Molecular bases of proliferation of Francisella tularensis in arthropod vectors

Abstract

Arthropod vectors are important vehicles for transmission of Francisella tularensis between mammals, but very little is known about the F. tularensis-arthropod vector interaction. Drosophila melanogaster has been recently developed as an arthropod vector model for F. tularensis. We have shown that intracellular trafficking of F. tularensis within human monocytes-derived macrophages and D. melanogaster-derived S2 cells is very similar. Within both evolutionarily distant host cells, the Francisella-containing phagosome matures to a late endosome-like phagosome with limited fusion to lysosomes followed by rapid bacterial escape into the cytosol where the bacterial proliferate. To decipher the molecular bases of intracellular proliferation of F. tularensis within arthropod-derived cells, we screened a comprehensive library of mutants of F. tularensis ssp. novicida for their defect in intracellular proliferation within D. melanogaster-derived S2 cells. Our data show that 394 genes, representing 22% of the genome, are required for intracellular proliferation within D. melanogaster-derived S2 cells, including many of the Francisella Pathogenicity Island (FPI) genes that are also required for proliferation within mammalian macrophages. Functional gene classes that exhibit growth defect include metabolic (25%), FPI (2%), type IV pili (1%), transport (16%) and DNA modification (5%). Among 168 most defective mutants in intracellular proliferation in S2 cells, 80 are defective in lethality and proliferation within adult D. melanogaster. The observation that only 135 of the 394 mutants that are defective in S2 cells are also defective in human macrophages indicates that F. tularensis utilize common as well as distinct mechanisms to proliferate within mammalian and arthropod cells. Our studies will facilitate deciphering the molecular aspects of F. tularensis-arthropod vector interaction and its patho-adaptation to infect mammals.

Fig 1. Functional groups of mutants defective in intracellular growth

The S2 cells were infected with mutants of F. tularensis subsp novicida at MOI of 10 for 1 h followed by 1 h of gentamicin treatment. Growth of the mutants was compared to the wild type strain at 24 h post-infection and the relative reduction in growth relative to the wild type strain was determined. Mutants were considered defective if they exhibited ≥10 fold reduction in growth. The growth defective mutants were grouped according to the function of the genes.

Fig 2. Distribution of functional categories of mutants defective for growth in S2 cells compared to mutants defective in both human U937 macrophages and S2 cells

Growth of the mutants was compared to the wild type strain and the relative reduction in cfu relative to the wild type strain at 24 h post-infection was determined. The mutants were divided into two groups depending on whether they showed growth defect in S2 cells only or both S2 cells and U937 macrophages. The defective mutants in each group were categorized according to the function of the genes.

Fig 3. Lethality to D. melanogaster by F. tularensis subsp novicida mutants

D. melanogaster was infected and survival curves of representative F. tularensis subsp novicida mutants are shown. Wild type F. tularensis subsp novicida U112 and its isogenic iglC mutant are positive and negative controls, respectively. Data are represented as a daily mean % survival of 20 flies divided into two groups of 10. Error bar are standard deviation (SD) of the two groups.