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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, purification and characterization ofCsACAT. (A) rCsACAT was identified by 12% SDS-PAGE. Protein molecular weight markers (lane M), lysate of E. coli containing pET28a (+) without induction (lane 1) and with induction by IPTG (lane 2), lysate of E. coli containing pET28a (+)-CsACAT without induction (lane 3) and with induction by IPTG (lane 4), supernatant (lane 5) and precipitant (lane 6) of lysate of E. coli containing the recombinant plasmid after induction, the purified recombinant CsACAT protein (lane 7). (B) Western blotting analysis of CsACAT. Lane 1–4, rCsACAT probed with naive serum, anti-His tag monoclonal antibody, rat anti-rCsACAT serum and serum from C. sinensis-infected rat; lane 5–6, CsESPs probed with naive serum and rat anti-rCsACAT serum; lane 7–8, total proteins of adult worm probed with naive serum and rat anti-rCsACAT serum; lane 9, purified rCsACAT blotted with rat anti-CsESPs serum.
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Fig1: Expression, purification and characterization ofCsACAT. (A) rCsACAT was identified by 12% SDS-PAGE. Protein molecular weight markers (lane M), lysate of E. coli containing pET28a (+) without induction (lane 1) and with induction by IPTG (lane 2), lysate of E. coli containing pET28a (+)-CsACAT without induction (lane 3) and with induction by IPTG (lane 4), supernatant (lane 5) and precipitant (lane 6) of lysate of E. coli containing the recombinant plasmid after induction, the purified recombinant CsACAT protein (lane 7). (B) Western blotting analysis of CsACAT. Lane 1–4, rCsACAT probed with naive serum, anti-His tag monoclonal antibody, rat anti-rCsACAT serum and serum from C. sinensis-infected rat; lane 5–6, CsESPs probed with naive serum and rat anti-rCsACAT serum; lane 7–8, total proteins of adult worm probed with naive serum and rat anti-rCsACAT serum; lane 9, purified rCsACAT blotted with rat anti-CsESPs serum.

Mentions: The ORF of CsACAT was cloned into pET-28a (+) expression vector and the recombinant plasmids were confirmed by sequencing. The IPTG induced rCsACAT was purified and analyzed by SDS-PAGE, showing a single band with molecular mass of approximately 30 kDa (Figure 1A). Western blotting (Figure 1B) indicated that rCsACAT could be probed by mouse anti-His tag monoclonal antibody, rat anti-CsACAT serum, rat anti-CsESPs serum, and serum from C. sinensis-infected rat at a clear band about 30 kDa, while rCsACAT could not be blotted with serum from pre-immunized rat. CsESPs and total proteins of worm could also be probed by rat anti-CsACAT serum but not by serum from pre-immunized rat. In addition, the optimum enzymatic activity of rCsACAT was at pH 7.5 and 37°C (Additional file 2: Figure S2).Figure 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, purification and characterization ofCsACAT. (A) rCsACAT was identified by 12% SDS-PAGE. Protein molecular weight markers (lane M), lysate of E. coli containing pET28a (+) without induction (lane 1) and with induction by IPTG (lane 2), lysate of E. coli containing pET28a (+)-CsACAT without induction (lane 3) and with induction by IPTG (lane 4), supernatant (lane 5) and precipitant (lane 6) of lysate of E. coli containing the recombinant plasmid after induction, the purified recombinant CsACAT protein (lane 7). (B) Western blotting analysis of CsACAT. Lane 1–4, rCsACAT probed with naive serum, anti-His tag monoclonal antibody, rat anti-rCsACAT serum and serum from C. sinensis-infected rat; lane 5–6, CsESPs probed with naive serum and rat anti-rCsACAT serum; lane 7–8, total proteins of adult worm probed with naive serum and rat anti-rCsACAT serum; lane 9, purified rCsACAT blotted with rat anti-CsESPs serum.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Expression, purification and characterization ofCsACAT. (A) rCsACAT was identified by 12% SDS-PAGE. Protein molecular weight markers (lane M), lysate of E. coli containing pET28a (+) without induction (lane 1) and with induction by IPTG (lane 2), lysate of E. coli containing pET28a (+)-CsACAT without induction (lane 3) and with induction by IPTG (lane 4), supernatant (lane 5) and precipitant (lane 6) of lysate of E. coli containing the recombinant plasmid after induction, the purified recombinant CsACAT protein (lane 7). (B) Western blotting analysis of CsACAT. Lane 1–4, rCsACAT probed with naive serum, anti-His tag monoclonal antibody, rat anti-rCsACAT serum and serum from C. sinensis-infected rat; lane 5–6, CsESPs probed with naive serum and rat anti-rCsACAT serum; lane 7–8, total proteins of adult worm probed with naive serum and rat anti-rCsACAT serum; lane 9, purified rCsACAT blotted with rat anti-CsESPs serum.
Mentions: The ORF of CsACAT was cloned into pET-28a (+) expression vector and the recombinant plasmids were confirmed by sequencing. The IPTG induced rCsACAT was purified and analyzed by SDS-PAGE, showing a single band with molecular mass of approximately 30 kDa (Figure 1A). Western blotting (Figure 1B) indicated that rCsACAT could be probed by mouse anti-His tag monoclonal antibody, rat anti-CsACAT serum, rat anti-CsESPs serum, and serum from C. sinensis-infected rat at a clear band about 30 kDa, while rCsACAT could not be blotted with serum from pre-immunized rat. CsESPs and total proteins of worm could also be probed by rat anti-CsACAT serum but not by serum from pre-immunized rat. In addition, the optimum enzymatic activity of rCsACAT was at pH 7.5 and 37°C (Additional file 2: Figure S2).Figure 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