Clade specific virulence patterns of M. tuberculosis complex strains in human macrophages: Molecular mechanisms underlying phagosomal escape and induction of cell death.

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The outcome of tuberculosis is determined by both the virulence of the infecting M. tuberculosis complex (MTC) strain and the killing capacity of the host macrophage. In the current study we will examine the impact of pathogen variability and host cell signaling variability on the molecular composition of the MTC strain harboring compartment, the phagosome. The study comprises (i) the comparative analysis of the protein and lipid composition of MTC clade I and clade II containing phagosomes from human primary macrophages, (ii) the identification of critical host cell factors that govern control of M. tuberculosis growth in macrophages and (iii) the differential analysis of phagosomes isolated from human and murine macrophages to define “core proteome components” which are present and engaged in both species and “individual proteome components” which are present or exclusively recruited to the phagosome in one of the two species. A new lipid-based procedure to label bacterial surfaces and a rapid immunomagnetic technique will be used to isolate intact bacteria-containing compartments from mouse and human primary cells in order to further characterize them by electron and atomic force microscopy, flow cytometry, Western blot, and mass spectrometry. The newly developed method facilitates comparative studies across the kingdom of intracellular pathogens and will be made available to other members of the priority program.