title: Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos.
abstract: Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions. The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model. In this respect, the present manuscript describes the use of zebrafish as an in vivo model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.
authors.affiliation: Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS, UMR 535, Université Montpellier; Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé, CNRS, FRE 3689, Université Montpellier.
authors.forename: Audrey
authors.initials: A
authors.lastname: Bernut
authors.affiliation: Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS, UMR 535, Université Montpellier; Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé, CNRS, FRE 3689, Université Montpellier.
authors.forename: Christian
authors.initials: C
authors.lastname: Dupont
authors.affiliation: Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS, UMR 535, Université Montpellier.
authors.forename: Alain
authors.initials: A
authors.lastname: Sahuquet
authors.affiliation: Unité de Formation et de Recherche des Sciences de la Santé, EA3647-EPIM, Université Versailles St Quentin.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Herrmann
authors.affiliation: Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS, UMR 535, Université Montpellier; lutfalla@univ-montp2.fr.
authors.forename: Georges
authors.initials: G
authors.lastname: Lutfalla
authors.affiliation: Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS, UMR 535, Université Montpellier; Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé, CNRS, FRE 3689, Université Montpellier; laurent.kremer@cpbs.cnrs.fr.
authors.forename: Laurent
authors.initials: L
authors.lastname: Kremer
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D004625:Embryo, Nonmammalian; D005260:Female; D054884:Host-Pathogen Interactions; D008264:Macrophages / Q000276:immunology; D008297:Male; D009161:Mycobacterium / Q000276:immunology* / Q000472:pathogenicity*; D061848:Optical Imaging / Q000379:methods; D014774:Virulence; D015027:Zebrafish / Q000276:immunology* / Q000382:microbiology*