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.
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.
['Hildebrandt|Petra|P|', 'Surmann|Kristin|K|', 'Salazar|Manuela Gesell|MG|', 'Normann|Nicole|N|', 'Völker|Uwe|U|', 'Schmidt|Frank|F|']
D000328:Adult D000369:Aged, 80 and over D001426:Bacterial Proteins D002460:Cell Line D004269:DNA, Bacterial D004847:Epithelial Cells D005260:Female D005434:Flow Cytometry D005456:Fluorescent Dyes D054884:Host-Pathogen Interactions D006801:Humans D008297:Male D020543:Proteome D040901:Proteomics D013194:Staining and Labeling D013203:Staphylococcal Infections D013211:Staphylococcus aureus
flow cytometry proteomics S. aureus internalization gram-positive bacteria cell counting and sorting fluorescence labeling of S. aureus fluorescence microscopy fluorescent vancomycin conjugate