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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 level and enzymatic activity ofCsACAT in adult worms incubated in lecithin. (A) mRNA levels of CsACAT in worms cultured with different concentrations of lecithin were evaluated by quantitative real-time PCR. (B) Crude proteins extracted from worms cultured in 6 mM (Lane 1), 0.6 mM (Lane 2), 0.06 mM (Lane 3) of lecithin and control group (Lane 4) were probed with rat anti-rCsACAT serum. (C) Optical density analysis of the protein levels. The optical density was calculated by Tanon Gis software and analyzed by Student’s t test, *p < 0.05.
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Fig4: Expression level and enzymatic activity ofCsACAT in adult worms incubated in lecithin. (A) mRNA levels of CsACAT in worms cultured with different concentrations of lecithin were evaluated by quantitative real-time PCR. (B) Crude proteins extracted from worms cultured in 6 mM (Lane 1), 0.6 mM (Lane 2), 0.06 mM (Lane 3) of lecithin and control group (Lane 4) were probed with rat anti-rCsACAT serum. (C) Optical density analysis of the protein levels. The optical density was calculated by Tanon Gis software and analyzed by Student’s t test, *p < 0.05.

Mentions: Living worms were collected 48 h after incubation in lecithin and subjected to crude extraction to examine enzymatic activity of CsACAT. The CsACAT activity in newly recovered worms taken from rats was set as 100%. The enzymatic activity of CsACAT from worms cultured in 6 mM lecithin showed a highest activity of 40.19% compared with other groups (Table 1). Both mRNA and protein levels of CsACAT were higher in 6 mM lecithin and 0.6 mM lecithin groups compared with those of the control group by Q-PCR (Figure 4A) and Western blotting (Figure 4B-C).Table 1


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 level and enzymatic activity ofCsACAT in adult worms incubated in lecithin. (A) mRNA levels of CsACAT in worms cultured with different concentrations of lecithin were evaluated by quantitative real-time PCR. (B) Crude proteins extracted from worms cultured in 6 mM (Lane 1), 0.6 mM (Lane 2), 0.06 mM (Lane 3) of lecithin and control group (Lane 4) were probed with rat anti-rCsACAT serum. (C) Optical density analysis of the protein levels. The optical density was calculated by Tanon Gis software and analyzed by Student’s t test, *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Expression level and enzymatic activity ofCsACAT in adult worms incubated in lecithin. (A) mRNA levels of CsACAT in worms cultured with different concentrations of lecithin were evaluated by quantitative real-time PCR. (B) Crude proteins extracted from worms cultured in 6 mM (Lane 1), 0.6 mM (Lane 2), 0.06 mM (Lane 3) of lecithin and control group (Lane 4) were probed with rat anti-rCsACAT serum. (C) Optical density analysis of the protein levels. The optical density was calculated by Tanon Gis software and analyzed by Student’s t test, *p < 0.05.
Mentions: Living worms were collected 48 h after incubation in lecithin and subjected to crude extraction to examine enzymatic activity of CsACAT. The CsACAT activity in newly recovered worms taken from rats was set as 100%. The enzymatic activity of CsACAT from worms cultured in 6 mM lecithin showed a highest activity of 40.19% compared with other groups (Table 1). Both mRNA and protein levels of CsACAT were higher in 6 mM lecithin and 0.6 mM lecithin groups compared with those of the control group by Q-PCR (Figure 4A) and Western blotting (Figure 4B-C).Table 1

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