RNAi Screen Reveals Host Cell Kinases Specifically Involved in Listeria monocytogenes Spread from Cell to Cell 英文参考文献.docVIP

RNAiScreenRevealsHostCellKinasesSpecifically InvolvedinListeriamonocytogenesSpreadfromCellto Cell RyanChong,RaynalSquires,RachelSwiss,Herve′ Agaisse* SectionofMicrobialPathogenesis,BoyerCenterforMolecularMedicine,YaleUniversitySchoolofMedicine,NewHaven,Connecticut,UnitedStatesofAmerica Abstract Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereasthemechanismssupportingcytosolicactin-basedmotilityarefairlywellunderstood,itisunclearwhetherspecific hostfactorsmayberequiredforsupportingtheformationandresolutionofmembraneprotrusions.Toaddressthisgapin knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library coveringthehumankinomeandidentified7candidatekinaseswhosedepletionledtoseverespreadingdefectsincells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlatedwiththeformationofprotrusionthatdidnotresolveintodouble-membranevacuoles.Moreover,wedeveloped sendingandreceivingcell-specificRNAiproceduresandshowedthatCSNK1Awasrequiredinthesendingcells,butwas dispensableinthereceivingcells,forprotrusionresolution.Finally,weshowedthattheobserveddefectswerespecificto Listeriamonocytogenes,asRickettsiacono