Autophagy-mediated reentry of Francisella tularensis
into the endocytic compartment after
Claire Checroun*, Tara D. Wehrly*, Elizabeth R. Fischer†, Stanley F. Hayes†, and Jean Celli*‡
*Tularemia Pathogenesis Section, Laboratory of Intracellular Parasites, and†Microscopy Core Unit, Rocky Mountain Laboratories, National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
Edited by E. Peter Greenberg, University of Washington School of Medicine, Seattle, WA, and approved August 3, 2006 (received for review March 6, 2006)
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 macro-
phages the intracellular life cycle of Francisella tularensis, a highly
infectious bacterium that survives and replicates within mamma-
lian 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 subse-
quently 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
results demonstrate that, via autophagy, F. tularensis reenters the
endocytic pathway after cytoplasmic replication, a process thus far
undescribed for intracellular pathogens.
macrophage ? pathogenesis ? tularemia ? trafficking
lian cells, in order to survive and multiply intracellularly (1, 2).
The two canonical strategies used by these pathogens are (i)
physical escape from the degradative endocytic compartments
by means of lysis of the phagosomal membrane and replication
within the cytoplasm, often accompanied by actin-based motil-
ity, as exemplified by Listeria, Shigella, or Rickettsia spp.; or (ii)
maintenance inside a pathogen-tailored, membrane-bound com-
partment stalled along, or segregated from, the endocytic com-
partment, as is the case for Salmonella, Mycobacterium, Chla-
mydia, Brucella, or Legionella spp. (1–3).
Francisella tularensis is a Gram-negative, highly infectious,
facultative intracellular pathogen that causes tularemia, a zoo-
notic disease affecting humans and other mammals with signif-
icant mortality (4). Given its high infectivity and lethality, F.
tularensis has raised concerns as a potential bioterrorism agent;
however, little is known about its pathogenesis. F. tularensis is
capable of infecting various mammalian cell types, among which
to the virulence of this bacterium. Early studies in rodent
inside a phagosome that does not fuse with lysosomes and whose
acidification is essential for intracellular survival (5, 6). How-
ever, recent evidence has challenged these results by demon-
strating the phagosomal escape of virulent and attenuated
Francisella strains into the cytoplasm of various murine or
ntracellular bacterial pathogens have devised various strate-
gies for circumventing the microbicidal functions of mamma-
human primary macrophages or macrophage-like cells, followed
by bacterial replication (7–9). Phagosomal escape occurs 2–4 h
postinfection (p.i.), suggesting some early interactions with the
endocytic pathway. After replication, bacterial egress is thought
to occur via the induction of programmed cell death (4, 10). It
has also recently been proposed that Francisella-induced cell
death is an innate immune macrophage response to cytoplasmic
bacteria aimed at restricting bacterial multiplication (11). Here,
we have investigated Francisella interactions with the endocytic
compartment in a synchronized infection model of murine
limited interactions with the endocytic pathway, phagosomal
disruption occurs rapidly. Moreover, we describe a postreplica-
tion stage of Francisella intracellular trafficking whereby bacte-
ria reenter the endocytic compartment via an autophagy-
mediated process to reside in large fusogenic vacuoles. This
finding represents a unique trafficking event for an intracellular
bacterium and suggests that bacterial pathogens can cycle
through different host cell compartments during their intracel-
Results and Discussion
Francisella Rapidly Disrupts Its Phagosomal Membrane to Access the
Macrophage Cytoplasm. To examine early interactions of Fran-
cisella with the endocytic compartment of murine BMMs, we
synchronized bacterial uptake by BMMs as described in Mate-
rials and Methods. Five minutes after infection, intracellular
bacteria colocalized with the early endosome marker early
endosome antigen-1 (EEA-1; Fig. 1A), indicating interactions
with early endosomes. Such interactions were transient and were
rapidly followed by the acquisition of the late endosomal?
lysosomal marker lysosomal-associated membrane protein 1
(LAMP-1; Fig. 1A), indicating a progressive maturation of the
Francisella-containing phagosome along the endocytic pathway.
The percentage of LAMP-1-positive phagosomes peaked at 20
min p.i. (52 ? 5.7%; all data are given as mean ? SD) and then
decreased to 7.7 ? 2.9% at 60 min p.i. (Fig. 1A), suggesting
significant phagosomal escape by 60 min p.i., earlier than
previously reported in murine (7) or human (8) phagocytes. To
determine whether the loss of LAMP-1 colocalization with
Author contributions: C.C. and J.C. designed research; C.C., T.D.W., E.R.F., S.F.H., and J.C.
performed research; C.C., E.R.F., S.F.H., and J.C. analyzed data; and C.C. and J.C. wrote the
The authors declare no conflict of interest.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: BMM, bone marrow-derived macrophage; FCV, Francisella-containing vac-
uole; LAMP-1, lysosomal-associated membrane protein 1; LVS, live vaccine strain; MDC,
monodansylcadaverine; p.i., postinfection; TEM, transmission electron microscopy; 3-MA,
‡To whom correspondence should be addressed at: Laboratory of Intracellular Parasites,
Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Na-
tional Institutes of Health, 903 South Fourth Street, Hamilton, MT 59840. E-mail:
September 26, 2006 ?
vol. 103 ?
intracellular bacteria was due to phagosomal disruption, we
developed a fluorescence microscopy assay of phagosomal in-
tegrity, based on the sequential use of digitonin and saponin to
differentially label bacteria that are cytoplasmic or within a
compromised phagosome and those enclosed within an intact
phagosome (see Supporting Materials and Methods, which is
published as supporting information on the PNAS web site). In
36 ? 5.7% and 95 ? 3.1% of intracellular bacteria were
detectable at 20 min and 60 min p.i., respectively, by cytoplas-
mically delivered antibodies (Fig. 1B; and Fig. 6E, which is
published as supporting information on the PNAS web site),
indicating that these bacteria had significantly escaped from
their phagosome or compromised their phagosomal membrane.
Consistently, infected BMMs analyzed by transmission electron
microscopy (TEM) contained an increasing percentage of bac-
teria surrounded by degraded membranes from 30 min to 2 h p.i.
(55% at 30 min, 75% at 1h, and 90% at 2 h p.i.; Fig. 1 B and C).
Most phagosomes displayed ?75% of degraded membranes by
1 h p.i. This finding demonstrates that F. tularensis LVS rapidly
disrupts its phagosome after uptake by murine BMMs and
reaches the cytoplasm. Thereafter (Fig. 1B and data not shown),
?95% of the intracellular bacteria were cytoplasmic, and rep-
lication occurred from 4 h onward, as reported previously (7–9).
Intracellular Francisella Localize to Large Vacuoles After Intracyto-
plasmic Replication. While examining the fate of replicating
cytoplasmic bacteria, we unexpectedly detected clusters of bac-
teria that were not accessible to cytoplasmically delivered anti-
bodies at 24 h p.i. (Fig. 2A). Immunostaining of infected BMMs
for the late endosomal?lysosomal marker LAMP-1 revealed that
these bacterial clusters were surrounded by a LAMP-1-positive
membrane (Fig. 2B), demonstrating that bacteria were enclosed
within large endocytic vacuoles. These organelles, named ‘‘Fran-
cisella-containing vacuoles’’ (FCVs), were juxtanuclear, hetero-
geneous in size (2–15 ?m in diameter) and numbers of enclosed
bacteria (data not shown) and were present in cells that also
contained cytoplasmic bacteria. Formation of FCVs increased
with time, with the percentage of FCV-containing BMMs reach-
ing 46 ? 8.1% at 24 h p.i. and 81 ? 8.4% at 36 h p.i. (Fig. 2C).
The kinetics and extent of FCV formation did not depend
significantly on the apparent moi used, although higher infection
rates favored FCV formation (Fig. 7, which is published as
supporting information on the PNAS web site). FCV formation
occurred after the net intracellular growth of Francisella (20 h
p.i.; Fig. 2C), suggesting that FCV formation is a postreplication
event requiring a specific intracellular bacterial load. Impor-
tantly, the virulent F. tularensis subsp. holarctica strain FSC200
and F. tularensis subsp. tularensis strain Schu S4 also formed
FCVs in murine BMMs, with comparable kinetics after cyto-
plasmic replication (Fig. 8, which is published as supporting
information on the PNAS web site), indicating that this phe-
nomenon is not due to the attenuation of LVS.
We next quantified the proportion of cytoplasmic and vacu-
olar bacteria recovered from infected BMMs by flow cytometry
analysis. In these experiments, digitonin permeabilization was
used to specifically label cytoplasmic bacteria, while all bacteria
were detectable through GFP expression, as described in Mate-
rials and Methods. We first controlled for the specificity of
labeling cytoplasmic bacteria. In the absence of cytoplasmic
delivery of Alexa Fluor 647-conjugated anti-Francisella antibod-
ies, no intracellular bacteria were labeled (Fig. 3A), whereas the
majority of intracellular bacteria (92%) were labeled after BMM
lysis (Fig. 3B). At 16 h p.i., 81% of intracellular GFP-expressing
LVS were detected by cytoplasmically delivered Alexa Fluor
647-conjugated anti-Francisella antibodies (Fig. 3C) and hence
were cytoplasmic, in agreement with the low frequency of FCV
were inaccessible to cytoplasmically delivered antibodies at 24 h
p.i. (Fig. 3D) and hence were vacuolar, demonstrating that the
majority of intracellular Francisella are located within FCVs at
this time point.
To assess whether FCV formation requires metabolically
active bacteria, we blocked bacterial protein synthesis by adding
chloramphenicol at 14 h p.i. and examined FCV formation
thereafter. Chloramphenicol treatment stopped bacterial mul-
tiplication without significant killing (Fig. 2D) and significantly
prevented FCV formation (22 ? 6.1% at 28 h p.i.) compared
with untreated BMMs (64 ? 7.1%, P ? 0.01; Fig. 2D). This
phenomenon was reversible, inasmuch as bacterial replication
and FCV formation resumed when chloramphenicol was chased
BMMs. (A) Confocal microscopy images and quantitation of endocytic marker
acquisition by phagosomes during early trafficking events. BMMs were in-
fected with LVS for the indicated times, fixed, and processed for immunoflu-
orescence using anti-Francisella and either EEA-1 or LAMP-1 antibodies. Co-
localization of bacteria with either EEA-1 or LAMP-1 was scored for 100
bacteria per condition. Arrows indicate areas magnified in Insets. (Scale bars:
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 digi-
tonin permeabilization (filled circles, cytoplasmic bacteria) or as the percent-
age of bacteria surrounded by degraded membranes (open squares, repre-
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 ?m.)
Phagosomal escape of F. tularensis LVS occurs rapidly in murine
Checroun et al.
September 26, 2006 ?
vol. 103 ?
no. 39 ?
at 20 h p.i. (data not shown). Hence, bacterial protein synthesis,
or the full extent of replication, is required for FCV formation.
Induction of programmed cell death has been associated with
Francisella replication (10). To examine whether FCVs origi-
nated from phagocytosed bodies from neighboring Francisella-
infected BMMs that had undergone cell death, we measured cell
death in LVS-infected BMMs via the lactate dehydrogenase
release assay and the TUNEL assay. Under our infection
conditions, cell death was very low at 24 h p.i. (? 15%; Fig. 9,
which is published as supporting information on the PNAS web
site). We also quantitated FCV formation either in C57BL?6
BMMs treated with the pan-caspase inhibitor Z-VAD (Biomol,
Plymouth Meeting, PA) to inhibit programmed cell death or in
BMMs derived from ASC??? mice, which do not undergo
programmed cell death when infected with Francisella because
(11). In both conditions, FCV formation was not affected
compared with untreated C57BL?6 BMMs (Fig. 9). Collectively,
these results rule out an exogenous, cell-death-related origin for
Next, FCV ultrastructure was examined by TEM. At 24 h p.i.,
many individual bacteria or groups of bacteria were enclosed in
membrane-bound compartments (Fig. 4 A and B), a feature that
was not observed at earlier time points (data not shown). In most
cases, FCVs were large membrane-bound vacuoles filled with
undegraded bacteria (Fig. 4 B and E). Interestingly, these
compartments displayed double-membrane structures reminis-
cent of autophagic vacuoles (Fig. 4 C and D, arrows).
FCV Formation Requires Autophagy. Because the ultrastructure of
FCVs suggested an autophagic nature, we sought to determine
whether these organelles displayed additional autophagic fea-
tures. We first examined whether FCVs accumulated the auto-
phagic probe monodansylcadaverine (MDC). At 24 h p.i., 74 ?
8.9% of the LAMP-1-positive FCVs strongly labeled with MDC
(Fig. 5 A and C), indicating an autophagic origin for FCVs. To
confirm this result, we expressed in BMMs a GFP fusion with the
autophagosomal membrane-associated protein LC3 (12) that
specifically labels autophagosomes and examined GFP-LC3
recruitment on FCVs. GFP-LC3 was enriched on the majority of
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 detec-
tion 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. Bacte-
rial clusters (green) are enclosed in LAMP-1-positive, membrane-bound com-
partments (red) termed Francisella-containing vacuoles (FCVs). Arrows indi-
cate FCVs. (Scale bars: 10 ?m.) (C) Kinetics of intracellular replication and FCV
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 ?g?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 chloram-
phenicol-treated BMMs at 28 h p.i. (P ? 0.05, two-tailed unpaired Student’s t
Intracellular Francisella become enclosed in large vacuoles after
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
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
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.
Flow-cytometry-based quantitation of cytoplasmic and vacuolar
www.pnas.org?cgi?doi?10.1073?pnas.0601838103Checroun et al.
FCVs at 24 h p.i. (62 ? 9.6% of LAMP-1-positive FCVs; Fig. 5
B and C). GFP-LC3 enrichment was dependent on LC3 associ-
ation with membranes because the GFP-LC3?C22, G120Amutant
form, which cannot be processed and conjugated to autophago-
somal membranes (12), was not significantly recruited to FCVs
(8.8 ? 1.9%; Fig. 5 B and C). Taken together, these results
demonstrate the autophagic origin of FCVs. To extend these
findings, we examined the effect of autophagy inhibition on FCV
formation. Infected BMMs were treated at 14 h p.i. with 5 mM
3-methyladenine (3-MA), and FCV formation was analyzed
at 24 h p.i. In uninfected cells, such treatment inhibited
autophagosome formation by 76% upon amino acid starvation
(Fig. 5D). 3-MA significantly reduced FCV formation such that
only 18 ? 4.5% of BMMs contained FCVs, compared with
did not significantly affect the intracellular yield of bacteria (Fig.
10, which is published as supporting information on the PNAS
web site). Collectively, these results demonstrate that FCVs
display autophagic features and require autophagy for their
formation but are not involved in either intracellular prolifera-
tion or killing.
FCVs Interact with Late Endocytic?Lysosomal Compartments. Given
the presence of LAMP-1 on FCV membranes, we tested whether
FCVs are mature, fusogenic autolysosomes. With live cell im-
aging, 74 ? 2.9% of FCVs formed by GFP-expressing LVS at
24 h p.i. accumulated Alexa Fluor 568-dextran, a fluorescent
teria. 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 en-
closed 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
?m; C and D, 0.2 ?m.)
FCVs are double membrane-bound vacuoles containing intact bac-
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-
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-
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 un-
treated or pretreated with 3-MA for 1 h, then starved for 4 h to induce
autophagosome formation. (Right) Autophagy was then scored as the per-
centage 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).
FCVs display autophagic features, and their biogenesis requires
Checroun et al.
September 26, 2006 ?
vol. 103 ?
no. 39 ?
fluid-phase marker preloaded into lysosomes (Fig. 11 A and C,
which is published as supporting information on the PNAS web
site). Consistently, 76 ? 11% of FCVs contained the luminal
lysosomal hydrolase cathepsin D (Fig. 11 B and C), confirming
that FCVs fuse with lysosomes, and 73 ? 5.6% of FCVs also
accumulated the acidotropic probe LysoTracker Red DND-99
(Fig. 11 C and D), indicating that they are acidic organelles.
Hence, FCVs are fusogenic, matured autolysosomes.
Autophagy in mammalian cells has been associated with
innate defense mechanisms against intracellular pathogens, re-
trieving cytoplasmic bacteria for degradation (13, 14) or resum-
ing the maturation of phagosomes stalled along the endocytic
pathway (15). Predictably, some cytoplasmic pathogens, such as
Shigella, have evolved strategies to avoid autophagy (16),
whereas membrane-bound pathogens such as Legionella pneu-
abortus may take advantage of this mammalian process (17–20).
Here, we show that, after cytoplasmic replication, F. tularensis
reenters the endocytic pathway via the autophagic machinery of
primary murine macrophages. In addition to uncovering a
distinctive stage of the Francisella intracellular cycle, we dem-
onstrate that an intracellular pathogen with a cytoplasmic rep-
lication phase can subsequently resume interactions with the
FCV formation could be a host- or bacterial-driven process.
Given the role of autophagy in innate defense against intracel-
lular pathogens, FCVs could result from a macrophage response
to the intracellular bacterial load, aimed at restricting bacterial
multiplication. This hypothesis is, nonetheless, questioned by the
fact that autophagy is not induced earlier when Francisella
initially reach the cytoplasm, inasmuch as autophagy occurs
rapidly in response to other cytoplasmic bacteria, including
Group A Streptococcus (13), Salmonella (14), or Listeria ren-
dered metabolically inactive with chloramphenicol (21). In
contrast, chloramphenicol treatment of Francisella-infected
macrophages during (Fig. 2D) or before replication (4 h p.i., data
not shown) did not induce the autophagic uptake of bacteria.
Instead, Francisella remained cytoplasmic and intact for up to
16 h posttreatment (data not shown). Because FCV biogenesis
requires active bacterial protein synthesis and?or substantial
replication of bacteria, FCV formation could be a temporally
regulated, bacterially driven process. Francisella appears to
prevent a macrophage autophagic response before and during
bacterial replication. Once replication is completed, the macro-
phage autophagic response develops or is actively triggered by
formation. The biogenesis of such organelles, which is obviously
a significant event of the Francisella intracellular cycle, involves
the majority of bacteria and occurs with both attenuated and
highly virulent strains. Additionally, the report of bacteria
enclosed by double membranes within peritoneal cells of mice
infected with LVS (6) potentiates the significance of FCVs by
suggesting that they form in vivo.
The reentry of Francisella into the endocytic degradative
pathway after cytoplasmic replication is a unique phenomenon
in the trafficking of intracellular pathogens. Because FCVs
possess features of autolysosomes, intravacuolar Francisella
likely encounter a lysosomal environment. Yet no sign of
bacterial degradation was observed in most FCVs, suggesting
once it reenters the endocytic pathway. The role of FCVs in the
intracellular cycle and pathogenesis of Francisella remains to be
elucidated. FCV formation does not affect intracellular survival
and replication, suggesting that the vacuoles are not involved in
bacterial proliferation. As a postreplication stage, FCVs may
subject the bacteria to environmental cues required to induce a
interactions with the endocytic compartment, FCVs might also
provide intracellular Francisella with access to the macrophage
membrane trafficking functions, promoting bacterial egress
through exocytosis. Studies of the biogenesis and dynamics of
FCVs could address the role of these intracellular organelles in
Francisella pathogenesis. Given the increasing prominence of
autophagy in bacterial pathogenesis, future studies of FCVs may
also provide much-needed information about this important
Materials and Methods
Bacterial Strains and Culture Conditions. F. tularensis subsp. holarc-
tica strains LVS (ATCC 29684) and FSC200 (22) were obtained
from Francis Nano (University of Victoria, Victoria, BC, Can-
ada) and Anders Sjo ¨stedt (Umea University, Umea, Sweden),
respectively. F. tularensis subsp. tularensis strain Schu S4 (23) was
obtained from Rick Lyons (University of New Mexico, Albu-
querque, NM). To generate GFP-expressing bacteria, the plas-
mid pFNLTP6groE-gfp (24) was introduced by electroporation
into the LVS strain, as described in ref. 24. Bacteria were grown
on cysteine heart agar (Becton Dickinson, Sparks, MD) supple-
mented with 9% heated sheep blood (CHAB), and kanamycin
(10 ?g?ml) when required, for 3 days at 37°C in 7% CO2. For
infections with LVS, two to three fresh colonies were inoculated
into tryptic soy broth (Becton Dickinson) supplemented with
0.1% L-cysteine (TSB-C) and grown overnight at 37°C with
shaking to an OD600 ? 1.5. Virulent holarctica or tularensis
strains were scraped off freshly streaked CHAB plates and
resuspended in TSB-C before infection. To enumerate viable
intracellular bacteria, infected macrophages were lysed in sterile
distilled water, and serial dilutions were plated on CHAB plates.
Macrophage Culture and Infection. To generate BMMs, bone
marrow cells were collected from dissected femurs of 6- to
12-week-old C57BL?6 female mice (Harlan, Indianapolis, IN),
and macrophages were derived in 150-mm non-tissue-culture-
treated dishes, as described in ref. 25. After 5 days, loosely
adherent BMMs were washed with PBS, harvested by incubation
in chilled cation-free PBS on ice for 10 min, resuspended in
complete medium, and seeded onto 12-mm glass coverslips in
24-well plates (immunofluorescence, 1 ? 105per well) or
WillCo-dish glass-bottomed 35-mm dishes (live cell imaging, 1 ?
105per dish; WillCo Wells BV, Amsterdam, The Netherlands).
BMMs were further cultured for 2 days before infection. BMMs
derived from ASC??? knockout mice were kindly provided by
David Weiss and Denise Monack (Stanford University, Stan-
BMM infections were performed at an moi of 50 by centri-
fuging bacteria suspended in complete medium onto prechilled
macrophages at 400 ? g for 10 min at 4°C. BMMs were then
rapidly warmed to 37°C for 2 min in a water bath to trigger
phagocytosis and further incubated for a total of 20 min at 37°C
in 7% CO2. BMMs were extensively washed with DMEM to
remove extracellular bacteria and incubated for 40 min in
complete medium and then for 60 min in 100 ?g?ml gentamicin-
containing medium to kill the remaining extracellular bacteria.
Thereafter, infected BMMs were incubated in gentamicin-free
medium. Such conditions allowed for a synchronized entry of
bacteria, leading to 29 ? 3.8% of BMMs infected with one to two
bacteria (Fig. 7). At each time point, BMMs were washed three
icol (Sigma, St. Louis, MO) was added (10 ?g?ml). Autophagy
was induced by incubating cells in Hank’s balanced salt solution
(Mediatech, Herndon, VA) supplemented with 1 g?liter D-
glucose for 4 h to mimic amino acid starvation conditions.
Autophagy was inhibited by treating BMMs with 5 mM 3-MA
(Fluka?Sigma–Aldrich, St. Louis, MO), and the percentage of
GFP-LC3-expressing BMMs containing LC3-positive vesicles
www.pnas.org?cgi?doi?10.1073?pnas.0601838103Checroun et al.
Fluorescence Microscopy. BMMs were fixed with 3% paraformal-
dehyde in PBS, pH 7.4, for 10 min at 37°C and processed for
immunofluorescence staining as described in ref. 20. The pri-
mary antibodies used were mouse monoclonal anti-F. tularensis
LPS (US Biologicals, Swampscott, MA), rat monoclonal anti-
mouse LAMP-1 (1D4B; developed by J. T. August; obtained
from the Developmental Studies Hybridoma Bank; developed
under the auspices of the National Institute of Child Health and
Human Development; and maintained by the Department of
Biological Sciences, University of Iowa, Iowa City, IA), rabbit
polyclonal anticalnexin and mouse monoclonal antiprotein di-
Canada), rabbit polyclonal anti-human cathepsin D (provided by
Stuart Kornfeld, Washington University, St. Louis, MO), goat
polyclonal anti-EEA1 (N-19; Santa Cruz Biotechnologies, Santa
Cruz, CA), and rabbit polyclonal anti-GFP (Molecular Probes,
Eugene, OR). The secondary antibodies used were Alexa Fluor
488-conjugated and Alexa Fluor 568-conjugated (Molecular
Probes) and cyanin-5-conjugated (Jackson ImmunoResearch,
West Grove, PA) donkey anti-mouse, anti-rat, anti-rabbit, and
anti-goat antibodies. To label autophagosomes, BMMs were
incubated with 50 ?M MDC for 1 h, followed by a 30-min chase
before fixation. Samples were observed on either a Nikon
(Melville, NY) Eclipse E800 epifluorescence microscope for
quantitative analysis or a Carl Zeiss (Thornwood, NY) LSM 510
confocal laser scanning microscope for quantitative analysis and
image acquisition. MDC fluorescence was imaged by epifluo-
rescence using a DAPI filter and a Carl Zeiss Axiocam digital
camera mounted on the LSM 510 confocal microscope, con-
comitant with the confocal acquisitions of the other fluorescent
and assembled using Adobe Photoshop CS software (Adobe
Systems, San Jose, CA).
proportions of cytoplasmic and vacuolar Francisella, BMMs in
six-well plates (5 ? 105per well) were infected with GFP-
expressing LVS and washed with KHM buffer (110 mM potas-
sium acetate?20 mM Hepes?2 mM MgCl2, pH 7.3), and their
plasma membranes were selectively permeabilized with 50
?g?ml digitonin in KHM buffer for 1 min at room temperature.
After washing with KHM buffer, mouse anti-Francisella LPS
antibodies conjugated to Alexa Fluor 647 (Molecular Probes)
were specifically delivered to the macrophage cytoplasm (see
Supporting Materials and Methods) for 15 min at 37°C to label
lysed in water and lysates were centrifuged at 200 ? g for 5 min
to remove cellular debris. Supernatants containing all intracel-
lular bacteria were analyzed by using a FACSCalibur flow
cytometer (BD Biosciences, San Jose, CA) for GFP and Alexa
Fluor 647 fluorescence. Data were analyzed with FlowJo soft-
ware, version 6.3.2 (Tree Star, Ashland, OR). Under these
Alexa Fluor 647-positive, whereas vacuolar bacteria were only
GFP-positive. At least 90% of intracellular bacteria were GFP-
positive after a 24-h infection (data not shown), confirming that
the analysis was performed on the majority of the bacterial
TEM. Infected BMMs on 12-mm Aclar coverslips were processed
as described in ref. 26, except that BMMs were fixed for 24 h and
postfixed in 1% OsO4. Samples were viewed in a Hitachi (Tokyo,
Japan) H7500 TEM at 80 kV, fitted with a Hamamatsu (Bridge-
water, NJ) CCD camera C4742–95 and Advantage HR?HR-B
digital image software (AMT, Danvers, MA) for imaging. To
assess phagosomal membrane integrity, 50–100 phagosomes
were analyzed per condition in two independent experiments.
FCV ultrastructural analysis was performed on ?50 vacuoles in
three independent experiments.
Statistical Analyses. All data are given as mean ? SD from at least
three independent experiments, unless otherwise stated. Statis-
with Tukey posttest or an unpaired, two-tailed Student t test. A
P value ? 0.05 was considered significant.
We thank Leigh Knodler, Rey Carabeo, and Samantha Gruenheid for
critical reading of the manuscript; Francis Nano, Anders Sjo ¨stedt, Rick
Lyons, Tamotsu Yoshimori, Stuart Kornfeld, David Weiss, and Denise
Monack for providing strains, plasmids, antibodies and macrophages;
Holger Lorenz for technical advice on digitonin permeabilization; and
Ron Messer for advice and help with flow cytometry. This work was
of Health, National Institute of Allergy and Infectious Diseases.
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