Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 Reveals a Tight Link between Tyrosine Phosphorylation and Virulence

• DOI: 10.1074/mcp.m900276-mcp200
• Publication Date: 2009-08-21T00:35:23Z
• Authors: Miao-Hsia Lin; Tung-Li Hsu; Shu-Yu Lin; Yi-Jiun Pan; Jia-Tsrong Jan; Jin-Town Wang; Kay-Hooi Khoo; Shih-Hsiung Wu
• Keywords: Phosphoproteomics

Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. surface was determined as key to virulence. Although regulation biosynthesis largely unclear, it found protein-tyrosine kinases and phosphatases are involved. Therefore, identification characterization such kinases, phosphatases, their substrates would advance our knowledge underlying mechanism in capsule formation could contribute development new therapeutic strategies. Here, we analyzed phosphoproteome NTUH-K2044 with a shotgun approach identified 117 unique phosphopeptides along 93 vivo phosphorylated sites corresponding 81 proteins. Interestingly, three tyrosine proteins, namely kinase (Wzc), phosphomannomutase (ManB), undecaprenyl-phosphate glycosyltransferase (WcaJ), were be distributed cps locus thus speculated involved converging signal transduction biosynthesis. Consequently, decided focus lesser studied ManB WcaJ for mutation analysis. polysaccharides mutant (WcaJY5F) dramatically reduced quantitatively, LD50 increased by 200-fold mouse peritonitis model compared wild-type strain. However, (ManBY26F) showed no difference quantity, merely 6-fold mice test. Our study provided clear trend phosphorylation can regulate result pathogenicity NTUH-K2044. Protein one most biologically relevant ubiquitous post-translational modifications both eukaryotic prokaryotic organisms. It best known protein reversible enzyme-catalyzed process controlled various phosphatases. aberrant functions irregular ultimately lead serious states malignant transformation, immune disorders, pathogenic infections mammals (1Cozzone A.J. 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Diagn Lab. 393-399Crossref study, wanted prove mediated through subset An MS-based K2044 identify associated We validated those site-directed mutagenesis, quantification, serum killing, lethality assay.RESULTSPhosphoproteomics extract based classical LC-MS/MS 17Macek 18Soufi 19Aivaliotis Due abundance phosphoproteome, anti-phosphotyrosine (Tyr(P)) antibody enrich Tyr(P) population. First all, grown mid-log phase 37 °C before harvesting. Both total proteome extraction anti-Tyr(P) enriched digested separately. non-Tyr(P) sample subjected SCX chromatography fractionation subsets peptide level TiO2. captured eluted peptides nanoscale LC coupled LTQ-Orbitrap individually preparations increase coverage confidence positive hits (52Reed L.J. Muench simple method estimating fifty percent endpoints.Am. Hyg. 1938; 27: 493-497Google resulting MS/MS data searched against composite target-decoy database constructed NCBI (accession numbers AP006725 AP006726), all confident compiled into reported Table I supplemental Data 1, manually annotated spectra presented 2.Table IThe phosphoproteins, phosphopeptides, studyGene namePhosphopeptide sequenceGene sequenceacnBIpDEVFIGSCMTNIGHFRgldAp[SLAD]AGLAAEIAPFGGECSHNEINRahpCAAQpYVASHPGEVCPAKglpKAVTpSTHGLLTTIACGPRargDQLCpDQHNALLIFDEVQTGVGRAVpTSTHGLLTTIACGPRatpAIpHGLAECMQGEMISLPGNRAVp[TSTH]GLLTTIACGPRatpDAAPp[SY]EELSSSQELLETGIKgltAp[DSH]PMAVMCGITGALAAFYHDSLDVNNPRcsrAIQAEKSQQSSpYDSHPMAVMCGIpTGALAAFYHDSLDVNNPRIQAEKSQQSp[SY]gpmApHGESQWNNENRdeoBFGpDVGADTLGHIAEACAKgroELQIVSNAGEEPpSVVANNVKdeoCAAIApYGADEVDVVFPYRhflBREVRPPAGWEEPGp[SSNNS]DNNGTPRefpATpYYSNDFRhisSApHFAGLCALLDDAGIRfabFGPpSISIATACTSGVHNIGQAARhutHpHLLTDDSAISQSHHNCSKfbaAHNLPHNSLNFVFHGGp[SGSS]AQEIKinfBNWpSETSDSPEDSSDYHVTTSQHARfeaBQpHGLYIDGAPCAAQSENRkblGpSHEYCDVMGRfimAGEVVNAACAVpDAGSIDQTVQLGQVRkdsAApSNNSPVIFDVTHALQCRLATAGSTSpSAVGFNIQLDDCDTTVATKmanBGELGEELNEDIApYRLApTAGSTSSAVGFNIQLDDCDTTVATKmlcDALpYNGSLLIRYpYATGAATAGIANADATFKmsrAEVCpSGQTGHAEAVRLATAGSp[TS]SAVGFNIQLDDCDTTVATKnagAKPpDAALVDFLCDNADVITKLAp[TAGS]TSSAVGFNIQLDDCDTTVATKnarGFpSEVCVGHLGKfimDDVpTFQADAQGHGLSPCLTRnuoFpTFCAHAPGAVEPLQSAIKfrdAEPIPVRPp[TAH]YTMGGIETDQQCETRompAGMGEpSNPVTGNTCDNVKftsApHGCALGSIVGKDENVEVPSVGGRPPRompCNGpSVSGEGATNNGRHISCQNEIGMVPIpSEEEVTQDDVENVVHTAKpckAGIApSMHCSANVGEKpHISCQNEIGMVPISEEEVTQDDVENVVHTAKpepDpTPNIQIIHAGLECGLFKgabTALCpDEHGIMLIADEVQSGAGRpepNpHDGSAATCDDFVQAMEDASNVDLSHFRgalUGLGHAVLCAHPVVGDEPVAVILPDVILDEpYESDLSRHDGSAATCDDFVQAMEDApSNVDLSHFRgcvTAQQpTPLYEQHTLCGARpgkpYAALCDVFVMDAFGTAHRFApDVACAGPLLAAELDALGKpgmKGGPLADGIVITPpSHNPPEDGGIKwzcYGEYAYpYEpYEpYKSKEKGGPLADGIVIpTPSHNPPEDGGIKYGEYAYYEYEpYKphoCIRPFAFYGVSTCNTTEQpDKpYGEYAYYEpYEYKppsApTCHAAIIARYGEYApYYEYEpYKGGRpTCHAAIIARpYGEYAYYEYEpYKLpYTSLGDAAVGRYGEYApYYEpYEYKSKEpurLpDNPACADQEHQAKYGEYAYpYEYEpYKSKEpyrEpDYGCQVISIITLKp[YGEY]AYYEpYEpYKSKErncSGEAHDQEFp[TIH]CQVSGLSEPVVGTGSSRYGEYApYpYEpYEpYKSKErplBDGAYVpTLRpYGEYApYYEpYEpYKSKErplJAAAFEGELIPApSQIDRYGEYApYYEpYEp[YKS]KErplNFpDGNACVILNNNSEQPIGTRybbNpSQHCEQLTPVLERrplUMpYAVFQSGGKyceFFPNp[HLIIGSD]QVCVLDGEITGKPHTEENARrpmEpSTVGHDLNLDVCGKygaGp[DH]LNGNGVEIIDISPMGCRGIpHPNYDEITATCSCGNVMKyhaHFGPDPKPFSpYrpoBp[STGSY]SLVTQQPLGGKU/NKPIPLGVpSESSVPAPKrpoCNMpDLEQECEQLREELNETNSETKU/NFNALpYEAIHVQGAKrpsApSNPWQQFAETHNKSSNNpYLGAARrpsBpDAALSCDQFFVNHRU/NpHASIVPHCAETFAHLVRrpsIAENQpYYGTGRU/NALpTICTVSDHIRrpsLpSNVPALEACPQKRU/NISpSTAVRrpsQLpHVHDENNECGIGDKU/NAAHDFpYterZSpSCGSVVHSGDNLTGEGDGDDEIIHVNLLKU/NLLNNpSELTHAPCAPGTLETLARtufBEGGRpTVGAGVVAKGpYLIPKpHYAHVDCPGHADYVKpSPVNDHPLCLFNPQEDAQILQKuxaCpDQPIFDYHCHLPPQQVAENYRU/NVCVpSPGIGFGDYGDTHVRwcaJATLpYHRU/NEpTGACNVQVIGKwzcQQNSpSVDLTMEAKU/NIApYNVEAARINNLPEpTQQEVLRSGIGPVTAADIp[THD]GDVEIVKPQHVICHLTDENAAISMRNNILMISGPpSPEIGKp[SGIGPVTAADIT]HDGDVEIVKPQHVICHLTDENAAISMRTFVp[ST]NLAGVVAQAGQKU/NVRHDVFEpSYPEELHGK Open table tab Mid-log Phase pneumoniaeA combination false discovery estimated less than 1%. those, distinct (Table I): 29 Ser (31.2%), 14 Thr (15.1%), 24 Tyr (25.8%). indicates proportion significantly higher, phosphoproteomic composition 17 13 selectively subset. Consistent previous His Asp Mascot searches, 12 (12.9%) His11 phosphoglycerate mutase (gpmA), homologue (53Puttick Baker E.N. Delbaere L.T. Histidine systems.Biochim. 100-105Crossref (63) observed. Manual inspection (supplemental 2) revealed y ions consistent overall assigned mostly fell short unambiguously identifying inferred Asp/His sites. unusual forms same residual increments cannot ruled should considered caution.Phosphoproteins derived I) according Blast2GO (shown Fig. 1). 75 (excluding six hypothetical annotated) 3512 5107 genes, selected ontology annotations, reference. Those housekeeping pathways significant over-representation (Fig. 1A) translation protein/carbohydrate metabolism. enzymes fructose-1,6-bisphosphate aldolase (fbaA), 2-deoxyribose-5-phosphate (deoC), (pgk), phosphoglucomutase (pgm), phosphoenolpyruvate synthase (ppsA) glycolysis pathway phosphorylated, accord Mi