Restricted cytosolic growth of Francisella tularensis subsp. tularensis by IFN-{gamma} activation of macrophages

1 Tularemia Pathogenesis Section, Laboratory of Intracellular Parasites, Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA 2 Electron Microscopy Unit, Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA The intracellular bacterium Francisella tularensis ensures its survival and proliferation within phagocytes of the infected host through phagosomal escape and cytosolic replication, to cause the disease tularemia. The cytokine interferon- (IFN- ) is important in controlling primary infections in vivo , and in vitro intracellular proliferation of Francisella in macrophages, but its actual effects on the intracellular cycle of the bacterium are ambiguous. Here, we have performed an extensive analysis of the intracellular fate of the virulent F. tularensis subsp. tularensis strain Schu S4 in primary IFN- -activated murine and human macrophages to understand how this cytokine controls Francisella proliferation. In both murine bone marrow-derived macrophages (muBMMs) and human blood monocyte-derived macrophages (MDMs), IFN- controlled bacterial proliferation. Schu S4 growth inhibition was not due to a defect in phagosomal escape, since bacteria disrupted their phagosomes with indistinguishable kinetics in both muBMMs and MDMs, regardless of their activation state. Rather, IFN- activation restricted cytosolic replication of Schu S4 in a manner independent of reactive oxygen or nitrogen species. Hence, IFN- induces phagocyte NADPH oxidase Phox- and inducible nitric oxide synthase (iNOS)-independent cytosolic effector mechanisms that restrict growth of virulent Francisella in macrophages. Correspondence Jean Celli jcelli{at}niaid.nih.gov Abbreviations: FAC, ferric ammonium citrate; IFN- , interferon- ; iNOS, inducible nitric oxide synthase; LVS, live vaccine strain; MDM, human blood monocyte-derived macrophage; muBMM, murine bone marrow-derived macrophage; NAC, N -acetyl- L -cysteine; NMMLA, N 5-[imino(methylamino)methyl]- L -ornithine; PEC, peritoneal exudate cells; pi, post-infection; RNS, reactive nitrogen species; ROS, reactive oxygen species; TEM, transmission electron microscopy; TNF- , tumour necrosis factor These authors contributed equally to this work. Present address: Department of Microbiology, Ohio State University, Colu