Autophagy-mediated reentry of Francisella tularensis into the endocytic compartment after cytoplasmic replication

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Abstract
Intracellular bacterial pathogens evade the bactericidal functions of mammalian cells by physical escape from their phagosome and replication into the cytoplasm or through the modulation of phagosome maturation and biogenesis of a membrane-bound replicative organelle. Here, we detail in murine primary macrophages the intracellular life cycle of Francisella tularensis, a highly infectious bacterium that survives and replicates within mammalian cells. After transient interactions with the endocytic pathway, bacteria escaped from their phagosome by 1 h after infection and underwent replication in the cytoplasm from 4 to 20 h after infection. Unexpectedly, the majority of bacteria were subsequently found to be enclosed within large, juxtanuclear, LAMP-1-positive vacuoles called Francisella-containing vacuoles (FCVs). FCV formation required intracytoplasmic replication of bacteria. Using electron and fluorescence microscopy, we observed that the FCVs contained morphologically intact bacteria, despite fusing with lysosomes. FCVs are multimembranous structures that accumulate monodansylcadaverine and display the autophagy-specific protein LC3 on their membrane. Formation of FCVs was significantly inhibited by 3-methyladenine, confirming a role for the autophagic pathway in the biogenesis of these organelles. Taken together, our results demonstrate that, via autophagy, F. tularensis reenters the endocytic pathway after cytoplasmic replication, a process thus far undescribed for intracellular pathogens.