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Infection and Immunity, May 1999, p. 2225-2232, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Functional Expression of Nramp1 In Vitro in the Murine Macrophage Line RAW264.7

Gregory Govoni,1 François Canonne-Hergaux,1 Cheryl G. Pfeifer,2 Sandra L. Marcus,2 Scott D. Mills,2 David J. Hackam,3 Sergio Grinstein,3 Danielle Malo,4 B. Brett Finlay,2 and Philippe Gros1,*

Department of Biochemistry, McGill University,1 and Montreal General Hospital Research Institute,4 Montreal, Quebec, Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia,2 and Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario,3 Canada

Received 11 November 1998/Returned for modification 14 January 1999/Accepted 18 February 1999

Mutations at the Nramp1 locus in vivo cause susceptibility to infection by unrelated intracellular microbes. Nramp1 encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-type Nramp1G169 cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1D169 allele and thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane of Salmonella typhimurium and Yersinia enterocolitica containing phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication of S. typhimurium. Studies with a replication-defective S. typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant of S. typhimurium. Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele. Together, these results indicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.

* Corresponding author. Mailing address: Department of Biochemistry, McGill University, 3655 Drummond, Room 907, Montreal, Quebec, Canada H3G 1Y6. Phone: (514) 398-7291. Fax: (514) 398-2603. E-mail: gros{at}med.mcgill.ca.

Infection and Immunity, May 1999, p. 2225-2232, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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