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Since transmission ofFrancisella tularensisinto the mammalian host occurs via arthropod vectors such as ticks, mosquitoes, horseflies and deerflies, recent studies have establishedDrosophila melanogasteras an arthropod vector model system. Nothing is known about the intracellular fate ofF. tularensiswithin arthropod-derived cells, and the role of this host-parasite adaptation in the evolution of this pathogen to infect mammals. In this report, we explored intracellular trafficking ofF. tularensisssp.novicidainD. melanogaster-derived S2 cells. First, we show that similar to theF. tularensisssp.holarctica-derived LVS strain,F. tularensisssp.novicidais highly infectious, replicates exponentially within S2 cells and within adult flies, and is fatal to adult fruit flies in a dose-dependent manner, while theiglC, iglDandmglAmutants are defective. Using electron and fluorescence microscopy-based phagosome integrity assays, we show that the wild-type strain escapes into the cytosol of S2 cells within 30-60 min post infection and by 6 h, 90% were cytosolic. In contrast, approximately 40-50% of theiglCandiglDmutants escape into the cytosol by 6 h while the other subpopulation becomes enclosed within multilamellar vesicles (MLVs). Pre-treatment of S2 cells with the autophagy inhibitor methyl adenine blocks formation of the MLVs and all the vacuolar subpopulation of theiglCandiglDmutant bacteria become enclosed within single membrane-surrounded vacuoles. Endocytic trafficking studies ofF. tularensiswithin S2 cells show transient colocalization of the bacterial phagosome withD. melanogasterLAMP2-GFP fusion but not with lysosomes pre-loaded with fluorescent dextran. Our data show that MLVs harbouring theiglCmutant acquire Lamp2 and dextran while MLVs harbouring theiglDmutant exclude these late endosomal and lysosomal markers. Our data indicate crucial differences in the role of the pathogenicity island-encoded proteins in modulating intracellular trafficking within human macrophages and arthropod vector-derived cells.