title: Screening and identification of ClpE interaction proteins in Streptococcus pneumoniae by a bacterial two-hybrid system and co-immunoprecipitation.
abstract: Hsp100/Clp proteins have crucial functions in the protein quality control, stress tolerance, and virulence of many pathogenic bacteria. ClpE is an important virulence factor involved in adherence and invasion in Streptococcus pneumoniae. To explore the underlying mechanism, we screened ClpE interaction proteins using a bacterial two-hybrid system and co-immunoprecipitation. We used ClpE as bait and constructed the pBT-ClpE bait plasmid for two-hybrid screening. Then, we constructed ClpE::GFP fusion for co-immunoprecipitation screening using anti-GFP monoclonal antibody. We obtained eight potential ClpE interaction proteins, including carbamoyl-phosphate synthase, pyruvate oxidase (SpxB), phosphoenolpyruvate-protein phosphotransferase, aminopeptidase N (pepN), L-lactate dehydrogenase, ribosomal protein S4, sensor histidine kinase (SPD_2019), and FtsW (a cell division protein). FtsW, SpxB, pepN, and SPD_2019 were confirmed to interact with ClpE using Bacterial Two-hybrid or Co-immunoprecipitation. Morphologic observations found that ΔclpE strain existed in abnormal division. β-Galactosidase activity assay suggested that ClpE contributed to the degradation of FtsW. Furthermore, FtsW could be induced by heat shock. The results suggested that ClpE might affect cell division by regulating the level of FtsW. These data may provide new insights in studying the role of ClpE in S. pneumoniae.
authors.affiliation: Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, P. R. China.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Yan
authors.forename: Yingying
authors.initials: Y
authors.lastname: Cai
authors.forename: Qun
authors.initials: Q
authors.lastname: Zhang
authors.forename: Yusi
authors.initials: Y
authors.lastname: Liu
authors.forename: Wenchun
authors.initials: W
authors.lastname: Xu
authors.forename: Yibing
authors.initials: Y
authors.lastname: Yin
authors.forename: Yujuan
authors.initials: Y
authors.lastname: He
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Xuemei
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D017353:Gene Deletion; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D059748:Proteolysis; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; C061684:FtsW protein, Bacteria; D000251:Adenosine Triphosphatases; C118340:ClpE protein, bacteria