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A novel role for Stat1 in phagosome acidification and natural host resistance to intracellular infection by Leishmania major.

Späth GF, Schlesinger P, Schreiber R, Beverley SM - PLoS Pathog. (2009)

Bottom Line: Intracellular parasites of the genus Leishmania generate severe diseases in humans, which are associated with a failure of the infected host to induce a protective interferon gamma (IFNgamma)-mediated immune response.We tested the role of the JAK/STAT1 signaling pathway in Leishmania pathogenesis by utilizing knockout mice lacking the signal transducer and activator of transcription 1 (Stat1) and derived macrophages.This novel Stat1 function may have important implications to studies of other pathogens, as the acidic phagolysosomal pH plays an important role in antigen processing and the uncoating process of many viruses.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University Medical School, St. Louis, Missouri, USA.

ABSTRACT
Intracellular parasites of the genus Leishmania generate severe diseases in humans, which are associated with a failure of the infected host to induce a protective interferon gamma (IFNgamma)-mediated immune response. We tested the role of the JAK/STAT1 signaling pathway in Leishmania pathogenesis by utilizing knockout mice lacking the signal transducer and activator of transcription 1 (Stat1) and derived macrophages. Unexpectedly, infection of Stat1-deficient macrophages in vitro with promastigotes from Leishmania major and attenuated LPG1 knockout mutants (lpg(-)) specifically lacking lipophosphoglycan (LPG) resulted in a twofold increased intracellular growth, which was independent of IFNgamma and associated with a substantial increase in phagosomal pH. Phagosomes in Stat1-/- macrophages showed normal maturation as judged by the accumulation of the lysosomal marker protein rab7, and provided normal vATPase activity, but were defective in the anion conductive pathway required for full vesicular acidification. Our results suggest a role of acidic pH in the control of intracellular Leishmania growth early during infection and identify for the first time an unexpected role of Stat1 in natural anti-microbial resistance independent from its function as IFNgamma-induced signal transducer. This novel Stat1 function may have important implications to studies of other pathogens, as the acidic phagolysosomal pH plays an important role in antigen processing and the uncoating process of many viruses.

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pH measurement on isolated phagosomes.Intra-vesicular pH of phagosomes containing FITC-labeled zymosan was determined in untreated control preparations (left panel), untreated preparations from Stat1−/− BMMs (middle panel), and control preparations treated with 93 mM K2SO4 (right panel). Acidification was initiated by addition of 2.5 mM ATP and 1 mM MgSO4 (closed arrow). Subsequent addition of 20 µM NH4Cl increased the pH of all phagosome preparations (open arrow). The addition of 10 µM nigericin increased the pH to 7 (not shown). Each time course was done in triplicate with the standard deviation indicated by the error bars.
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ppat-1000381-g005: pH measurement on isolated phagosomes.Intra-vesicular pH of phagosomes containing FITC-labeled zymosan was determined in untreated control preparations (left panel), untreated preparations from Stat1−/− BMMs (middle panel), and control preparations treated with 93 mM K2SO4 (right panel). Acidification was initiated by addition of 2.5 mM ATP and 1 mM MgSO4 (closed arrow). Subsequent addition of 20 µM NH4Cl increased the pH of all phagosome preparations (open arrow). The addition of 10 µM nigericin increased the pH to 7 (not shown). Each time course was done in triplicate with the standard deviation indicated by the error bars.

Mentions: Phagosomes containing FITC-conjugated zymosan were isolated from control and Stat1-deficient bone marrow-derived macrophages (BMM), diluted into the reaction mixture containing ATP and reactions were started by the addition of MgSO4 (Figure 5, closed arrows). Phagosomes from control mice showed a rapid but transient decrease in vesicular pH by 0.3 pH units to 5.95 (s.d. 0.04) during the first minute after MgSO4 addition (Figure 5, left panel). Phagosomes from Stat1−/− BMMs were able to initiate phagosome acidification (Figure 5, middle panel) but showed a pH decrease of only 0.15 pH units to 6.13 (s.d.0.04). This acidification profile indicates the presence of a functional H+-ATPase that provides limited activity most likely due to a defect in charge neutralization compared to the control (p<0.002 for the difference observed one minute after ATP addition). This hypothesis was further sustained in K2SO4-treated control preparations. Replacement of chloride with impermeant anion sulfate eliminates the charge neutralization normally conferred by the chloride channel, a treatment that resulted in partial acidification of Stat1+/+-preparations similar to the one observed in Stat1−/− preparations (Figure 5, right panel).


A novel role for Stat1 in phagosome acidification and natural host resistance to intracellular infection by Leishmania major.

Späth GF, Schlesinger P, Schreiber R, Beverley SM - PLoS Pathog. (2009)

pH measurement on isolated phagosomes.Intra-vesicular pH of phagosomes containing FITC-labeled zymosan was determined in untreated control preparations (left panel), untreated preparations from Stat1−/− BMMs (middle panel), and control preparations treated with 93 mM K2SO4 (right panel). Acidification was initiated by addition of 2.5 mM ATP and 1 mM MgSO4 (closed arrow). Subsequent addition of 20 µM NH4Cl increased the pH of all phagosome preparations (open arrow). The addition of 10 µM nigericin increased the pH to 7 (not shown). Each time course was done in triplicate with the standard deviation indicated by the error bars.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2663844&req=5

ppat-1000381-g005: pH measurement on isolated phagosomes.Intra-vesicular pH of phagosomes containing FITC-labeled zymosan was determined in untreated control preparations (left panel), untreated preparations from Stat1−/− BMMs (middle panel), and control preparations treated with 93 mM K2SO4 (right panel). Acidification was initiated by addition of 2.5 mM ATP and 1 mM MgSO4 (closed arrow). Subsequent addition of 20 µM NH4Cl increased the pH of all phagosome preparations (open arrow). The addition of 10 µM nigericin increased the pH to 7 (not shown). Each time course was done in triplicate with the standard deviation indicated by the error bars.
Mentions: Phagosomes containing FITC-conjugated zymosan were isolated from control and Stat1-deficient bone marrow-derived macrophages (BMM), diluted into the reaction mixture containing ATP and reactions were started by the addition of MgSO4 (Figure 5, closed arrows). Phagosomes from control mice showed a rapid but transient decrease in vesicular pH by 0.3 pH units to 5.95 (s.d. 0.04) during the first minute after MgSO4 addition (Figure 5, left panel). Phagosomes from Stat1−/− BMMs were able to initiate phagosome acidification (Figure 5, middle panel) but showed a pH decrease of only 0.15 pH units to 6.13 (s.d.0.04). This acidification profile indicates the presence of a functional H+-ATPase that provides limited activity most likely due to a defect in charge neutralization compared to the control (p<0.002 for the difference observed one minute after ATP addition). This hypothesis was further sustained in K2SO4-treated control preparations. Replacement of chloride with impermeant anion sulfate eliminates the charge neutralization normally conferred by the chloride channel, a treatment that resulted in partial acidification of Stat1+/+-preparations similar to the one observed in Stat1−/− preparations (Figure 5, right panel).

Bottom Line: Intracellular parasites of the genus Leishmania generate severe diseases in humans, which are associated with a failure of the infected host to induce a protective interferon gamma (IFNgamma)-mediated immune response.We tested the role of the JAK/STAT1 signaling pathway in Leishmania pathogenesis by utilizing knockout mice lacking the signal transducer and activator of transcription 1 (Stat1) and derived macrophages.This novel Stat1 function may have important implications to studies of other pathogens, as the acidic phagolysosomal pH plays an important role in antigen processing and the uncoating process of many viruses.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University Medical School, St. Louis, Missouri, USA.

ABSTRACT
Intracellular parasites of the genus Leishmania generate severe diseases in humans, which are associated with a failure of the infected host to induce a protective interferon gamma (IFNgamma)-mediated immune response. We tested the role of the JAK/STAT1 signaling pathway in Leishmania pathogenesis by utilizing knockout mice lacking the signal transducer and activator of transcription 1 (Stat1) and derived macrophages. Unexpectedly, infection of Stat1-deficient macrophages in vitro with promastigotes from Leishmania major and attenuated LPG1 knockout mutants (lpg(-)) specifically lacking lipophosphoglycan (LPG) resulted in a twofold increased intracellular growth, which was independent of IFNgamma and associated with a substantial increase in phagosomal pH. Phagosomes in Stat1-/- macrophages showed normal maturation as judged by the accumulation of the lysosomal marker protein rab7, and provided normal vATPase activity, but were defective in the anion conductive pathway required for full vesicular acidification. Our results suggest a role of acidic pH in the control of intracellular Leishmania growth early during infection and identify for the first time an unexpected role of Stat1 in natural anti-microbial resistance independent from its function as IFNgamma-induced signal transducer. This novel Stat1 function may have important implications to studies of other pathogens, as the acidic phagolysosomal pH plays an important role in antigen processing and the uncoating process of many viruses.

Show MeSH
Related in: MedlinePlus