title: Alternative fluorescent labeling strategies for characterizing gram-positive pathogenic bacteria: Flow cytometry supported counting, sorting, and proteome analysis of Staphylococcus aureus retrieved from infected host cells.
abstract: Staphylococcus aureus is a Gram-positive opportunistic pathogen that is able to cause a broad range of infectious diseases in humans. Furthermore, S. aureus is able to survive inside nonprofessional phagocytic host cell which serve as a niche for the pathogen to hide from the immune system and antibiotics therapies. Modern OMICs technologies provide valuable tools to investigate host-pathogen interactions upon internalization. However, these experiments are often hampered by limited capabilities to retrieve bacteria from such an experimental setting. Thus, the aim of this study was to develop a labeling strategy allowing fast detection and quantitation of S. aureus in cell lysates or infected cell lines by flow cytometry for subsequent proteome analyses. Therefore, S. aureus cells were labeled with the DNA stain SYTO®9, or Vancomycin BODIPY®FL (VMB), a glycopeptide antibiotic binding to most Gram-positive bacteria which was conjugated to a fluorescent dye. Staining of S. aureus HG001 with SYTO 9 allowed counting of bacteria from pure cultures but not in cell lysates from infection experiments. In contrast, with VMB it was feasible to stain bacteria from pure cultures as well as from samples of infection experiments. VMB can also be applied for histocytochemistry analysis of formaldehyde fixed cell layers grown on coverslips. Proteome analyses of S. aureus labeled with VMB revealed that the labeling procedure provoked only minor changes on proteome level and allowed cell sorting and analysis of S. aureus from infection settings with sensitivity similar to continuous gfp expression. Furthermore, VMB labeling allowed precise counting of internalized bacteria and can be employed for downstream analyses, e.g., proteomics, of strains not easily amendable to genetic manipulation such as clinical isolates. © 2016 International Society for Advancement of Cytometry.
authors.affiliation: Department Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany.|ZIK-FunGene University Medicine Greifswald, Department Functional Genomics, Greifswald, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Hildebrandt
authors.affiliation: Department Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany.|ZIK-FunGene University Medicine Greifswald, Department Functional Genomics, Greifswald, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Surmann
authors.affiliation: Department Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Manuela Gesell
authors.initials: MG
authors.lastname: Salazar
authors.affiliation: Department Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany.|ZIK-FunGene University Medicine Greifswald, Department Functional Genomics, Greifswald, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Normann
authors.affiliation: Department Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany.|ZIK-FunGene University Medicine Greifswald, Department Functional Genomics, Greifswald, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Völker
authors.affiliation: Department Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany. Frank.Schmidt@uni-greifswald.de.|ZIK-FunGene University Medicine Greifswald, Department Functional Genomics, Greifswald, Germany. Frank.Schmidt@uni-greifswald.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Schmidt
mesh_terms: D000328:Adult; D000369:Aged, 80 and over; D001426:Bacterial Proteins / Q000378:metabolism; D002460:Cell Line; D004269:DNA, Bacterial / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism / Q000382:microbiology; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism*; D054884:Host-Pathogen Interactions / Q000502:physiology*; D006801:Humans; D008297:Male; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*; D013203:Staphylococcal Infections / Q000378:metabolism / Q000382:microbiology*; D013211:Staphylococcus aureus / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D020543:Proteome
keywords: S. aureus; cell counting and sorting; flow cytometry; fluorescence labeling of S. aureus; fluorescence microscopy; fluorescent vancomycin conjugate; gram-positive bacteria; internalization; proteomics