Natural resistance to intracellular infections: natural resistance-associated macrophage protein 1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane - PubMed (original) (raw)

Natural resistance to intracellular infections: natural resistance-associated macrophage protein 1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane

N Jabado et al. J Exp Med. 2000.

Abstract

Mutations at the natural resistance-associated macrophage protein 1 (Nramp1) locus cause susceptibility to infection with antigenically unrelated intracellular pathogens. Nramp1 codes for an integral membrane protein expressed in the lysosomal compartment of macrophages, and is recruited to the membrane of phagosomes soon after the completion of phagocytosis. To define whether Nramp1 functions as a transporter at the phagosomal membrane, a divalent cation-sensitive fluorescent probe was designed and covalently attached to a porous particle. The resulting conjugate, zymosan-FF6, was ingested by macrophages and its fluorescence emission was recorded in situ after phagocytosis, using digital imaging. Quenching of the probe by Mn(2+) was used to monitor the flux of divalent cations across the phagosomal membrane in peritoneal macrophages obtained from Nramp1-expressing (+/+) and Nramp1-deficient (-/-) macrophages. Phagosomes from Nramp1(+/+) mice extrude Mn(2+) faster than their Nramp(-/-) counterparts. The difference in the rate of transport is eliminated when acidification of the phagosomal lumen is dissipated, suggesting that divalent metal transport through Nramp1 is H(+) dependent. These studies suggest that Nramp1 contributes to defense against infection by extrusion of divalent cations from the phagosomal space. Such cations are likely essential for microbial function and their removal from the phagosomal microenvironment impairs pathogenesis, resulting in enhanced bacteriostasis or bactericidal activity.

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Figures

Figure 1

Synthesis of the fluorescent probe zymosan–FF6. Step a, activation of COOH-FF6(COOEt)4 using dicyclohexylcarbodiimide and _N_-hydroxysuccinimide. Step b, coupling of the activated intermediate to zymosan. Step c, deprotection of the conjugate by silanolate-induced ester hydrolysis.

Figure 2

(A–E) Spectral properties of zymosan–FF6 particles in suspension. (A) Zymosan–FF6 particles were added to medium containing 140 mM KCl, 20 mM Hepes-Na (pH 7.4), and 500 μM EGTA, and the excitation spectrum was acquired with emission at 510 nm. Increasing amounts of Ca2+ were then added (total concentrations indicated) and fluorescence was recorded as above. (B–E) Zymosan–FF6 particles were added to medium containing 500 μM EGTA and 500 μM Ca2+ buffered at pH 7.4 or 4.5, as indicated, and excitation spectra were acquired (emission = 510 nm). Increasing amounts of Mn2+ (B), Fe2+ (C), Cu2+ (D), or Zn2+ (E) were then added (total concentrations indicated) and the effects of the divalent cations on zymosan–FF6 fluorescence were recorded. (F) Fluorescence properties of zymosan–FF6 particles ingested by macrophages. Peritoneal macrophages plated on coverslips were bathed in Ca2+-free medium and allowed to ingest zymosan–FF6 particles for 30 min at 37°C. Coverslips were mounted in thermoregulated chambers and examined using differential interference contrast optics (left) and epifluorescence with excitation at 360 nm and emission at 535 nm (right). The location of zymosan–FF6 particles is indicated by arrows.

Figure 3

Nramp1 association with zymosan–FF6-containing phagosomes. Macrophages from normal mice (129sv, +/+; A and B) and from mutant mice with a null mutation at Nramp1 (Nramp1 − _/_−, −/−; C and D) were harvested and plated on glass coverslips. After 48 h, the cells were allowed to ingest zymosan–FF6 for 1 h at 37°C. The cells were washed free of uningested particles and fixed before indirect immunofluorescence with an affinity-purified rabbit anti-Nramp1 antibody. Phase–contrast images (A and C) and the corresponding immunofluorescence micrographs (B and D) are shown.

Figure 4

Quenching of cytosolic Fura-2 by Mn2+ in normal (+/+) and mutant (−/−) macrophages after treatment with thapsigargin. Peritoneal macrophages from normal (+/+) and Nramp1 mutant (−/−) mice were plated on coverslips and loaded with Fura-2 by incubation with the precursor acetoxymethyl ester for 30 min at 37°C. Coverslips were mounted in thermoregulated chambers, the cells were bathed in Ca2+-free medium containing 250 μM EGTA, and fluorescence was visualized with excitation at 360 nm. After recording baseline fluorescence, thapsigargin (thaps; 100 nM) was added to deplete calcium stores and open store–operated channels. Finally, 500 μM Mn2+ was added and recording was resumed. Data are representative of three similar experiments.

Figure 5

The effect of Nramp1 expression on Mn2+-induced quenching of internalized zymosan–FF6 fluorescence. Peritoneal macrophages from normal (+/+) and Nramp1 mutant (−/−) mice were plated for 48 h on coverslips, and allowed to ingest zymosan–FF6 particles for 30 min at 37°C in Ca2+-free medium containing 250 μM EGTA. Coverslips were mounted in thermoregulated chambers and examined using differential interference contrast optics to locate internalized particles. Fluorescence was measured with excitation at 360 nm before and after addition of 100 nM thapsigargin (thaps). Mn2+ (500 μM) was added to the cells where indicated and recording was resumed. A illustrates a typical experiment, and B summarizes the fractional quenching (as percentage of initial fluorescence) as a function of time. Data in B are means ± SE of 15 individual experiments for −/− macrophages (filled symbols) and 17 experiments for +/+ macrophages (open symbols).

Figure 5

Figure 6

Effect of pH on Mn2+ transport across the phagosomal membrane. (A) Phagosomal pH was measured by ratio imaging in peritoneal macrophages from normal (+/+; open symbols) and Nramp1 mutant (−/−; filled symbols) mice, using zymosan covalently conjugated to FITC and Oregon green 514, as described in Materials and Methods. Fluorescence at 535 nm was measured with alternating excitation at 440 and 490 nm. Where indicated, the cells were treated with 100 nM thapsigargin (thaps), followed by 100 nM concanamycin. For calibration, the cells were permeabilized with 1% Triton X-100 and sequentially perfused with solutions at pH 7.5, 7, 6.5, 5.5, 4.5, and 4. Results are representative of three determinations for each type of macrophage. (B) Effect of the vacuolar H+-ATPase inhibitor concanamycin on the Mn2+-induced quenching of fluorescence of phagosomal zymosan–FF6 in normal (+/+; open symbols) and Nramp1 mutant (−/−; filled symbols) macrophages. The experiment was carried out as described in the legend to Fig. 5 B, except that the cells were pretreated with 100 nM of concanamycin for 25 min to allow inhibition dissipation of the phagosomal acidification, before the addition of thapsigargin and finally Mn2+. The data are means ± SE of eight individual experiments.

Figure 6

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Natural resistance to intracellular infections: natural resistance-associated macrophage protein 1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane - PubMed (original) (raw)