title: Surface-affinity profiling to identify host-pathogen interactions.
abstract: Proteolytic treatment of intact bacterial cells has proven to be a convenient approach for the identification of surface-exposed proteins. This class of proteins directly interacts with the outside world, for instance, during adherence to human epithelial cells. Here, we aimed to identify host receptor proteins by introducing a preincubation step in which bacterial cells were first allowed to capture human proteins from epithelial cell lysates. Using Streptococcus gallolyticus as a model bacterium, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteolytically released peptides yielded the identification of a selective number of human epithelial proteins that were retained by the bacterial surface. Of these potential receptors for bacterial interference, (cyto)keratin-8 (CK8) was verified as the most significant hit, and its surface localization was investigated by subcellular fractionation and confocal microscopy. Interestingly, bacterial enolase could be assigned as an interaction partner of CK8 by MS/MS analysis of cross-linked protein complexes and complementary immunoblotting experiments. As surface-exposed enolase has a proposed role in epithelial adherence of several Gram-positive pathogens, its interaction with CK8 seems to point toward a more general virulence mechanism. In conclusion, our study shows that surface-affinity profiling is a valuable tool to identify novel adhesin-receptor pairs, which advocates its application in other hybrid biological systems.
authors.affiliation: Department of Laboratory Medicine, Nijmegen Institute for Infection, Inflammation and Immunity and Radboud University Centrefor Oncology of the Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
authors.forename: Annemarie
authors.initials: A
authors.lastname: Boleij
authors.forename: Coby M
authors.initials: CM
authors.lastname: Laarakkers
authors.forename: Jolein
authors.initials: J
authors.lastname: Gloerich
authors.forename: Dorine W
authors.initials: DW
authors.lastname: Swinkels
authors.forename: Harold
authors.initials: H
authors.lastname: Tjalsma
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism; D001422:Bacterial Adhesion / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D004847:Epithelial Cells / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D054884:Host-Pathogen Interactions; D006801:Humans; D053533:Keratin-8 / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D013291:Streptococcus / Q000166:cytology / Q000502:physiology*; D053719:Tandem Mass Spectrometry
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D053533:Keratin-8