Cytokine activation leads to acidification and increases maturation of M. avium-containing phagosomes in murine macrophages

Department of Molecular Microbiology, Washington University, School of Medicine, St. Louis, MO 63110, USA.
The Journal of Immunology (Impact Factor: 4.92). 03/1998; 160(3):1290-6.
Source: PubMed

ABSTRACT Mycobacterium avium (MAC) organisms multiply in phagosomes that have restricted fusigenicity with lysosomes, do not acidify due to a paucity of vacuolar proton-ATPases, yet remain accessible to recycling endosomes. During the course of mycobacterial infections, IFN-gamma-mediated activation of host and bystander macrophages is a key mechanism in the regulation of bacterial growth. Here we demonstrate that in keeping with earlier studies, cytokine activation of host macrophages leads to a decrease in MAC viability, demonstrable by bacterial esterase staining with fluorescein diacetate as well as colony-forming unit counts from infected cells. Analysis of the pH of MAC phagosomes demonstrated that the vacuoles in activated macrophages equilibrate to pH 5.2, in contrast to pH 6.3 in resting phagocytes. Biochemical analysis of MAC phagosomes from both resting and activated macrophages confirmed that the lower intraphagosomal pH correlated with an increased accumulation of proton-ATPases. Furthermore, the lower pH is reflected in the transition of MAC phagosomes to a point no longer accessible to transferrin, a marker of the recycling endosomal system. These alterations parallel the coalescence of bacterial vacuoles from individual bacilli in single vacuoles to communal vacuoles with multiple bacilli. These data demonstrate that bacteriostatic and bactericidal activities of activated macrophages are concomitant with alterations in the physiology of the mycobacterial phagosome.

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    • "IFN-γ and TNF are key cytokines that lead to cell activation and enhanced phagosome–lysosome fusion in mycobacteria-infected macrophages (Schaible et al., 1998; Via et al., 1998). Phagolysosomes are formed upon fusion of phagosomes with intracellular organelles, in particular lysosomes. "
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    ABSTRACT: Infection of humans with Mycobacterium tuberculosis remains frequent and may still lead to death. After primary infection, the immune system is often able to control M. tuberculosis infection over a prolonged latency period, but a decrease in immune function (from HIV to immunosenescence) leads to active disease. Available vaccines against tuberculosis are restricted to BCG, a live vaccine with an attenuated strain of M. bovis. Immunodeficiency may not only be associated with an increased risk of tuberculosis, but also with local or disseminated BCG infection. Genetic deficiency in the reactive oxygen species (ROS)-producing phagocyte NADPH oxidase NOX2 is called chronic granulomatous disease (CGD). CGD is among the most common primary immune deficiencies. Here we review our knowledge on the importance of NOX2-derived ROS in mycobacterial infection. A literature review suggests that human CGD patient frequently have an increased susceptibility to BCG and to M. tuberculosis. In vitro studies and experiments with CGD mice are incomplete and yielded - at least in part - contradictory results. Thus, although observations in human CGD patients leave little doubt about the role of NOX2 in the control of mycobacteria, further studies will be necessary to unequivocally define and understand the role of ROS.
    Cellular Microbiology 06/2014; 16(8). DOI:10.1111/cmi.12322 · 4.92 Impact Factor
    • "Interferon- γ (IFN-γ) is a key cytokine that plays multiple roles during the activation and modulation of intracellular trafficking, especially in macrophages (Gordon, 2003; Montaner et al., 1999; Schroder et al., 2004). IFN-γ endows macrophages with the ability to control the immune function of phagosomes by stimulating phagosome maturation (Nathan et al., 1983; Santic et al., 2005a; Santic et al., 2005b; Schaible et al., 1998; Via et al., 1998). However, in the same cells, IFN-γ also delays phagosome maturation and acidification at early time points (Jutras et al., 2008; Trost et al., 2009; Tsang et al., 2000; Yates et al., 2007). "
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    ABSTRACT: 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. IFN-γ stimulated Rab20 association with early phagosomes in macrophages. Using single phagosome imaging in live cells, we found that Rab20 induced an early delay in phagosome maturation and extended the time Rab5a and phosphatidylinositol 3-phosphate (PI3P) remains associated with phagosomes. Moreover, Rab20 depletion in macrophages abrogated the phagosome maturation delay induced by IFN-γ. Finally, we demonstrate that Rab20 interacts with the Rab5 guanine nucleotide exchange factor Rabex-5 and that Rab20 knockdown impaired the IFN-γ-dependent Rabex-5/Rab5a recruitment into phagosomes. Altogether, we uncover here Rab20 as a key player in the Rab cascade by which IFN-γ induces a delay in phagosome maturation in macrophages.
    Journal of Cell Science 02/2014; 127(9). DOI:10.1242/jcs.144923 · 5.43 Impact Factor
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    • "In non-activated macrophages, Mtb is capable of blocking the fusion of phagosomes with lysosomes and avoids a degradative state [3]. Activation of macrophages with interferon-gamma (IFNγ) overcomes the block in phagosome maturation and leads to acidification of the compartment to pH∼4.5 [4], [5]. "
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    ABSTRACT: Bacterial pathogens like Mycobacterium tuberculosis (Mtb) encounter acidic microenvironments in the host and must maintain their acid-base homeostasis to survive. A genetic screen identified two Mtb strains that cannot control intrabacterial pH (pHIB) in an acidic environment; infection with either strain led to severe attenuation in mice. To search for additional proteins that Mtb requires to survive at low pH, we introduced a whole-cell screen for compounds that disrupt pHIB, along with counter-screens that identify ionophores and membrane perturbors. Application of these methods to a natural product library identified four compounds of interest, one of which may inhibit novel pathway(s). This approach yields compounds that may lead to the identification of pathways that allow Mtb to survive in acidic environments, a setting in which Mtb is resistant to most of the drugs currently used to treat tuberculosis.
    PLoS ONE 08/2013; 8(7):e68942. DOI:10.1371/journal.pone.0068942 · 3.23 Impact Factor
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