Host and Pathogen Copper-Transporting P-Type ATPases Function Antagonistically during Salmonella Infection

Abstract

Copper is an essential yet potentially toxic trace element that is required by all aerobic organisms. A key regulator of copper homeostasis in mammalian cells is the copper-transporting P-type ATPase ATP7A, which mediates copper transport from the cytoplasm into the secretory pathway, as well as copper export across the plasma membrane. Previous studies have shown that ATP7A-dependent copper transport is required for killing phagocytosed Escherichia coli in a cultured macrophage cell line. In this investigation, we expanded on these studies by generating Atp7a^LysMcre mice, in which the Atp7a gene was specifically deleted in cells of the myeloid lineage, including macrophages. Primary macrophages isolated from Atp7a^LysMcre mice exhibit decreased copper transport into phagosomal compartments and a reduced ability to kill Salmonella enterica serovar Typhimurium compared to that of macrophages isolated from wild-type mice. The Atp7a^LysMcre mice were also more susceptible to systemic infection by S Typhimurium than wild-type mice. Deletion of the S Typhimurium copper exporters, CopA and GolT, was found to decrease infection in wild-type mice but not in the Atp7a^LysMcre mice. These studies suggest that ATP7A-dependent copper transport into the phagosome mediates host defense against S Typhimurium, which is counteracted by copper export from the bacteria via CopA and GolT. These findings reveal unique and opposing functions for copper transporters of the host and pathogen during infection.

Keywords: ATP7A copper transporter; Salmonella Typhimurium; bacterial copper tolerance; bacterial infection; copper toxicity; host-pathogen interactions; immunology and infection; macrophages; nutritional immunity.

FIG 1

ATP7A traffics to the phagosomal compartment in activated macrophages. (A) Immunofluorescence analysis of the ATP7A protein in primary mouse peritoneal macrophages that were stimulated with LPS (100 ng/ml) or IFN-γ (25 ng/ml) for 16 h. Cells were labeled with antibodies against ATP7A protein (green) and the Golgi marker, GM130 (red). Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI; blue). (B) Colocalization of ATP7A with phagocytosed E. coli. Peritoneal macrophages were stimulated with IFN-γ (25 ng/ml) for 16 h and exposed to fluorescently labeled E. coli (red) to allow phagocytosis. Cells were washed to remove extracellular bacteria and processed to detect ATP7A by immunofluorescence as described above. Regions of colocalization between ATP7A and E. coli are shown in yellow in the merge panel (arrows).

FIG 2

Deletion of Atp7a in macrophages of Atp7a^LysMcre mice. (A) Immunoblot analysis of ATP7A protein levels in primary peritoneal macrophages derived from Atp7a^WT and Atp7a^LysMcre mice. Each lane represents a different mouse. (B) Immunofluorescence analysis of the ATP7A protein levels in peritoneal macrophages from Atp7a^WT and Atp7a^LysMcre mice. Cells were labeled with antibodies against ATP7A protein (green) and the Golgi marker, GM130 (red). Nuclei were labeled with DAPI (blue). (C) Immunoblot analysis of ATP7A protein levels in the indicated tissues derived from Atp7a^WT and Atp7a^LysMcre mice.

FIG 3

Loss of ATP7A in macrophages impairs copper loading of the phagosomal compartment and enhances survival of phagocytosed S. Typhimurium. (A) Survival of wild-type S. Typhimurium in murine peritoneal macrophages derived from Atp7a^WT or Atp7a^LysMcre mice. Macrophages were pretreated overnight with 25 ng/ml of IFN-γ and then incubated with wild-type S. Typhimurium for 30 min to allow phagocytosis. The percentage of surviving bacteria in macrophages was determined after 1 h and 2 h (means ± standard deviation [SD]; n = 3; Student t test). (B) Loss of ATP7A reduces copper delivery by macrophages to phagocytosed S. Typhimurium. S. Typhimurium was grown overnight in M9 minimal medium to deplete copper content and then preloaded with the 4 μM CF4. The bacteria were then exposed to peritoneal macrophages derived from Atp7a^WT or Atp7a^LysMcre mice to allow for phagocytosis. Macrophages were then washed extensively in PBS and imaged. Each panel is representative of macrophages isolated from a different mouse. (C) Fluorescence measurements of phagocytosed bacteria within Atp7a^WT or Atp7a^LysMcre macrophages (means ± SD; n = 15 cells in at least 5 different fields; Student t test). Experiments were repeated three times, with essentially the same results.

FIG 4

Survival of S. Typhimurium is dependent on the CopA/GolT copper exporters in Atp7a^WT, but not Atp7a^LysMcre, macrophages. Peritoneal macrophages were pretreated overnight with 25 ng/ml of IFN-γ and then infected at an MOI of 10:1 with the wild-type (SL1344) or ΔcopA ΔgolT strain of S. Typhimurium. Percent survival was calculated as the proportion of bacteria recovered at the indicated time points relative to the initial uptake of bacteria (mean ± SD; n = 5; Student t test). Note that the ΔcopA ΔgolT mutant exhibits reduced survival in wild-type but not Atp7a^LysMcre macrophages.

FIG 5

Copper export via CopA/GolT is required for S. Typhimurium survival in Atp7a^WT mice but not Atp7a^LysMcre mice. Equal amounts of wild-type (SL1344) and ΔcopA ΔgolT S. Typhimurium were intraperitoneally injected into Atp7a^WT or Atp7a^LysMcre mice. At 4 days postinfection, the competitive index was calculated in the liver and spleen, defined as the ratio of wild-type to ΔcopA ΔgolT bacteria recovered from each organ divided by the ratio of wild-type to ΔcopA ΔgolT bacteria injected (mean ± SD; n = 5; Student t test). Note the competitive advantage of wild-type S. Typhimurium over the ΔcopA ΔgolT strain in wild-type mice.