Molecular and Functional Characterization of Inhibitor of Apoptosis Proteins (IAP, BIRP) in Echinococcus granulosus

Abstract

The larval stage of Echinococcus granulosus sensu lato, resulting in cystic echinococcosis, a parasitic zoonosis, causes huge economic losses to the livestock industry and poses a threat to public health. Inhibitor of apoptosis proteins (IAPs) is a class of endogenous anti-apoptotic family, which plays a significant functional role in the regulation of organism's development. Herein, to explore potential functions of IAPs in E. granulosus, two members of IAPs from E. granulosus (Eg-IAP and Eg-BIRP) were cloned, expressed, and molecularly characterized. Eg-IAP and Eg-BIRP encoded putative 331 and 168 residue proteins, respectively. Bioinformatic analysis showed that both proteins contained a type II BIR domain-the essential functional domain of IAPs. Fluorescence immunohistochemistry revealed that both proteins were ubiquitously localized in all life-cycle stages of E. granulosus. Our fluorescent quantitative PCR (RT-qPCR) results revealed relatively higher transcription levels of two Eg-IAPs in protoscoleces (PSCs) compared to the 18-day strobilated worms. We further used different concentrations of LCL161, a Smac-mimetic pan-IAPs inhibitor, to induce the apoptosis in PSCs in vitro, and revealed that the survival rate of PSCs and transcription levels of both genes were negatively correlated with the concentration of LCL161. While the results of light microscopy, transmission electron microscopy (TEM), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay also showed a higher apoptotic rate in PSCs with the increasing concentrations of LCL161. Taken together, our findings provide the reasonable evidence that both Eg-IAP and Eg-BIRP have potential implication in critical anti-apoptotic roles during the development of E. granulosus.

Molecular characterization and expression analysis of annexin B3 and B38 as secretory proteins in Echinococcus granulosus

“…granulosus were obtained from naturally infected sheep in Sichuan Province, China. PSCs and the germinal layer were separated aseptically as described previously [ 25 , 26 ]. The 28-day strobilated worms were acquired by artificially infecting beagles.…”

“…The preparation process of the polyclonal antibodies of recombinant Eg AnxB3 (r Eg AnxB3) and r Eg AnxB38 was the same as previously reported [ 26 ]. Briefly, rabbit sera were collected as the negative control before immunization.…”

“…Immunofluorescence localization was performed as described previously [ 26 ]. The sections (PSCs, germinal layer from fertile/infertile cysts, 18-day strobilated worms and 45-day adult worms) were incubated with purified anti-r Eg AnxB3/anti-r Eg AnxB38 rabbit IgG or negative rabbit sera (1:200 v/v dilutions) for about 14 h at 4 °C.…”

Molecular Characterization and Expression Analysis of Annexin B3 and B38 as Secretory Proteins in Echinococcus Granulosus

“…Cysts of E. granulosus were seperated from naturally infected sheep in Sichuan Province, China. Protoscoleces (PSCs) and germinal layer were separated aseptically as described previously [22,23]. The 28-day strobilated worms were acquired by arti cially infected beagles.…”

“…The preparation process of the polyclonal antibodies of rEgAnxB3 and rEgAnxB38 was the same as previously reported [23]. Brie y, rabbit sera were collected as a negative control before immunization.…”