Natural resistance to intracellular infections: Natural resistance-associated macrophage protein 1 (NRAMP1) functions as a pH-dependent manganese transporter at the phagosomal membrane

Mutations at the natural resistance-associated macrophage protein 1 (Nramp1 ) locus cause susceptibility to infection with antigenically unrelated intr acellular pathogens. Nramp1 codes for an integral membrane protein expresse d in the lysosomal compartment of macrophages, and is recruited to the memb rane of phagosomes soon after the completion of phagocytosis. To define whe ther Nramp1 functions as a transporter at the phagosomal membrane, a divale nt 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 phago cytosis, using digital imaging. Quenching of the probe by Mn2+ was used to monitor the flux of divalent cations across the phagosomal membrane in peri toneal macrophages obtained from Nramp1-expressing (+/+) and Nramp1-deficie nt (-/-) macrophages. Phagosomes from Nramp1(+/+) mice extrude Mn2+ faster than their Nramp-/- counterparts. The difference in the rate of transport i s eliminated when acidification of the phagosomal lumen is dissipated, sugg esting that divalent metal transport through Nramp1 is H+ dependent. These studies suggest that Nramp1 contributes to defense against infection by ext rusion of divalent cations from the phagosomal space. Such cations are like ly essential for microbial function and their removal from the phagosomal m icroenvironment impairs pathogenesis, resulting in enhanced bacteriostasis or bactericidal activity.