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

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.

Fig. 1.

Phagosomal escape of F. tularensis LVS occurs rapidly in murine BMMs. (A) Confocal microscopy images and quantitation of endocytic marker acquisition by phagosomes during early trafficking events. BMMs were infected with LVS for the indicated times, fixed, and processed for immunofluorescence using anti-Francisella and either EEA-1 or LAMP-1 antibodies. Colocalization of bacteria with either EEA-1 or LAMP-1 was scored for 100 bacteria per condition. Arrows indicate areas magnified in Insets. (Scale bars: 10 μm; Insets, 2 μm.) (B) Quantitation of Francisella escape into the cytoplasm. LVS-infected BMMs were processed for the phagosomal integrity assay, TEM, or LAMP-1 and Francisella immunofluorescence staining. Phagosomal escape was measured as the percentage of intracellular bacteria labeled after digitonin permeabilization (filled circles, cytoplasmic bacteria) or as the percentage of bacteria surrounded by degraded membranes (open squares, representative TEM analysis), and LAMP-1 colocalization with 100 bacteria per time point was scored (open circles). At least 50 bacteria per time point were analyzed by TEM in each experiment. (C) Representative TEM micrograph of an LVS-infected BMM at 1 h p.i. The bacterium is surrounded by degraded membranes, which indicates phagosomal disruption. (Scale bar: 0.5 μm.)

Fig. 2.

Intracellular Francisella become enclosed in large vacuoles after intracytoplasmic replication. (A) Confocal micrographs of an LVS-infected BMM at 24 h p.i., subjected to the phagosomal integrity assay. Cytoplasmic bacteria (red and green, appearing yellow in the overlay) are labeled after digitonin permeabilization, whereas clustered bacteria are detected only after saponin permeabilization (red). Calnexin staining (blue) allows detection of digitonin-permeabilized cells. (B) Confocal micrographs of an LVS-infected BMM at 24 h p.i. BMMs were infected with LVS, fixed, and processed for immunofluorescence with Francisella LPS and LAMP-1 antibodies. Bacterial clusters (green) are enclosed in LAMP-1-positive, membrane-bound compartments (red) termed Francisella-containing vacuoles (FCVs). Arrows indicate FCVs. (Scale bars: 10 μm.) (C) Kinetics of intracellular replication and FCV formation. BMMs were infected with LVS for the indicated times. Intracellular bacteria were enumerated from cfus, and FCV formation was measured as the percentage of infected cells harboring LAMP-1-positive FCVs. (D) Effect of inhibition of bacterial protein synthesis on FCV formation and replication. BMMs were infected with LVS and left untreated or treated at 14 h p.i. with 10 μg/ml chloramphenicol (indicated by arrow), and FCV formation (filled shapes) or intracellular growth (open shapes; cfu) was measured. The asterisk indicates statistically significant differences between control and chloramphenicol-treated BMMs at 28 h p.i. (P < 0.05, two-tailed unpaired Student’s t test).

Fig. 3.

Flow-cytometry-based quantitation of cytoplasmic and vacuolar Francisella at 16 and 24 h p.i. BMMs were infected with GFP-expressing LVS, and cytoplasmic bacteria were labeled using AlexaFluor 647-conjugated anti-Francisella antibodies after digitonin permeabilization. (A) Negative control of AlexaFluor 647 labeling of cytoplasmic, GFP-expressing bacteria when BMMs infected for 24 h were processed without digitonin permeabilization. (B) Positive control of AlexaFluor 647 labeling of GFP-expressing bacteria recovered from BMMs infected for 24 h. The majority (92%) were labeled after total lysis. (C) Analysis of GFP-expressing bacteria recovered from BMMs infected for 16 h, showing that the majority (81%) were labeled with Alexa Fluor 647 and hence are cytoplasmic. (D) Analysis of GFP-expressing bacteria recovered from BMMs infected for 24 h, showing that the majority (72%) were not labeled with AlexaFluor 647 and hence are vacuolar. Percentages shown in red refer to the proportions of vacuolar bacteria. Data are from one experiment representative of three.

Fig. 4.

FCVs are double membrane-bound vacuoles containing intact bacteria. BMMs were infected with LVS and processed for TEM at 24 h p.i. (A and B) TEM micrographs showing individual bacteria or groups of bacteria enclosed by double membranes (indicated by arrows). (C and D) Magnifications of the boxed areas in A and B, respectively, showing double membranes (arrows) surrounding bacteria. (E) Ultrastructure of a typical FCV showing clustered, intact bacteria enclosed by a membrane. (Scale bars: A, B, and E, 0.5 μm; C and D, 0.2 μm.)

Fig. 5.

FCVs display autophagic features, and their biogenesis requires autophagy. (A) LVS-infected BMMs were labeled with MDC (blue) before fixation at 24 h p.i. and immunofluorescence staining of Francisella (green) and LAMP-1 (red). (B) BMMs were transduced to express GFP-LC3 or GFP-LC3^{ΔC22, G120A} (GFP-LC3ΔC22) and infected with LVS for 24 h p.i. before fixation and immunostaining of Francisella (blue), GFP (green), and LAMP-1 (red). Insets are single-channel fluorescence images of FCVs. Arrows indicate FCVs. (C) Quantitation of MDC accumulation and recruitment of GFP-LC3 or GFP-LC3^{ΔC22, G120A} on LAMP-1-positive FCVs at 24 h p.i. For each condition, 100 FCVs were scored per experiment. (D) Effect of autophagy inhibition on FCV formation. (Left) LVS-infected BMMs were treated with 5 mM 3-MA at 14 h p.i., and FCV formation was scored at 24 h p.i. As a positive control for autophagy inhibition, uninfected, GFP-LC3-expressing BMMs were left untreated or pretreated with 3-MA for 1 h, then starved for 4 h to induce autophagosome formation. (Right) Autophagy was then scored as the percentage of cells containing GFP-LC3-positive vesicles. Asterisks indicate data significantly different from untreated controls (P < 0.05, two-tailed unpaired Student’s t test).