title: Live imaging of the genetically intractable obligate intracellular bacteria Orientia tsutsugamushi using a panel of fluorescent dyes.
abstract: Our understanding of the molecular mechanisms of bacterial infection and pathogenesis are disproportionally derived from a small number of well-characterised species and strains. One reason for this is the enormous time and resources required to develop a new organism into experimental system that can be interrogated at the molecular level, in particular with regards to the development of genetic tools. Live cell imaging by fluorescence microscopy is a powerful technique to study biological processes such as bacterial motility, host cell invasion, and bacterial growth and division. In the absence of genetic tools that enable exogenous expression of fluorescent proteins, fluorescent chemical probes can be used to label and track living cells. A large number of fluorescent chemical probes are commercially available, but these have overwhelmingly been applied to the study of eukaryotic cell systems. Here, we present a methodical analysis of four different classes of probes, which can be used to delineate the cytoplasm, nucleic acids, cell membrane or peptidoglycan of living bacterial cells. We have tested these in the context of the important but neglected human pathogen Orientia tsutsugamushi but expect that the methodology would be broadly applicable to other bacterial species.
authors: Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors: Sharanjeet
authors: S
authors: Atwal
authors: Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
authors: Suparat
authors: S
authors: Giengkam
authors: Indiana University, Bloomington, IN, USA.
authors: Michael
authors: M
authors: VanNieuwenhze
authors: Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom. Electronic address: jeanne.salje@ndm.ox.ac.uk.
authors: Jeanne
authors: J
authors: Salje
mesh_terms: D000818:Animals; D001419:Bacteria; D001431:Bacteriological Techniques / Q000379:methods*; D018929:Cell Culture Techniques; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane; D002470:Cell Survival; D003593:Cytoplasm / Q000382:microbiology*; D005456:Fluorescent Dyes / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D009696:Nucleic Acids; D012285:Orientia tsutsugamushi / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism / Q000472:pathogenicity; D010457:Peptidoglycan; D012612:Scrub Typhus / Q000382:microbiology; D013194:Staining and Labeling / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D009696:Nucleic Acids; D010457:Peptidoglycan
keywords: Fluorescence microscopy; Fluorescent dyes; Intracellular bacteria; Neglected bacterial pathogens; Rickettsia