Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Dec;76(12):5488-99.
doi: 10.1128/IAI.00682-08. Epub 2008 Oct 13.

The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression

Audrey Chong  1 , Tara D Wehrly, Vinod Nair, Elizabeth R Fischer, Jeffrey R Barker, Karl E Klose, Jean Celli
Affiliations

The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression

Audrey Chong et al. Infect Immun. 2008 Dec.

Abstract

Francisella tularensis is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway, F. tularensis rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the Francisella intracellular cycle, we have characterized early trafficking events of the F. tularensis subsp. tularensis strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early Francisella-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further, Francisella pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Early intracellular trafficking of Schu S4 in BMMs and effect of inhibition of endosomal acidification. BMMs were left untreated or were treated with either BAF or ConA for 1 h prior to infection and infected with Schu S4. At various times p.i., samples were fixed and processed for immunolabeling of bacteria and either EEA-1 or LAMP-1. (A) Quantification of colocalization of intracellular Schu S4 with EEA-1. Data are means ± SD from three independent experiments. (B) Representative confocal micrographs of untreated or BAF-treated BMMs that were infected with Schu S4 for either 5 or 60 min. Bacteria (green) are within EEA-1-positive FCPs (red) at 5 min p.i. Arrows indicate bacteria of interest in whole images or EEA-1-positive FCPs in insets. Scale bars, 10 or 2 μm. (C) Quantification of colocalization of intracellular Schu S4 with LAMP-1. Data are means ± SD from three independent experiments. (D) Representative confocal micrographs of untreated or BAF-treated BMMs that were infected with Schu S4 for either 20 or 60 min. Bacteria (green) are within LAMP-1-positive FCPs (red) at 20 min p.i. in both untreated and BAF-treated cells and at 60 min p.i. in BAF-treated cells. Arrows indicate bacteria of interest in whole images or LAMP-1-positive FCPs in insets. Scale bars, 10 or 2 μm.
FIG. 2.
FIG. 2.
Early FCPs are acidified prior to phagosomal escape. BMMs were infected with either live or PFA-killed, GFP-expressing Schu S4 for various time periods and loaded with Lysotracker Red DND-99 before live cell imaging analysis. (A) Quantification of Lysotracker-positive FCPs at 20, 40, and 60 min p.i. Data are means ± SD from three independent experiments. Asterisks indicate a statistically significant difference (P < 0.01, two-tailed unpaired Student's t test). (B) Representative live-cell micrographs of either live or PFA-killed Schu S4-infected BMMs that have been loaded with Lysotracker Red DND-99. Live bacteria (green) colocalize with Lysotracker signals (red) at 20 but not at 60 min p.i., while PFA-killed bacteria colocalized with Lysotracker at both 20 and 60 min p.i. Arrows indicate bacteria of interest in whole images or Lysotracker-positive FCPs in insets. Scale bars, 10 or 2 μm.
FIG. 3.
FIG. 3.
Inhibition of endosomal acidification delays Schu S4 phagosomal escape. (A) Inhibition of endosomal acidification blocks early phagosomal disruption. BMMs were left untreated or were treated with either DMSO, BAF, or ConA for 1 h prior to infection, infected with Schu S4 (or PFA-killed Schu S4), and subjected at various times p.i. to a phagosomal integrity assay, as described in Materials and Methods. The percentage of cytoplasmically accessible bacteria was scored using epifluorescence microscopy. Data are means ± SD from three independent experiments. (B) Representative confocal micrographs of infected BMMs subjected to a phagosomal integrity assay at 60 min p.i. Calnexin staining (blue) indicates cytoplasmic delivery of antibodies to digitonin-permeabilized cells. Upper insets show bacterial staining following digitonin permeabilization (bacteria-DIG). Middle insets show total bacterial staining following saponin permeabilization (bacteria-SAP). Lower insets show overlays of fluorescence, where yellow bacteria are cytoplasmic and red bacteria are phagosomal. Live bacteria in either BAF- or ConA-treated cells remained phagosomal, like killed bacteria in untreated cells. Arrows indicate bacteria of interest. (C) Phagosomal escape of Schu S4 is only delayed by BAF pretreatment of BMMs. BMMs were left untreated or were treated with BAF for various time periods (−1 to 4 h p.i., green curve; −1 to 8 h p.i., red curve; 2 to 8 h p.i., blue curve), infected with Schu S4, and subjected at various times p.i. to a phagosomal integrity assay. The percentage of cytoplasmically accessible bacteria was scored using epifluorescence microscopy. Data are means ± SD from three independent experiments. (D) Phagosomal escape of Schu S4 is only delayed by ConA pretreatments of BMMs. BMMs were left untreated or were treated with ConA for various time periods (−1 to 4 h p.i., green curve; −1 to 8 h p.i., red curve; 2 to 8 h p.i., blue curve), infected with Schu S4, and subjected at various times p.i. to a phagosomal integrity assay. The percentage of cytoplasmically accessible bacteria was scored using epifluorescence microscopy. Data are means ± SD from three independent experiments.
FIG. 4.
FIG. 4.
Inhibition of phagosomal acidification delays degradation of the phagosomal membrane. BMMs were left untreated or were treated with either BAF or ConA for 1 h prior to infection, infected with Schu S4, and processed for transmission electron microscopy (TEM) at 30 min (A to C), 1 h (D to F) or 2 h (G to I) p.i. TEM micrographs show representative FCPs for all conditions. Insets show a magnification of boxed areas on the whole images. Small arrows indicate intact phagosomal membranes (B and C). Arrowheads indicate phagosomal membrane disruptions (E and F). Asterisks indicate a lack of phagosomal membrane (panels A, D, and G to I). Scale bars, 100 or 10 nm.
FIG. 5.
FIG. 5.
Inhibition of phagosomal acidification delays cytosolic replication of Schu S4. BMMs were left untreated or were treated with either BAF or ConA for various time periods (−1 to 4 h p.i., green curve; −1 to 12 h p.i., red curve; 2 to 12 h p.i., blue curve) and infected with Schu S4, and viable intracellular bacteria were enumerated at various times p.i. (A) Representative growth curves of Schu S4 in untreated or BAF-treated BMMs. Data are from a representative experiment, performed in triplicate, out of three independent experiments. (B) Representative growth curves of Schu S4 in untreated or ConA-treated BMMs. Data are from a representative experiment performed in triplicate out of three independent experiments. (C) Representative confocal micrographs of BMMs left untreated (upper panels) or treated with BAF from either 1 h prior to infection to 4 h p.i. (middle panels) or from 2 to 8 h p.i. (lower panels) and infected with Schu S4 before being subjected to a phagosomal integrity assay at 8 h p.i. Staining of cytoplasmically accessible bacteria (digitonin) appears in green, while staining of all bacteria (saponin) appears in red and calnexin staining in blue. Cytoplasmic bacteria appear yellow on the merged images, and phagosomal bacteria appear red. Scale bar, 10 μm. (D) Intracellular doubling times of Schu S4 in either untreated BMMs or BMMs that were pretreated with either BAF or ConA for various time periods. Doubling times were calculated between 8 and 12 h p.i., when intracellular growth was obvious under all conditions tested. Data are means ± SD from three independent experiments.
FIG. 6.
FIG. 6.
Inhibition of phagosomal acidification delays phagosomal escape of F. tularensis subsp. novicida. (A) Western blot analysis of IglC and IglD expression in F. tularensis subsp. novicida U112, U112 ΔiglC::ermC, and U112 ΔiglC::ermC(piglCD). Bacteria grown in tryptic soy broth supplemented with 0.1% l-cysteine to an optical density at 600 nm of 0.6 were harvested, and lysates equivalent to 2 × 107 CFU were processed for immunoblotting using mouse monoclonal antibodies against either IglC or IglD. (B and C) BMMs were left untreated (B) or were treated with BAF (C) either from 1 h prior to infection to 8 h p.i. or from 2 to 8 h p.i., infected with either live or PFA-killed U112 or U112 ΔiglC::ermC or U112 ΔiglC::ermC(piglCD), and subjected at various times p.i. to a phagosomal integrity assay. The percentage of cytoplasmically accessible bacteria was scored using epifluorescence microscopy. Data are means ± SD from three independent experiments. (D) Representative transmission electron microscopy micrographs of untreated BMMs infected with either U112 (wild type, upper panel) or U112 ΔiglC::ermC (lower panel) for 8 h. The wild-type bacterium is not surrounded by any visible membrane, while the iglC mutant is contained within a phagosome that shows distinct foci of membrane disruption (arrows). Scale bar, 500 nm.
FIG. 7.
FIG. 7.
FPI protein expression is induced early but does not depend upon FCP acidification. Untreated or BAF-treated BMMs were infected with Schu S4, and samples were processed for CFU enumeration and Western blotting at 0, 1, 2, and 4 h p.i., as described in Materials and Methods. Sample loading for SDS-PAGE was normalized to CFU. CFU equivalents loaded per lane were as follows: 1.4 × 106 for IglC analysis in untreated BMMs, 7.5 × 105 for IglC analysis in BAF-treated BMMs, and 6.3 × 105 for PdpC analysis in both untreated and BAF-treated BMMs. (A) Intrabacterial accumulation of IglC in untreated or BAF-treated BMMs. (B) Intrabacterial accumulation of PdpC in untreated or BAF-treated BMMs. Western blots are representative of three independent experiments, and relative band intensities are means ± SD from three independent experiments. Asterisks indicate a statistically significant difference from results at time zero (one-way ANOVA followed by Bonferroni's posthoc test, P < 0.05). “(−)” indicates an untreated, uninfected sample.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Beauregard, K. E., K. D. Lee, R. J. Collier, and J. A. Swanson. 1997. pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes. J. Exp. Med. 1861159-1163. - PMC - PubMed
    1. Boschiroli, M. L., S. Ouahrani-Bettache, V. Foulongne, S. Michaux-Charachon, G. Bourg, A. Allardet-Servent, C. Cazevieille, J. P. Liautard, M. Ramuz, and D. O'Callaghan. 2002. The Brucella suis virB operon is induced intracellularly in macrophages. Proc. Natl. Acad. Sci. USA 991544-1549. - PMC - PubMed
    1. Checroun, C., T. D. Wehrly, E. R. Fischer, S. F. Hayes, and J. Celli. 2006. Autophagy-mediated reentry of Francisella tularensis into the endocytic compartment after cytoplasmic replication. Proc. Natl. Acad. Sci. USA 10314578-14583. - PMC - PubMed
    1. Cirillo, D. M., R. H. Valdivia, D. M. Monack, and S. Falkow. 1998. Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Mol. Microbiol. 30175-188. - PubMed
    1. Clague, M. J., S. Urbe, F. Aniento, and J. Gruenberg. 1994. Vacuolar ATPase activity is required for endosomal carrier vesicle formation. J. Biol. Chem. 26921-24. - PubMed

Publication types

LinkOut - more resources

-