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 V R 9, or Vancomycin BODIPY V R 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. V C 2016 International Society for Advancement of Cytometry

Construction and validation of EGFP-expressingStaphylococcus aureusclinical strains for adhesion and internalization assays on epithelial cells

“…By contrast, fluorescent labelling methods using dye (e.g. Vancomycin Bodipy FL) or antibodies that interact with the bacterial cell wall could modify interactions with cell host receptors [15]. In this study, we show that EGFP expressed inside the bacteria does not modify adhesion and internalization capacities of S. aureus .…”

“…In addition, the EGFP expression enables live imaging of S. aureus clinical isolates because the EGFP expression driven by pBSU101 does not alter the bacterial viability [12] whereas vancomycin-based labelling has been found to exhibit a bacteriostatic activity that limits its use over the time [15]. By contrast to staining methods using peptidoglycan-binding or DNA-binding dyes, plasmid-encoded fluorescent reporters are highly specific since they are expressed only in transformed strains.…”

“…The ability of some strains to form clump could also have an impact for automated quantification system. Nevertheless, the use of EGFP-expressing S. aureus strains may be easier, more specific and more accurate than live labelling of bacteria with fluorescent dyes or cell-permeable nuclear probes [15,19]. Although only EGFP was tested in this study, the pBSU101 plasmid gives opportunity to replace the egfp gene by another gene coding for other fluorescent proteins such as yellow fluorescent protein or mCherry that have been found suitable to label different S. aureus laboratory strains (RN 4220, SH1000 and SH1001) [20,21].…”

Improving Proteome Coverage for Small Sample Amounts: An Advanced Method for Proteomics Approaches with Low Bacterial Cell Numbers

“…In order to allow flow cytometry‐based cell counting, a green fluorescent protein (GFP) expressing L. pneumophila Corby strain was used. For the analysis of S. aureus NCTC8325‐4 and S. suis the bacterial cultures were stained using SYTO9 (Thermo Fisher Scientific, MA, USA) as described before . After determining the bacterial cell numbers in each culture using a Guava easyCyte flow cytometer (Merck‐Millipore, MA, USA), 2 × 10 6 or 5 × 10 6 bacteria, respectively, were collected on 0.22 µm filters (96‐Well Durapore membrane filtration plate Multiscreen HTS, Merck, Germany) using a vacuum pump (≈200 mbar, KNF Laboport, NJ, USA).…”

Deconvolution models for a better understanding of natural microbial communities enumerated by flow‐cytometry

“…THE application of flow cytometry is well-established for the characterization of aquatic microbial communities in natural systems (1)(2)(3). These microbial assemblages are of fundamental importance for the so called "Earth's Critical Zone" (the planet surface including rivers, lakes, and oceans) as they are the main drivers of all biochemical cycles in waters and are responsible for more than half of the global production of oxygen.…”