Study On Transcriptional Regulators Of Cps Locus In Streptococcus Pneumoniae: Isolation,Identification,and Regulation Of Come On Capsular

Objective Streptococcus pneumoniae（pneumococcus）,a Gram-positive opportunistic pathogen residing on the human upper respiratory tract,remains a leading cause of morbidity and mortality worldwide in young children and in immunocompromised elderly.The capsular polysaccharide（CPS）of S.pneumoniae is the critical virulence factor required for effective colonization in the nasopharyngeal of host,for resist phagocytosis and for invasive infection in the blood and lungs.S.pneumoniae must require a coordinated regulation in the expression of capsular to survive in different niches within the host.All capsule types but 3 and 37 serotypes are synthesized by the Wzy-dependent pathway,in which the CPS synthesis loci as a gene cluster are located at the same region of the chromosome between the dex B and ali A genes.Our previous studies have demonstrated that the mutation in the-10 box of the cps promoter from the consensus TATAAT to TACAAT or TATAAC results in reducing powerfully promoter strength and transcription of the cps genes associated with CPS formation.However,hitherto,the transcription factors of cps promoter remain uncharacterized,and the transcription regulation of CPS production is still poorly understood.To better understand the transcription and regulation mechanism of CPS production,in this context,a 218 bp 5′-biotin labeled DNA probe（upstream of the cps2 A,contains 14 bp nucleotides downstream of cps2 A initiation codon）was utilized to isolate and identify the cps transcription factor by DNA affinity chromatography-pulldown,MALDI-TOF mass spectrometry（MS）and electrophoretic mobility shift assay（EMSA）.In this way,seven proteins were initially chosen to be further studied as candidate transcriptional regulator of cps locus.In present study,we have performed in-depth molecular analyses of the response regulator ComE.Methods Isolation and identification of transcriptional regulators of cps locus in S.pneumoniae were carried out by DNA affinity chromatography-pulldown,MALDI-TOF mass spectrometry（MS）and electrophoretic mobility shift assay（EMSA）.Deletion of transcriptional regulators of /in D39 was carried out by LFH-PCR,and real time PCR was used to detect the transcription level of cps2 A in D39 mutants.To detect the effect of transcriptional regulators on the transcription of cps promoter,β-galactosidase reporter for cps promoter region were prepared in the p EVP3 plasmid,andβ-galactosidase activity in D39 mutants were detected to assessed the transcription activity of cps promoter.D39Δcom E mutant was constructed by LFH-PCR.To compare the difference of capsule between D39-WT and D39Δcom E mutant,Neufeld test,immunofluorescence microscopy and transmission electron microscopy（TEM）were carried out.To exhibit the activity of ComE protein in vitro,a phosphorylated mimetic mutant,ComED58 E,was constructed by site-directed mutagenesis of com E.The p JWV25 plasmid was used to constructed the ComE complemented and over-expressed strains.The transcription level of cps2 A in D39 and its mutants was assessed by real time PCR,and the expression of CPS was detected by Western-blot and ELISA.To further verify the effect of com E on the transcription of cps locus promoter,the induction expression of ComE was carried out using exogenous CSP1 as inducer.The role of ComE in nasopharyngeal colonization and the virulence of S.pneumoniae D39 was investigated in mice pneumonia and bacteremia models.The identification of cps locus promoter binding sites for ComE was carried out by EMSA of truncation probe and the effect of ComE-mediated CPS regulation on transformation frequency was demonstrated by constructing un-marked deletion of binding site of ComE strain D39ΔJD3.Results Eight possible proteins had been isolated and identified as candidate transcriptional regulator by DNA affinity chromatography-pulldown,MALDI-TOF mass spectrometry（MS）using cps promoter DNA as bait: ComE（with the coverage rate of 78.4%）,Ccp A（72.02%）,hypothetical protein SPD₀410（67.61%）,DNA-binding protein HU（65.93%）,Gnt R（50.2%）,Plc R（48.43%）,Mar R（47.92%）and Tr-act（34.89%）.Seven proteins except of Plc R were demonstrated the specifically binding affinity for cps promoter by EMSA in vitro.We further demonstrated that ComE,Gnt R and Tr-act were transcription regulator of cps locus by detecting the transcriptional level of cps2 A and β-galactosidase activity in its D39 mutants.In present study,we have performed in-depth molecular analyses of the response regulator ComE.The EMSA showed that phosphorylated mimetic ComED58 E could specifically bind to the cps locus promoter in vitro,and ComE could regulate negatively cps locus transcription and CPS production of D39 in vivo.As compared with D39-WT strain,the level of transcription of cps2 A in D39△com E mutant increased approximately by 44.4%（***,p<0.0001）and the CPS production of D39△com E mutant increased significantly approximately by 80%（**,p=0.0018）,but the difference disappeared when the deleted com E gene was ectopic complemented in D39 △ com E::com ED58E（ns,p=0.1341）.The D39Δcom E mutant exhibited stronger immunofluorescence and more thicker capsule than that of D39-WT strain,and the mean capsules thickness for D39Δcom E mutant was 0.6271μm（for Neufeld test）and 37.57nm（for TEM）,and for D39-WT strain 0.4678μm（for Neufeld test）and 21.43nm（for TEM）,respectively.More importantly,we found that CSP-Com D/E competence system involved in regulating negatively the CPS production in competence D39.The CSP1 induction results showed that the expression of ComE in D39-p AE03-cps-promoter started to increase after CSP1 induction 5min,and reached peak value which increased 3.38-fold higher after CSP1 induction about 10 min,and then decreased promptly after CSP1 induction 15 min to 20 min.Corresponding to the expression of ComE,the expression of reporter protein GFP in D39-p AE03-cps-promoter decreased sharply by 83.3% after CSP1 induction about 10 min to 15 min,and then restored after about 25 min.However,the expression of GFP in D39Δcom E-p AE03-cps-promoter was almost unaffected by CSP1 in the development of competence from 0min to 25 min.Extracellular glucose concentrations could impact the expression of ComE and regulate positively the CPS production.In addition,D39Δcom E mutant exhibited attenuated nasopharyngeal colonization and enhanced virulence in both Balb/c mice pneumonia and bacteremia models.Hence,it can be concluded that ComE regulates negatively the transcription of the cps locus gene and decrease the CPS production,which leads to attenuate virulence.The binding site for ComE in cps locus was determined to be 33 bp sequence by EMSA of truncation probe: TGTCATGTTCTTATTTCATTTTACTATATTTTT.As compared with D39-WT strain,the production of CPS in D39ΔJD3 mutant increased significantly by 45.8%,but the transformation frequency in D39ΔJD3 mutant was only 29.6% of that in D39-WT.It implied that CPS regulation through ComE-mediated could impact on the bacterial transformation in the development of competence.Conclusion Our results show that ComE^P,as the transcriptional regulator of cps locus in S.pneumoniae D39,could regulate negatively the transcription of cps locus and impact the CPS production,which leads to attenuate the virulence of pneumococcal.Extracellular glucose concentration may affect the expression of ComE and regulate positively CPS production.CSP-Com D/E competence system involves in capsular regulation in the progress of natural genetic transformation of S.pneumoniae,which is benefit for the progress of bacterial transformation.