The mechanisms underlying the survival of intracellular parasites such as mycobacteria in host macrophages remain poorly understood. In mice, mutations at the Nramp1 gene (for natural resistance-associated macrophage protein), cause susceptibility to mycobacterial infections. Nramp1 encodes an integral membrane protein that is recruited to the phagosome membrane in infected macrophages. In this study, we used microfluorescence ratio imaging of macrophages from wild-type and Nramp1 mutant mice to analyze the effect of loss of Nramp1 function on the properties of phagosomes containing inert particles or live mycobacteria. The pH of phagosomes containing live Mycobacterium bovis was significantly more acidic in Nramp1- expressing macrophages than in mutant cells (pH 5.5 ± 0.06 versus pH 6.6 ± 0.05, respectively; P <0.005). The enhanced acidification could not be accounted for by differences in proton consumption during dismutation of superoxide, phagosomal buffering power, counterion conductance, or in the rate of proton “leak”, as these were found to be comparable in wild-type and Nramp1-deficient macrophages. Rather, after ingestion of live mycobacteria, Nramp1-expressing cells exhibited increased concanamycin-sensitive H+ pumping across the phagosomal membrane. This was associated with an enhanced ability of phagosomes to fuse with vacuolar-type ATPase–containing late endosomes and/or lysosomes. This effect was restricted to live M. bovis and was not seen in phagosomes containing dead M. bovis or latex beads. These data support the notion that Nramp1 affects intracellular mycobacterial replication by modulating phagosomal pH, suggesting that Nramp1 plays a central role in this process.
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20 July 1998
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July 20 1998
Host Resistance to Intracellular Infection: Mutation of Natural Resistance-associated Macrophage Protein 1 (Nramp1) Impairs Phagosomal Acidification
David J. Hackam,
David J. Hackam
From the *Division of Cell Biology, The Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada; the ‡Department of Surgery, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Ontario, Canada; and the §Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
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Ori D. Rotstein,
Ori D. Rotstein
From the *Division of Cell Biology, The Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada; the ‡Department of Surgery, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Ontario, Canada; and the §Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
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Wei-jian Zhang,
Wei-jian Zhang
From the *Division of Cell Biology, The Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada; the ‡Department of Surgery, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Ontario, Canada; and the §Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
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Samantha Gruenheid,
Samantha Gruenheid
From the *Division of Cell Biology, The Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada; the ‡Department of Surgery, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Ontario, Canada; and the §Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
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Philippe Gros,
Philippe Gros
From the *Division of Cell Biology, The Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada; the ‡Department of Surgery, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Ontario, Canada; and the §Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
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Sergio Grinstein
Sergio Grinstein
From the *Division of Cell Biology, The Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada; the ‡Department of Surgery, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Ontario, Canada; and the §Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
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David J. Hackam
From the *Division of Cell Biology, The Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada; the ‡Department of Surgery, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Ontario, Canada; and the §Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
Ori D. Rotstein
From the *Division of Cell Biology, The Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada; the ‡Department of Surgery, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Ontario, Canada; and the §Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
Wei-jian Zhang
From the *Division of Cell Biology, The Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada; the ‡Department of Surgery, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Ontario, Canada; and the §Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
Samantha Gruenheid
From the *Division of Cell Biology, The Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada; the ‡Department of Surgery, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Ontario, Canada; and the §Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
Philippe Gros
From the *Division of Cell Biology, The Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada; the ‡Department of Surgery, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Ontario, Canada; and the §Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
Sergio Grinstein
From the *Division of Cell Biology, The Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada; the ‡Department of Surgery, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Ontario, Canada; and the §Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
Address correspondence to Sergio Grinstein, Division of Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto, M5G 1X8, Ontario, Canada. Phone: 416-813-5727; Fax: 416-813-5028; E-mail: [email protected]
This research was funded by operating grants awarded to P. Gros from the National Institute of Allergy and Infectious Diseases (RO1 AI-35247-0352) and to S. Grinstein and O.D. Rotstein by the Medical Research Council of Canada and the National Sanatorium Association. S. Grinstein is cross-appointed to the Department of Biochemistry of the University of Toronto. P. Gros and S. Grinstein are International Scholars of the Howard Hughes Medical Institute. D.J. Hackam is the recipient of an Ethicon-Society of University Surgeons Surgical Research Award and a Medical Research Council of Canada Fellowship.
Received:
February 18 1998
Revision Received:
April 29 1998
Online ISSN: 1540-9538
Print ISSN: 0022-1007
1998
J Exp Med (1998) 188 (2): 351–364.
Article history
Received:
February 18 1998
Revision Received:
April 29 1998
Citation
David J. Hackam, Ori D. Rotstein, Wei-jian Zhang, Samantha Gruenheid, Philippe Gros, Sergio Grinstein; Host Resistance to Intracellular Infection: Mutation of Natural Resistance-associated Macrophage Protein 1 (Nramp1) Impairs Phagosomal Acidification . J Exp Med 20 July 1998; 188 (2): 351–364. doi: https://doi.org/10.1084/jem.188.2.351
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