conserved domains and spatial structure of CsACAT were identified and analyzed by bioinformatic tools. Recombinant CsACAT (rCsACAT) was obtained using a procaryotic expression system. The expression pattern of CsACAT was confirmed by quantitative real-time PCR, western blotting, and immunofluorescence. Gradients of lecithin were then set to culture C. sinensis adults in vitro and the survival rate of C. sinensis was analyzed, as well as the expression level and enzymatic activity of CsACAT in different lipid environments. Hypercholesteremia rabbit models were established by feeding with a hyperlipidemic diet and then infected intragastrically with C. sinensis. One and a half months later, the worm burdens and the expression level of CsACAT was detected.

Results: CsACAT was confirmed to be a member of the thiolase family and present in the excretory/secretory proteins of C. sinensis. CsACAT was specifically localized at the vitellarium and sub-tegumental muscle layer in adult worms. The mRNA level of CsACAT in eggs was higher than those in adult worms and metacercariae. When adult worms were cultured with higher concentration of lecithin, the expression level and enzyme activity of CsACAT were up-regulated. The survival rate of adult worms was higher than control group. More adult worms were recovered from hypercholesteremia rabbit models. The expression level of CsACAT in these worms was higher than control group.

Conclusions: Our results implied that C. sinensis might sense lipid levels and survive better in the bile environment with higher lipid levels. C. sinensis might modulate the expression and enzymatic activity of CsACAT, an enzyme involved in fatty acid metabolism, for energy or physical requirements to adapt to the host.

No MeSH data available.


Related in: MedlinePlus

Expression pattern ofCsACAT. (A) Immunolocalization of CsACAT in C. sinensis adult. Panel a and b, sections treated with anti-rCsACAT serum and specific fluorescences distributed in the vitellarium and sub-tegumental muscular layer of the adult worm; Panel c and d, sections treated with naive serum. No fluorescence was detected. M, sub-tegumental muscular layer; V, vitellarium. Magnification for the adult worm was × 100. (B) mRNA level of CsACAT at different developmental stages of C. sinensis by quantitative real-time PCR. The specific mRNA fragment of CsACAT was observed among the stages. The quantities were normalized with Cs β-actin and analyzed by means of the 2−ΔΔCt ratio.
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Fig2: Expression pattern ofCsACAT. (A) Immunolocalization of CsACAT in C. sinensis adult. Panel a and b, sections treated with anti-rCsACAT serum and specific fluorescences distributed in the vitellarium and sub-tegumental muscular layer of the adult worm; Panel c and d, sections treated with naive serum. No fluorescence was detected. M, sub-tegumental muscular layer; V, vitellarium. Magnification for the adult worm was × 100. (B) mRNA level of CsACAT at different developmental stages of C. sinensis by quantitative real-time PCR. The specific mRNA fragment of CsACAT was observed among the stages. The quantities were normalized with Cs β-actin and analyzed by means of the 2−ΔΔCt ratio.

Mentions: The specific fluorescences were specifically localized in the vitellarium and sub-tegumental muscle layer of the adult worm by using rat anti-rCsACAT serum, while no specific fluorescence was observed in sections treated with naive serum (Figure 2A). The result of quantitative real-time PCR demonstrated that mRNA of CsACAT was observed at life stages of adult worm, metacercaria and egg of C. sinensis. The mRNA level of CsACAT in egg was higher than that in metacercaria (2.93-fold, p < 0.05) or adult worm (10.16-fold, p < 0.01) (Figure 2B).Figure 2


Clonorchis sinensis acetoacetyl-CoA thiolase: identification and characterization of its potential role in surviving in the bile duct.

Lin J, Qu H, Chen G, He L, Xu Y, Xie Z, Ren M, Sun J, Li S, Chen W, Chen X, Wang X, Li X, Liang C, Huang Y, Yu X - Parasit Vectors (2015)

Expression pattern ofCsACAT. (A) Immunolocalization of CsACAT in C. sinensis adult. Panel a and b, sections treated with anti-rCsACAT serum and specific fluorescences distributed in the vitellarium and sub-tegumental muscular layer of the adult worm; Panel c and d, sections treated with naive serum. No fluorescence was detected. M, sub-tegumental muscular layer; V, vitellarium. Magnification for the adult worm was × 100. (B) mRNA level of CsACAT at different developmental stages of C. sinensis by quantitative real-time PCR. The specific mRNA fragment of CsACAT was observed among the stages. The quantities were normalized with Cs β-actin and analyzed by means of the 2−ΔΔCt ratio.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359446&req=5

Fig2: Expression pattern ofCsACAT. (A) Immunolocalization of CsACAT in C. sinensis adult. Panel a and b, sections treated with anti-rCsACAT serum and specific fluorescences distributed in the vitellarium and sub-tegumental muscular layer of the adult worm; Panel c and d, sections treated with naive serum. No fluorescence was detected. M, sub-tegumental muscular layer; V, vitellarium. Magnification for the adult worm was × 100. (B) mRNA level of CsACAT at different developmental stages of C. sinensis by quantitative real-time PCR. The specific mRNA fragment of CsACAT was observed among the stages. The quantities were normalized with Cs β-actin and analyzed by means of the 2−ΔΔCt ratio.
Mentions: The specific fluorescences were specifically localized in the vitellarium and sub-tegumental muscle layer of the adult worm by using rat anti-rCsACAT serum, while no specific fluorescence was observed in sections treated with naive serum (Figure 2A). The result of quantitative real-time PCR demonstrated that mRNA of CsACAT was observed at life stages of adult worm, metacercaria and egg of C. sinensis. The mRNA level of CsACAT in egg was higher than that in metacercaria (2.93-fold, p < 0.05) or adult worm (10.16-fold, p < 0.01) (Figure 2B).Figure 2

Bottom Line: CsACAT was confirmed to be a member of the thiolase family and present in the excretory/secretory proteins of C. sinensis.Our results implied that C. sinensis might sense lipid levels and survive better in the bile environment with higher lipid levels.C. sinensis might modulate the expression and enzymatic activity of CsACAT, an enzyme involved in fatty acid metabolism, for energy or physical requirements to adapt to the host.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, People's Republic of China. linjinsi1989@gmail.com.

ABSTRACT

Background: Clonorchis sinensis (C. sinensis) inhabits in bile duct of the host. However, the mechanisms involved in why C. sinensis can survive in the bile environment containing lipids have not yet been explored. In this study, C. sinensis acetoacetyl-CoA thiolase (CsACAT), a member of the thiolase family which has a key role in the beta oxidation pathway of fatty acid production, was identified and characterized to understand its potential role in adapting to the bile environment.

Methods: The encoding sequence, conserved domains and spatial structure of CsACAT were identified and analyzed by bioinformatic tools. Recombinant CsACAT (rCsACAT) was obtained using a procaryotic expression system. The expression pattern of CsACAT was confirmed by quantitative real-time PCR, western blotting, and immunofluorescence. Gradients of lecithin were then set to culture C. sinensis adults in vitro and the survival rate of C. sinensis was analyzed, as well as the expression level and enzymatic activity of CsACAT in different lipid environments. Hypercholesteremia rabbit models were established by feeding with a hyperlipidemic diet and then infected intragastrically with C. sinensis. One and a half months later, the worm burdens and the expression level of CsACAT was detected.

Results: CsACAT was confirmed to be a member of the thiolase family and present in the excretory/secretory proteins of C. sinensis. CsACAT was specifically localized at the vitellarium and sub-tegumental muscle layer in adult worms. The mRNA level of CsACAT in eggs was higher than those in adult worms and metacercariae. When adult worms were cultured with higher concentration of lecithin, the expression level and enzyme activity of CsACAT were up-regulated. The survival rate of adult worms was higher than control group. More adult worms were recovered from hypercholesteremia rabbit models. The expression level of CsACAT in these worms was higher than control group.

Conclusions: Our results implied that C. sinensis might sense lipid levels and survive better in the bile environment with higher lipid levels. C. sinensis might modulate the expression and enzymatic activity of CsACAT, an enzyme involved in fatty acid metabolism, for energy or physical requirements to adapt to the host.

No MeSH data available.


Related in: MedlinePlus