Functional expression of Nramp1 in vitro in the murine macrophage line RAW264.7

Abstract

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 Nramp1(G169) cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1(D169) 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.

FIG. 1

Effect of Nramp1 on in vivo replication of S. typhimurium in spleen and liver. Mouse strains 129sv (□) and 129sv.Nramp1^null (⧫) were infected intravenously with 0.8 × 10³ S. typhimurium Keller CFUs. At 1 h, 6 h, 1 day, 3 days, 4 days, 5 days, and 7 days postinfection, spleens (A) and livers (B) were removed, weighed, and homogenized for CFU counts. The results are expressed as CFU per gram of tissue. All 129sv.Nramp1^null mice died from infection prior to the 5-day time point, which is denoted by a cross. A minimum of three to five mice was used for each time point, and the results are shown as means ± standard deviations.

FIG. 2

Expression of recombinant Nramp1^G169-cMyc fusion protein in transfected RAW264.7 clones. Enriched membrane fractions were prepared from RAW264.7 cells (lane A), Nramp1-cMyc-transfected RAW264.7 clone 13 (lane B), clone 15 (lane C), clone 2.2 (lane D), and Nramp2-transfected RAW264.7 cells (lane E). Equal amounts of protein (20 μg per sample) were loaded on an SDS–7.5% polyacrylamide gel, followed by transfer to a nitrocellulose membrane and immunoblotting with an isoform-specific anti-Nramp1 polyclonal antibody (22). After washing, a mouse anti-rabbit secondary antibody conjugated with horseradish peroxidase was used to reveal specific immune complexes.

FIG. 3

The recombinant Nramp1-cMyc protein localizes to Y. enterocolitica- and S. typhimurium-containing phagosomes in RAW264.7 macrophages. Nramp1-cMyc-expressing RAW264.7 macrophages were infected with Y. enterocolitica (upper panels) or S. typhimurium (lower panels). Two hours postinfection, cells were fixed with paraformaldehyde and analyzed by immunofluorescence. Y. enterocolitica were identified by staining with an anti-Yersinia antibody plus Alexa-488 secondary antibody (upper left panel). S. typhimurium used in this experiment express GFP and were identifiable in the same channel as the Alexa-488 antibody (lower left panel). An anti-c-Myc monoclonal antibody (9E10) was used to identify the c-Myc tag fused in frame at the C terminus of Nramp1 (middle panels). The respective images are superimposed in the right panels to show colocalization (yellow staining) with red staining representing Nramp1 expression and green staining representing bacteria. Arrows indicate colocalized bacteria and Nramp1, while arrows plus ∗ indicate extracellular bacteria as determined by phase contrast (not shown).

FIG. 4

Effect of recombinant Nramp1-cMyc protein expression on antibacterial activity of RAW264.7 macrophages. Nontransfected RAW264.7 cells (sample 1), Nramp1-cMyc-transfected RAW264.7 macrophage clone 13 (sample 2), clone 2.2 (sample 3), and a Nramp2-transfected RAW264.7 clone (sample 4) were seeded at 5 × 10⁵ cells per well (5 wells per sample). Cells were infected with either S. typhimurium SL1344 (A) or the temperature-sensitive, replication-defective mutant TSΔ27 (B). After an initial 30-min phagocytosis period (T₀), cell cultures were lysed at predetermined time intervals, and CFU counts were determined. The level of infection was determined by dividing the number of CFU_t for each well at individual time points by the CFU₀ (at T₀) and is expressed as a percentage. The standard deviations for each time point are shown. The ranges in individual values for the samples at 24 h in panel A were as follows: sample 1, 4.4- to 3.0-fold increase; sample 2, 10 to 5%; sample 3, 81 to 57%; and sample 4, 4.2- to 3.1-fold increase. For panel B, the ranges for the samples at 24 h were as follows: sample 1, 11.5 to 8.5%; sample 2, 2.7 to 0.7%; sample 3, 1.6 to 0.3%; and sample 4, 4.2 to 2.1%.