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Identification of RanBMP interacting with Shigella flexneri IpaC invasin by two-hybrid system of yeast.

ABSTRACT: AIM:Bacillary dysentery caused by Shigella flexneri is still a threat to human health. Of four invasion plasmid antigen proteins (IpaA,B,C and D), IpaC plays an important role in the pathogenicity of this pathogen. The purpose of this study was to investigate the proteins interacting with IpaC in the host cell during the pathogenic process of this disease. METHODS:By applying two-hybrid system, the bait plasmid containing ipaC gene was constructed and designated pGBKT-ipaC. The bait plasmid was transformed AH109, and proved to express IpaC and then HeLa cDNA library plasmids were introduced into the above transformed AH109. The transformation mixture was plated on medium lacking Trp, Leu, and His in the initial screen, then restreaked on medium lacking Trp, Leu, His and Ade. Colonies growing on the selection medium were further assayed for beta-galactosidase activity. BLAST was carried out in the database after sequencing the inserted cDNA of the positive library plasmid. RESULTS:Among the 2X10(6) transformants, 64 positive clones were obtained as determined by activation of His, Ade and LacZ reporter genes. Sequence analysis revealed that cDNA inserts of two colonies were highly homologous to a known human protein, RanBPM. CONCLUSION:These results provide evidence that IpaC may be involved in the invasion process of S. flexneri by interacting with RanBPM, and RanBPM is most likely to be the downstream target of IpaC in the cascade events of S. flexneri infection.

SUBMITTER: Yao X

PROVIDER: S-EPMC4611814 | biostudies-literature | 2003 Jun

REPOSITORIES: biostudies-literature

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Identification of RanBMP interacting with Shigella flexneri IpaC invasin by two-hybrid system of yeast.

Yao Xiao X Wang Heng-Liang HL Shi Zhao-Xing ZX Yan Xiao-Yu XY Feng Er-Ling EL Yang Bo-Lun BL Huang Liu-Yu LY

World journal of gastroenterology 20030601 6

<h4>Aim</h4>Bacillary dysentery caused by Shigella flexneri is still a threat to human health. Of four invasion plasmid antigen proteins (IpaA,B,C and D), IpaC plays an important role in the pathogenicity of this pathogen. The purpose of this study was to investigate the proteins interacting with IpaC in the host cell during the pathogenic process of this disease.<h4>Methods</h4>By applying two-hybrid system, the bait plasmid containing ipaC gene was constructed and designated pGBKT-ipaC. The ba ...[more]

PMID: 12800254

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Project description:BackgroundTheileria annulata can infect monocytes/macrophages and B lymphocytes and causes severe lymphoproliferative disease in ruminants. Meanwhile, infection by T. annulata leads to the permanent proliferation of cell population through regulating signaling pathways of host cells. Cysteine proteinases (CPs) are one kind of protein hydrolase and usually play critical roles in parasite virulence, host invasion, nutrition and host immune response. However, the biological function of T. annulata CP (TaCP) is still unclear. In this study, a yeast-two-hybrid assay was performed to screen host proteins interacting with TaCP, to provide information to help our understanding of the molecular mechanisms between T. annulata and host cells.MethodsThe cDNA from purified bovine B cells was inserted into pGADT7-SfiI vector (pGADT7-SfiI-BcDNA, Prey plasmid) for constructing the yeast two-hybrid cDNA library. TaCP was cloned into the pGBKT7 vector (pGBKT7-TaCP) and was considered as bait plasmid after evaluating the expression, auto-activation and toxicity tests in the yeast strain Y2HGold. The yeast two-hybrid screening was carried out via co-transforming bait and prey plasmids into yeast strain Y2HGold. Sequences of positive preys were analyzed using BLAST, Gene Ontology, UniProt and STRING.ResultsTwo host proteins, CRBN (Bos taurus cereblon transcript variant X2) and Ppp4C (Bos indicus protein phosphatase 4 catalytic subunit) were identified to interact with TaCP. The results of functional analysis showed that the two proteins were involved in many cellular processes, such as ubiquitylation regulation, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs.ConclusionsThis study is the first to screen the host proteins of bovine B cells interacting with TaCP, and 2 proteins, CRBN and Ppp4C, were identified using yeast two-hybrid technique. The results of functional analysis suggest that the two proteins are involved in many cellular processes, such as ubiquitylation regulating, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs. The interaction with CRBN and Ppp4C indicate that TaCP possibly is involved in regulating signaling pathways and cell proliferation, which is helpful for understanding the interaction between T. annulata and host cells.

Dataset Information

Identification of RanBMP interacting with Shigella flexneri IpaC invasin by two-hybrid system of yeast.

Publications

Identification of RanBMP interacting with Shigella flexneri IpaC invasin by two-hybrid system of yeast.

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