title: Quantitative monitoring of the Chlamydia trachomatis developmental cycle using GFP-expressing bacteria, microscopy and flow cytometry.
abstract: Chlamydiae are obligate intracellular bacteria. These pathogens develop inside host cells through a biphasic cycle alternating between two morphologically distinct forms, the infectious elementary body and the replicative reticulate body. Recently, C. trachomatis strains stably expressing fluorescent proteins were obtained. The fluorochromes are expressed during the intracellular growth of the microbe, allowing bacterial visualization by fluorescence microscopy. Whether they are also present in the infectious form, the elementary body, to a detectable level has not been studied. Here, we show that a C. trachomatis strain transformed with a plasmid expressing the green fluorescent protein (GFP) accumulates sufficient quantities of the probe in elementary bodies for detection by microscopy and flow cytometry. Adhesion of single bacteria was detected. The precise kinetics of bacterial entry were determined by microscopy using automated procedures. We show that during the intracellular replication phase, GFP is a convenient read-out for bacterial growth with several advantages over current methods. In particular, infection rates within a non-homogenous cell population are easily quantified. Finally, in spite of their small size, individual elementary bodies are detected by flow cytometers, allowing for direct enumeration of a bacterial preparation. In conclusion, GFP-expressing chlamydiae are suitable to monitor, in a quantitative manner, progression throughout the developmental cycle. This will facilitate the identification of the developmental steps targeted by anti-chlamydial drugs or host factors.
authors.affiliation: Institut Pasteur, Unité de Biologie des Interactions Cellulaires, Paris, France; Centre National de la Recherche Scientifique, URA 2582, Paris, France; Université Pierre et Marie Curie, Cellule Pasteur UPMC, Paris, France.
authors.forename: François
authors.initials: F
authors.lastname: Vromman
authors.affiliation: Institut Pasteur, Unité de Biologie des Interactions Cellulaires, Paris, France; Centre National de la Recherche Scientifique, URA 2582, Paris, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Laverrière
authors.affiliation: Institut Pasteur, Unité de Biologie des Interactions Cellulaires, Paris, France; Centre National de la Recherche Scientifique, URA 2582, Paris, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Perrinet
authors.affiliation: Centre National de la Recherche Scientifique, URA 2582, Paris, France; Institut Pasteur, Unité d'Analyse d'images biologiques, Paris, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Dufour
authors.affiliation: Institut Pasteur, Unité de Biologie des Interactions Cellulaires, Paris, France; Centre National de la Recherche Scientifique, URA 2582, Paris, France.
authors.forename: Agathe
authors.initials: A
authors.lastname: Subtil
mesh_terms: D000048:Acanthamoeba / Q000382:microbiology*; D002690:Chlamydia Infections / Q000175:diagnosis* / Q000382:microbiology; D002692:Chlamydia trachomatis / Q000254:growth & development* / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: D049452:Green Fluorescent Proteins