Das M. tuberculosis-Phagosom: Dynamik der erregerinduzierten Modulation intrazellulärer Grenzflächen
Final Report Abstract
Bacterial infections are still a major cause of morbidity and mortality worldwide. Pathogenic microorganisms have developed various strategies to avoid detection and elimination by the immune system. For example, Listeria and Shigella disrupt the phagosomal membrane to escape into the cytoplasm, others like Leishmania and Coxiella survive and replicate within the lysosomal milieu. Many, like Legionella, Brucella, Ehrlichia, and Mycobacterium spp., effectively manipulate the normal progression of their phagosomal compartment and prevent it from fusing with or maturing into an active lysosomal organelle. Fundamental insights into the molecular composition and the dynamics of phagosomes have been obtained by the detailed analysis of latex bead phagosomes (LBP). However, to identify pathogen-specific virulence and persistence mechanisms it is indispensable to define the molecular composition of phagosomes containing viable infectious agents. In this project we have developed a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium (M.) avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system. Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay, and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen-containing compartments, in order to identify microbial and host cell targets for novel anti-infective strategies.
Publications
- Isolierung und Charakterisierung Mykobakterien-haltiger Phagosomen aus Primärzellen: Etablierung und Evaluierung eines neuen immunomagnetischen Verfahrens. Dissertation, Universität zu Lübeck
Steinhäuser C.
- Immunomagnetic isolation of tumor necrosis factor receptosomes.
Methods in Enzymology, Vol. 442. 2008, pp 101–123.
Tchikov V, Schütze S
(See online at https://dx.doi.org/10.1016/S0076-6879(08)01405-5) - Immunomagnetic Isolation of Subcellular Compartments.
Methods in Microbiology,Vol. 37. 2010, pp.21–33
Tchikov V, Fritsch J, Kabelitz D, Schütze S
(See online at https://dx.doi.org/10.1016/S0580-9517(10)37002-4) - Lipid-labeling facilitates a novel magnetic isolation procedure to characterize pathogen-containing phagosomes. Traffic, Vol. 14. 2013, Issue 3, pp. 321–336,
Steinhäuser C, Heigl U, Tchikov V, Schwarz J, Gutsmann T, Seeger K, Fritsch J, Schroeder J, Wiesmüller K-H, Rosenkrands I, Walther P, Pott J, Krause E, Ehlers S, Schneider-Brachert W, Schütze S, Reiling N
(See online at https://doi.org/10.1111/tra.12031)
