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

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. 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.

SUBMITTER: Pei G

PROVIDER: S-EPMC4004979 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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

Pei Gang G Repnik Urska U Griffiths Gareth G Gutierrez Maximiliano Gabriel MG

Journal of cell science 20140225 Pt 9

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 ...[more]

PMID: 24569883

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Project description:Candida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.

Dataset Information

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

Publications

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

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