The Role Of Pneumococcal DnaJ In Bacterial Virulence And Innate Immunity Induced By Streptococcus Pneumoniae

The gram-positive human opportunistic pathogen, Streptococcuspneumoniae（S.pn） can cause pneumonia, meningitis, sepsis and otherinvasive diseases which have high morbidity and mortality in the wholeworld. Researches in the past20years found that many of pneumococcalproteins played a crucial role in mediating inflammatory or damaged hosttissue directly, and pneumococcus has a signifcant attenuation of virulenceafter mutation of the genes encoding these proteins. Therefore, virulenceproteins are essential in the pathogenesis of pneumococcal infections.Spreading into host from the external environment,bacteria areinevitably faced with living condition changes, they will adjust genesexpression to adapt to environmental selection, heat shock protein is just aserial of the stress-inducing related protein to protect bacteria themself. Intheory, these heat shock proteins are often bacterial potential virulencefactors. Therefore, a comprehensive understanding of the interactionsbetween S.pn heat shock proteins and bacterial virulence or host immunecells is helpful to novel therapeutic agents against pneumococcal infection. The highly conserved and widely existing heat shock protein40, alsoknown as DnaJ protein, as one of the DnaK co-chaperones, mainly activateDnaK’s ATPase activity. DnaJ in pneumococcus is a bacterial superficialprotein, it was shown that DnaJ had the potential of vaccines,hosts couldgenerate acquired immune response to resist many pneumococcal serotypesafter abdominal or mucosal DnaJ inoculation. But whether DnaJ protein isinvolved in pneumococcal pathogenesis and the interaction between DnaJand innate immune cells are not clear.The purpose of this reaserch was to investigate the role ofpneumococcal DanJ in bactrial virulence and innate immunity，includingsecretion of proinflammatory cytokines, screening of the pattern recognitionreceptors （PRRs） recognizing DnaJ protein and activation of signalingpathways, induced by Streptococcus pneumoniae in vivo and in vitro.Thisstudy included the following three parts:1.Construction of dnaJ-deletion mutant of S. pn and investigation ofthe mutant growth in vitro under temperature stress, offering experimentalstrains and technology platform for the follow-up study of DnaJ’s role inbacteria and the interaction with hosts.A gene disruption construct was produced by Long flanking homologypolymerase chain reaction(LFH-PCR),which consisted of erythromycinresistance gene（erm）in the middle and the long flanking homology regions of dnaJ upstream and downstream gene sequences on both sides,then thePCR product was transformed directly to S.pn D39or R6strains,at last thednaJ-deletion mutant was screened by the blood agar containingerythromycin and identified by PCR and sequencing. The results showedthat dnaJ gene was completely replaced by erm gene, and there were noappearance changes after Gram staining in the mutants, while colonies of thednaJ-deletion strain became obviously smaller;The growth curve of thedeficient in vitro was broadly consistent with that of the wild at temperaturesof30℃and37℃,but the mutant growth were restrained and they almostcould not grow at20℃and40℃.2. Observation of DnaJ’s effect on the virulence of S.pn in vivo and invitro, respectively.Directly observed the survival status of mice infected the wild ordefective strains by abdominal challenge; Established a mouse pneumoniamodel by nasal attack, then count bacterial loads in susceptible organs tounderstand the effect of dnaJ-deletion on pneumococcal colonizationcapacity;Infected mouse lung epithelial cells MLE12with S.pn to comparetheir adhesion and invasion differences between the wild and defective, Thevirulence experiment showed mice challenged with mutants could alltolerate infection of lethal dose and survived in a long term; colonizationexperiment displayed pneumococcal CFUs of nasopharynxes and lungs were significantly lower in the mutational group than that in the wildgroup,and the mutants invading into host blood were cleared up quickly;dnaJ-defect significantly reduced adhesion and invasion ability of S.pn toMLE12, moreover,CFUs of wild strain adhered to MLE12pretreated withanti-DnaJ serum decreased notablely.3. Looking for the innate immunity changes of hosts and immune cells,and sieving the PRRs that recognized DnaJ protein to induce innate immuneresponse and the succedent active signal pathway by DnaJ stimulation.Established a mouse pneumococcal pneumonia model in vivo, tookinfective lung tissue, slicing and staining to observe inflammatory response,detecting the secretion of proinflammatory cytokines through quantitativePCR and ELISA; in vitro inoculated S.pn into murine macrophage cell lineRAW264.7to compare cellular phagocytic activity and inflammatoryfactors levels of the wild group with those of the defect one. Prokaryoticlyexpressed and purified full-length DnaJ protein and stimulated RAW264.7with it to measrue the pro-inflammatory cytokines, to screen TLRs identifiedDnaJ by real-time PCR and anti-TLRs mAb, to look for the active signalpathway involved in cytokine secretion though protein kinase inhibitor andwestern blot. Internal experiments indicated lung inflammation intensitydecreased, proinflammatorycytokine secretion peak was depressed and peaktime was delayed in mice infected with mutants; Exoteric experiments found RAW264.7phagocytized dnaJ-deletion mutants more than wild strains,andcytokine levels were reduced; DnaJ protein could excite RAW264.7to secretIL-6, activating PI3K and JNK pathway, independent of TLR2and TLR4.