Expression and Regulation of the Escherichia coli O157H7 Effector Proteins NleH1 and NleH2 英文参考文献.docVIP

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Expression and Regulation of the Escherichia coli O157H7 Effector Proteins NleH1 and NleH2 英文参考文献.doc

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Expression and Regulation of the Escherichia coli O157H7 Effector Proteins NleH1 and NleH2 英文参考文献

ExpressionandRegulationoftheEscherichiacoli O157:H7EffectorProteinsNleH1andNleH2 AshleighHolmes,CeciliaS.LindestamArlehamn,DaiWang,TimJ.Mitchell,TomJ.Evans,AndrewJ. Roe* InstituteofInfection,ImmunityandInflammation,CollegeofMedical,VeterinaryandLifeSciences,UniversityofGlasgow,Glasgow,UnitedKingdom Abstract Background: E. coli O157 carries two genes encoding the effector proteins NleH1 and NleH2 which are 87% identical. Despitethesimilaritybetweentheproteins,thepromoterregionsupstreamofthegenesencodingtheeffectorsaremore divergentsuggestingthattheactualexpressionofthegenesmaybedifferentiallyregulated.Thiswastestedbycreating reporterfusionsandexaminingtheirexpressionindifferentgeneticbackgrounds,mediaandoncontactwithhostcells.The functionoftheproteinswasalsotestedfollowingtransfectionintohostcells. PrincipalFindings: ExpressionofbothNleH1andNleH2wasenhancedwhenculturedunderconditions thatstimulated expression of the Type Three Secretion System (T3SS) and was influenced by the regulators Ler and GrlA. Maximal expressionofNleH1required531bpoftheupstreamuntranslatedregionbutNleH2requiredonly113bp.Interestingly, contact with host cells strongly repressed expression of both NleH1 and NleH2. Following transfection, both proteins produced only minor effects on NF-kB activation when assessed using a NF-kB luciferase reporter assay, a result that is consistentwiththerecentreportdemonstratingthedependenceonRPS3forNleH1modulationofNF-kB. Significance: This study demonstrates the importance of considering gene regulation when studying bacterial effector proteins.Despitetheirsequencesimilarity,NleH1andNleH2areexpresseddifferentiallyandmay,therefore,betranslocated atdistincttimesduringaninfection. Citation: Holmes A, Lindestam Arlehamn CS, Wang D, Mitchell TJ, Evans TJ, et al. (2012) Expression and Regulation of the Escherichia coli O157:H7 Effector ProteinsNleH1andNleH2.PLoSONE7(3):e33408.doi:10.1371/journal.pone.0033408 Editor:EdwardE.Schmidt,MontanaStateUniversity,Unite