Molecular complexity orchestrates modulation of phagosome biogenesis and escape to the cytosol of macrophages by Francisella tularensis

Abstract

Upon entry of Francisella tularensis to macrophages, the Francisella-containing phagosome (FCP) is trafficked into an acidified late endosome-like phagosome with limited fusion to the lysosomes followed by rapid escape into the cytosol where the organism replicates. Although the Francisella Pathogenicity Island (FPI), which encodes a type VI-like secretion apparatus, is required for modulation of phagosome biogenesis and escape into the cytosol, the mechanisms involved are not known. To decipher the molecular bases of modulation of biogenesis of the FCP and bacterial escape into the macrophage cytosol, we have screened a comprehensive mutant library of F. tularensis ssp. novicida for their defect in proliferation within human macrophages, followed by characterization of modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data show that at least 202 genes are required for intracellular proliferation within macrophages. Among the 125 most defective mutants in intracellular proliferation, we show that the FCP of at least 91 mutants colocalize persistently with the late endosomal/lysosomal marker LAMP-1 and fail to escape into the cytosol, as determined by fluorescence-based phagosome integrity assays and transmission electron microscopy. At least 34 genes are required for proliferation within the cytosol but do not play a detectable role in modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data indicate a tremendous adaptation and metabolic reprogramming by F. tularensis to adjust to the micro-environmental and nutritional cues within the FCP, and these adjustments play essential roles in modulation of phagosome biogenesis and escape into the cytosol of macrophages as well as proliferation in the cytosol. The plethora of the networks of genes that orchestrate F. tularensis-mediated modulation of phagosome biogenesis, phagosomal escape and bacterial proliferation within the cytosol is novel, complex and involves an unusually large portion of the genome of an intracellular pathogen.

Fig. 1. Functional groups of mutants defective in intra-macrophage growth

U937 macrophages were infected with each of the mutants of F. tularensis 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 the number of cfu relative to the wild type strain was determined. After the primary screen, 425 mutants were tested twice in triplicate.

Fig. 2. Co-localization of the FCP of selected mutants with LAMP-1 in U937 macrophages

The cells were infected with each of the 125 mutants defective in intra-macrophage proliferation to determine co-localization of the phagosome with LAMP-1 at 6h post-infection. Bacteria were labeled with goat polyclonal antibody (green) and LAMP-1 was labeled with mouse monoclonal antibody (red). Representative confocal images of phagosomes harboring representative mutants from each functional group showing co-localization with Lamp-1 similar to the IglC mutant (A) or no co-localization, similar to the Sua5 WT-like phenotype (B). Data analyses were based on 100 infected cells analyzed from two different coverslips and the data were reproducible in two independent experiments.

Fig. 3. Functional categories of mutants that co-localize with LAMP-1

U937 macrophages were infected with each of the 125 mutants severely defective in intra-macrophage proliferation to determine co-localization of the phagosome with LAMP-1 at 6h post-infection. Mutants were grouped according to the function of the mutated genes. Percentages of mutants in each functional group that co-localized persistently with LAMP-1 are shown.

Fig. 4. Representative mutants analyzed for their escape into the cytosol of U937 macrophages

U937 macrophages were infected with each of the 125 mutants of F. tularensis defective in intra-macrophage proliferation. Cytosolic bacteria were labeled with goat polyclonal antibody (red) loaded into the macrophage cytosol followed by permeabilization of all cellular membranes and labeling of all intracellular bacteria using mouse monoclonal antibody (green). Representative confocal images of F. tularensis mutant defective in phagosomal escape similar to the iglC mutant (A) and mutants that exhibit wild type-like Sua5 phenotype (B). Data analyses were based on 100 infected cells analyzed from two different coverslips and the data were reproducible in two independent experiments.

Fig. 5. Ultra-structural characterization of phagosomal escape

U937 macrophages were infected with F. tularensis for 1 h followed by 1 h of gentamicin treatment. After a total of 6 h, the infected cells were analyzed by TEM to determine whether bacteria were within intact or disrupted phagosomes. A) Six mutants were selected randomly to determine their phagosomal escape; B) Five mutants with discrepancy in the results of the fluorescence-based phagosome integrity assays and LAMP-1 co-localization were examined by TEM for their phagosomal escape.