Dataset Information

Screening and identification of SipC-interacting proteins in Salmonella enteritidis using Gal4 yeast two-hybrid system in duck.

ABSTRACT: The zoonotic pathogen Salmonella not only reduces the production performance in ducks, but also poses a serious threat to human health through eggs and pollutes water bodies through feces. SipC, an effector protein of type III secretion systems (T3SS) in Salmonella, mediates translocation of effectors into the eukaryotic host. However, the precise role of SipC effectors remains unknown in ducks. In this study, the SipC from duck granulosa cells (dGCs) was selected as bait, and the SipC-interacting proteins in Salmonella enteritidis (SE) were screened using Gal4 yeast two-hybrid system in duck. Twelve SipC-interacting proteins were identified. Among those, the p53-effector related to PMP-22 (PERP) and TGF-? activated kinase 1-binding protein 2 (TAB2) were selected to further confirm the function by GST pull-down in vitro. Over-expression of PERP resulted in not only increasing SE adhesion and invasion but also triggering the production of IL-1? and IFN-? in SE infected dGCs, while knock-down PERP showed the opposite tendency (P < 0.01). In addition, TAB2 significantly induced the production of IL-6, IL-1?, IFN-?, and INF-? in SE infected dGCs (P < 0.05), but did not cause obvious changes in SE adhesion and invasion. When the sipC in SE was deleted, the activities of duck PERP and TAB2 were abolished because they could not bind to SipC. Taken together, although the protein of PERP and TAB2 can interact with SipC, their mechanisms were different in duck challenged by SE. Therefore, PERP was involved in SE invasion and inflammatory response of dGC ovaries, and TAB2 only contributed to dGCs inflammatory response, which provided critical insights about the mechanism in host- bacterium protein interactions during Salmonella invasion in duck.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC6746301 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Screening and identification of SipC-interacting proteins in Salmonella enteritidis using Gal4 yeast two-hybrid system in duck.

Zhang Yu Y Gu Tiantian T Chen Yang Y Zhu Guoqiang G Vongsangnak Wanwipa W Xu Qi Q Chen Guohong G

PeerJ 20190913

The zoonotic pathogen Salmonella not only reduces the production performance in ducks, but also poses a serious threat to human health through eggs and pollutes water bodies through feces. SipC, an effector protein of type III secretion systems (T3SS) in Salmonella, mediates translocation of effectors into the eukaryotic host. However, the precise role of SipC effectors remains unknown in ducks. In this study, the SipC from duck granulosa cells (dGCs) was selected as bait, and the ...[more]

PMID: 31565575

Similar Datasets

Project description:The screening of a cDNA derived expression library of Salmonella Enteritidis 125109 expressed in E. coli using a fusion construct and specific HaloTag interaction to a modified surface is shown. Thus, 1536 different clones were screened including positive (fimA) and negative (argC, gapA) reference proteins. The goal of the screening was to identify potential novel immunogenic proteins from S. Enteritidis by selecting clones showing a high signal intensity in comparison to the known antigens used as positve markers. Afterwards, the most promising clones were sequenced to identify the gene and corresponding protein and these proteins were then investigated further. Consequently, 9 novel immunogenic proteins could be identified. In total 1536 different lysates were spotted on different microarray slides. Each slides contained 3600 distinct spots, seperated into two compartments of 1800 spots each. Each compartment comprised quadruplicate replicates of each sample lysate with controls being analysed with more replicates. As controls we used: fimA from 3 different samples (40 replicates) as positive reference proteins, as it has been described as immunogenic before. GapA from Klebsiella pneumoniae and Campylobacter jejuni (40 replicates) as negative reference proteins, additionally two sets of E.coli cell lysates without fusionprotein expressed, namely Acella electrocompetent cells (40 replicates) and KRX (32 replicates). Last but not least a buffer control (24 replicates) was included. Therefore, each set of replicate slides contained 384 different samples. Each screening was performed with three replicate slides. Consequently, a total number of 12 slides were screened (4 sets of samples x 3 replicates each). For identification rabbit polyclonal antibody to S. enterica (Abcam ab35156) as primary and Goat polyclonal to Rabbit IgG conjugated with ChromeoTM-546 (Abcam ab60317) as secondary antibody were used. The top compartment was incubated first with primary antibody, while the bottom compartment was incubated with PBS at the same time. Afterwards both compartments were incubated with secondary antibody.

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

Screening and identification of SipC-interacting proteins in Salmonella enteritidis using Gal4 yeast two-hybrid system in duck.

Publications

Screening and identification of SipC-interacting proteins in <i>Salmonella</i> enteritidis using Gal4 yeast two-hybrid system in duck.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets