Screening And Identification Of ClpE Interaction Proteins In Streotococcus Pneumoniae

ObjectiveHSP100/ClpATPase is a class of highly conserved and widespreadheat shock protein, which belongs to AAA+（ATPase associated withdiverse cellular activities） protein superfamily. They have many biologicalfunctions. They can form ATP-dependent protease complexes with ClpP topromote the hydrolysis of the specific substrate proteins. And ClpATPasedecides the proteolysis function of ClpP and the specificity of degradablesubstrate. At present many studies have confirmed that ClpATPase playedan important role in the pathogenic process of many pathogens. InStaphylococcus aureus, the virulence of ClpX deficient strain wassignificantly reduced, which was mainly due to the role of ClpX in theregulation of major virulence factors. In Listeria monocytogenes, ClpC wasinvolved in the adhesion and invasion of host cells by regulating theexpression of invasion related factors.The preliminary studies on ClpE of Streptococcus pneumoniae in ourlab found that the virulence of ClpE deficient strain was significantly reduced in a mouse intraperitoneal infection model of Streptococcuspneumoniae. So, How did ClpE contribute to the bacterial pathogenesis?Which virulence factors were affected by ClpE, resulting in thecorresponding characteristic of bacterial virulence? In order to clarify theseproblems, we should find the specific protein substrates of ClpE. In thisstudy, interacting protein of ClpE was screened using bacterial two-hybridsystem and co-immunoprecipitation experiments, which were currentlywidely used to study protein interaction. Further, the function of theinteraction protein was studied in order to reveal the regulation role ofClpE in Streptococcus pneumoniae.Methods1. Bacterial two-hybrid system screeningFirst, we expressed and purified rClpE and prepared the anti-ClpEpolyclonal antibody. We constructed a prokaryotic expression vectorpET28a-ClpE with His-tag, which was transformed into Escherichia coliBL21（DE3）. After adding IPTG to induce the expression of protein ClpE,the rClpE was purified using nickel affinity chromatography. Further, theSDS-PAGE was carried out to analysis the purity of the purified rClpE.Then, after dialyzing to remove the imidazole, the rClpE was used asantigen to immunize Balb/c mice and New Zealand white rabbits forpreparation of the anti-ClpE antibody.Next, we constructed the bait plasimd pBT-ClpE, which was successfully transformed into bacterial two-hybrid system strain （200192）after PCR and sequencing identification. Western blot experimentconfirmed that the ClpE could express normally in the plasmid after IPTGinducing. Then, the self-activation of pBT-ClpE was tested on NSSM orSSM plates by co-transformation of pBT-ClpE and pTRG into bacterialtwo-hybrid system strain （200192） to make sure that it could be used forthe screening experiments.Third, we construct the library plasmid pTRG-DNA. We used therestriction endonuclease Sau3Aâ… randomly to digest the genomic DNA ofStreptococcus pneumoniae, which was incubated overnight with plasmidpTRG digested with BamHâ… . Then, the product was transformed intobacterial two-hybrid system strain to get the library plasmid containingdifferent sizes of DNA fragments.Finally, bait plasmid pBT-ClpE and the library plasmid wasco-transformed into bacterial two-hybrid system strain. The positive cloneswere screened out on SSM plates.2. Co-immunoprecipitation screeningWe constructed the plasmid pAE03-ClpE containing GFP-tag, whichwas transformed into Streptococcus pneumoniae D39to get the strainD39（ClpE::GFP）. Then, the GFP monoclonal antibody was incubated withProtein G and the lysates of D39（ClpE::GFP） or D39overnight at4℃. TheClpE interacting protein might have been bound to the Protein G. The SDS-PAGE was used to detect the Protein G after washing5times withPBS. The different bands between these two groups were sent to BGI formass spectrometry analysis.3. Function studies of ClpE interacing proteinsIn the experiment, we found that compared with the wild strain themorphology of D39clpE was different, being rod shape withoutumbilication in the middle. Further Electron microscopy showed that theD39clpE was longer that the wild type strain. We speculated that ClpEmight be involved in the regulation of cell division. Bioinformatics analysisshowed that the ClpE interacting protein FtsW was related with celldivision, so we chose this protein for preliminary function studies. Theexpression of FtsW after heat shoch was tested using western blot. The roleof ClpE in the degradation of FtsW was tested using β-galactosidaseactivity detection.Results1. We successfully constructed vector pET28a-ClpE and got rClpE with thepurification greater than85%. The rClpE was used as an antigen to immunemice and rabbit. Finally, we got two different kind of anti-ClpE polyclonalantibody from mice and rabbit.2. Western blot experiment confirmed that the ClpE could express normally.Then, the self-activation assay confirmed that the pBT-ClpE could notactive the reporter gene by itself. Eventually, through the two-hybrid screening we obtained25positive clones, only two of which had correctexpression in the plasmid pTRG after PCR and sequencing. They were celldivision protein FtsW and putative sensor histidine kinase SPD₂019. Then,we cloned full-length SPD₂019or FtsW into pTRG for further validationof the interaction between ClpE and FtsW or SPD₂019by bacterialtwo-hybrid system.3. Through the co-immunoprecipitation screening we got six proteins, theywere pyruvate oxidase （SpxB）, aminopeptidase N （pepN）, L-lactatedehydrogenase（l-Ldh）, ribosomal protein S4（RpsD）, phosphoenolpyruvate-protein Phosphotransferase（PtsI）, carbamoyl-phosphate synthase （CarB）.It was reported that SpxB and PepN was related with bacterial virulence, sowe cloned the gene pepN or spxB to pTRG to validate their interaction withClpE using the bacterial two-hybrid assay.4. The preliminary study of FtsW showed that the expression of FtsW wasincreased when the bacteria was in heat shock. And the degradation ofFtsW was slower in D39clpE than in D39.ConclusionIn this study, we totally got eight ClpE interaction proteins, of whichFtsW, SPD₂019, PepN and SpxB were confirmed to interact with ClpE.These results prompted us that ClpE might contribute to bacterialmorphology and virulence through affecting the expression of theseproteins. The expression of cell division protein FtsW was induced by heatstress, and ClpE was involved in the degradation of FtsW. These resultsindicated that ClpE might affect cell division by regulating the level ofFtsW.