• Identification of an immune-regulated phagosomal Rab cascade in macrophages.

      Pei, Gang; Repnik, Urska; Griffiths, Gareth; Gutierrez, Maximiliano Gabriel; Helmholtz Centre for infection research, Inhoffenstr. 7 , D-38124 Braunschweig, Germany. (2014-05-01)
      Interferon-γ (IFN-γ) has been shown to regulate phagosome trafficking and function in macrophages, but the molecular mechanisms involved are poorly understood. Here, we identify Rab20 as part of the machinery by which IFN-γ controls phagosome maturation. We found that IFN-γ stimulates the association of Rab20 with early phagosomes in macrophages. By using imaging of single phagosomes in live cells, we found that Rab20 induces an early delay in phagosome maturation and extends the time for which Rab5a and phosphatidylinositol 3-phosphate (PI3P) remain associated with phagosomes. Moreover, Rab20 depletion in macrophages abrogates the delay in phagosome maturation induced by IFN-γ. Finally, we demonstrate that Rab20 interacts with the Rab5a guanine nucleotide exchange factor Rabex-5 (also known as RABGEF1) and that Rab20 knockdown impairs the IFN-γ-dependent recruitment of Rabex-5 and Rab5a into phagosomes. Taken together, here, we uncover Rab20 as a key player in the Rab cascade by which IFN-γ induces a delay in phagosome maturation in macrophages.
    • Internalization, phagolysosomal biogenesis and killing of mycobacteria in enucleated epithelial cells.

      de Souza Carvalho, Cristiane; Kasmapour, Bahram; Gronow, Achim; Rohde, Manfred; Rabinovitch, Michel; Gutierrez, Maximiliano Gabriel; Department of Vaccinology and Applied Microbiology, Research Group Phagosome Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany. (2011-08)
      Bacterial and parasitic intracellular pathogens or their secreted products have been shown to induce host cell transcriptional responses, which may benefit the host, favour the microorganism or be unrelated to the infection. In most instances, however, it is not known if the host cell nucleus is proximately required for the development of an intracellular infection. This information can be obtained by the infection of artificially enucleated host cells (cytoplasts). This model, although rather extensively used in studies of viral infection, has only been applied to few bacterial pathogens, which do not include Mycobacterium spp. Here, we investigate the internalization, phagosome biogenesis and survival of M. smegmatis in enucleated type II alveolar epithelial cells. Cytoplasts were infected with M. smegmatis, but the percentage of infection was significantly lower than that of nucleated cells. Scanning electron microscopy indicated that in both cells and cytoplasts, bacteria were internalized by a phagocytosis-like mechanism. Interestingly, phagosome fusion with lysosomes and mycobacterial killing were both more efficient in enucleated than in nucleated cells, a finding that may be correlated with the increased number of autophagic vesicles developed in cytoplasts. We provide evidence that although quantitative changes were observed, the full development of the infection, as well as mycobacterial killing did not require the presence of the host cell nucleus.