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Blackwell Munksgaard
# 2008 The Authors
Journal compilation # 2008 Blackwell Publishing Ltd
doi: 10.1111/j.1600-0854.2008.00718.x
Brucella Intracellular Replication Requires Trafficking
Through the Late Endosomal/Lysosomal
Compartment
Tregei Starr, Tony W. Ng, Tara D. Wehrly,
Leigh A. Knodler and Jean Celli*
Laboratory of Intracellular Parasites, Rocky Mountain
Laboratories, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Hamilton,
MT 59840, USA
*Corresponding author: Jean Celli, [email protected]
Upon entry into mammalian cells, the intracellular pathogen Brucella abortus resides within a membrane-bound
compartment, the Brucella-containing vacuole (BCV), the
maturation of which is controlled by the bacterium to
generate a replicative organelle derived from the endoplasmic reticulum (ER). Prior to reaching the ER, Brucella
is believed to ensure its intracellular survival by inhibiting
fusion of the intermediate BCV with late endosomes and
lysosomes, although such BCVs are acidic and accumulate the lysosomal-associated membrane protein (LAMP-1).
Here, we have further examined the nature of intermediate BCVs using confocal microscopy and live cell imaging.
We show that BCVs rapidly acquire several late endocytic
markers, including the guanosine triphosphatase Rab7
and its effector Rab-interacting lysosomal protein (RILP),
and are accessible to fluid-phase markers either delivered
to the whole endocytic pathway or preloaded to lysosomes, indicating that BCVs interact with late endosomes and lysosomes. Consistently, intermediate BCVs
are acidic and display proteolytic activity up to 12 h postinfection. Expression of dominant-negative Rab7 or overexpression of RILP significantly impaired the ability of
bacteria to convert their vacuole into an ER-derived
organelle and replicate, indicating that BCV maturation
requires interactions with functional late endosomal/
lysosomal compartments. In cells expressing dominantnegative Rab7[T22N], BCVs remained acidic, yet displayed decreased fusion with lysosomes. Taken together,
these results demonstrate that BCVs traffic along the
endocytic pathway and fuse with lysosomes, and such
fusion events are required for further maturation of BCVs
into an ER-derived replicative organelle.
Key words: Brucella, endocytic pathway, live cell imaging, pathogenesis, phagosome maturation, Rab7, vacuole
acidification
Received 30 September 2007, revised and accepted for
publication 31 January 2008, published online 18 March 2008
Bacteria of the genus Brucella are the etiological agent of
brucellosis, a worldwide zoonosis that affects a wide
variety of mammals including humans (1). Brucellae are
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intracellular pathogens that enter various cell types during
the infection process, including macrophages and epithelial cells (1). Once internalized, Brucella resides within
a membrane-bound compartment, the Brucella-containing
vacuole (BCV), a modified phagosome in which the bacterium survives and eventually proliferates. Brucella intracellular survival strategies have been elucidated from analysis
of BCV trafficking in either macrophage or epithelial cell
models (2–4). Based on immunofluorescence and electron
microscopy studies in both cell types, it is known that
newly formed BCVs interact with early compartments of
the endocytic pathway before acquiring and retaining the
late endosomal/lysosomal glycoprotein LAMP-1 for up to
12 h post-infection (p.i.) (2,4). Further maturation of BCVs
into replicative organelles is characterized by the progressive exclusion of LAMP-1 from the vacuolar membrane,
a process that is likely initiated when these vacuoles
intercept the secretory pathway at endoplasmic reticulum
(ER) exit sites (5), subsequently undergoing fusion with the
ER. This process leads to the biogenesis of a replicationpermissive ER-derived organelle (2,4) and is dependent
upon the functions of the Brucella type IV secretion
system VirB (2,3,6–8). In primary macrophages, monocytes and macrophage-like cells, the majority of internalized bacteria are killed during the first 4–8 h p.i. (2,9,10).
The small population of bacteria that survive this initial
killing event eventually replicate (2). This disparity in the
fate of intracellular Brucella has complicated interpretations of BCV trafficking in phagocytic cells. Although there
is evidence that replication-proficient bacteria traffic through
a LAMP-1-positive vacuole before reaching the ER (2), it
remains possible that these LAMP-1-positive BCVs contain
bacteria that will eventually be killed and the replicationproficient bacteria are contained within the small fraction of
LAMP-1-negative BCVs. Yet, a fraction of LAMP-1-positive
BCVs interact with the ER, while a small percentage of
LAMP-1-negative BCVs do not (2), suggesting that LAMP-1
is acquired by replication-proficient BCVs.
Despite the early and sustained accumulation of LAMP-1
on BCV membranes, the lack of detection of other late
endocytic markers on maturing BCVs has led to the proposal that BCVs have little to no interaction with late
endocytic compartments. Instead, it is believed that bacteria rapidly segregate themselves from the endocytic
pathway to avoid fusion with terminal lysosomes (2,4). In
support of this model, fluorescence microscopy studies
of fixed samples have shown that BCVs do not acquire
fluid-phase markers from preloaded lysosomes (9), and no
Brucella Trafficking Through Lysosomes
significant accumulation of lysosomal luminal enzymes,
such as cathepsin D, is detectable in BCVs in either
macrophages or epithelial cells (2–4). Additionally, electron
microscopy studies in the macrophage-like cell line
J774A.1 have concluded that BCVs do not significantly
fuse with gold-labeled BSA-loaded lysosomes nor do they
intercept incoming endocytic traffic (11). However, the
lack of detection of luminal antigens or fluid-phase markers
by fluorescence or electron microscopy after fixation and
permeabilization may simply result from the loss of soluble
antigens or markers during fixation procedures, as recently
demonstrated for fluorescent dextran in Salmonellacontaining vacuoles (12). In NIH3T3 fibroblast cells, BCV
trafficking and bacterial replication are affected by the
expression of the constitutively active allele of the small
guanosine triphosphatase (GTPase) Rab5, which controls
fusion with early endocytic compartments, but not by that
of the constitutively active Rab7 (Rab7[Q67L]), which
controls fusion with late endosomes and lysosomes (13).
However, expression of Rab7[Q67L] does not abrogate
the functionality of late endocytic compartments (14),
which could potentially skew the conclusions drawn from
these experiments. Altogether, the previous studies support a model of BCV trafficking through early endocytic
compartments, but because of the technical limitations
associated with these studies, a role for late endosomes/
lysosomes cannot be excluded.
The current model of BCV segregation from the endocytic
pathway does not account for the rapid and sustained
acidification of BCVs, which have a luminal pH similar
to that found in lysosomal compartments (pH 4–5)
(10,15,16), arguing for significant interactions with late
endosomal/lysosomal compartments. Moreover, BCV
acidification is required for bacterial survival and replication
(10,15) and has been linked to the intracellular induction of
the virB operon (17), suggesting that it is an essential trait
of maturing BCVs. Hence, the available data on the nature
of intermediate BCVs and their interactions with intracellular compartments are ambiguous and deserve further
investigation.
Here, we have used a combination of immunofluorescence microscopy and live cell imaging to further characterize the nature of intermediate BCVs and investigate
their potential interactions with late endocytic compartments in established models of Brucella infection of
murine bone marrow-derived macrophages (BMMs) and
HeLa cells. We show that BCVs acquire various late
endocytic markers, including Rab7 and its effector Rabinteracting lysosomal protein (RILP), as well as fluid-phase
markers preloaded into lysosomes, demonstrating that
BCVs traffic along the endocytic pathway and fuse with
lysosomes. More importantly, we show that BCV trafficking and bacterial replication are affected when the functionality of late endocytic compartments is compromised,
demonstrating that fusion with these compartments
during the intermediate stages of vacuolar maturation is
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requisite to trafficking to the ER and subsequent intracellular replication of the bacteria.
Results
BCV acidification is required for proper trafficking
and replication in macrophages and HeLa cells
A key process in the maturation of a newly formed
phagosome into a degradative phagolysosome is its progressive acidification through sequential interactions with
compartments of the endocytic pathway (16). The rapid
acidification of phagosomes containing Brucella suis and
its importance for Brucella survival and eventual replication
in J774A.1 macrophage-like cells (10,15) prompted us to
further examine the role of BCV acidification in Brucella
abortus intracellular trafficking. Using a model of murine
BMM infection with B. abortus (2), we first tested the
effect of blocking phagosomal acidification on Brucella
intracellular survival. BMMs were left untreated or treated
with the vacuolar adenosine triphosphatase (ATPase)
inhibitor bafilomycin A1 (BAF), infected with the virulent
B. abortus strain 2308 and intracellular viable bacteria were
quantified at various times p.i. The number of intracellular
viable bacteria in untreated BMMs initially decreased,
consistent with previous observations (2), and then
increased from 12 to 24 h as a result of bacterial replication
(Figure 1A,C). When BAF was added to BMMs 1 h prior to
infection and maintained up to 8 h p.i., the number of
recoverable bacteria decreased over 24 h, indicating that
internalized bacteria did not survive in BAF-treated BMMs
(Figure 1A,C). By contrast, treatment of infected BMMs
with BAF from 8 to 24 h p.i. did not significantly affect
the ability of intracellular Brucella to survive and replicate,
as evidenced from the kinetics of intracellular replication
in these cells or untreated BMMs (Figure 1A). Taken together, these results confirm and extend previous observations (15) that BCV acidification is essential to B. abortus
survival and replication and is required during early, but not
late, trafficking events in phagocytic cells.
To expand upon these findings, we examined the vacuolar
trafficking of B. abortus in BAF-treated BMMs by monitoring the presence of LAMP-1 on BCVs. As previously
reported (2), LAMP-1 was rapidly acquired and then progressively excluded from BCVs in untreated cells (Figure
1B,C), concomitant with bacterial replication. By contrast,
LAMP-1 in BAF-treated BMMs was retained on BCVs up to
24 h p.i., even after removal of the drug (Figure 1B,C),
indicating that BCVs were unable to mature into ERderived, replicative organelles (2). Hence, inhibition of
phagosomal acidification during the early stages of BCV
maturation leads to a defect in trafficking of bacteria
toward the ER. As a consequence, bacteria are degraded
within LAMP-1-positive BCVs.
The acidification of BCVs in macrophage-like cells (10,15)
suggests a sustained interaction of early vacuoles with late
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Figure 1: Early acidification of BCVs is required for proper trafficking, survival and replication of Brucella in BMMs. A) Effect of
inhibition of BCV acidification upon bacterial survival and replication. BMMs were left untreated (open circles), treated with 100 nM BAF
from 1 h prior to infection to 8 h p.i. (closed squares) or from 8 to 24 h p.i. (open squares), infected with Brucella abortus 2308 and the
viability of intracellular bacteria was monitored over 24 h by enumeration of colony-forming units (CFU). Data are means SD from
a representative experiment performed in triplicate. B) Effect of inhibition of BCV acidification upon BCV maturation. BMMs were left
untreated (open circles) or treated with 100 nM BAF from 1 h prior to infection to 8 h p.i. (closed circles), infected with DsRedm-expressing
B. abortus 2308 and processed for immunostaining of LAMP-1. BCV maturation was monitored over 24 h by measuring colocalization of
bacteria with LAMP-1 using confocal microscopy. Data are means SD from three independent experiments. C) Representative confocal
micrographs of untreated (control) or BAF-treated BMMs infected with DsRedm-expressing B. abortus 2308 at 4 h p.i. (left-hand panels)
and 24 h p.i. (right-hand panels). LAMP-1 was detected using Alexa Fluor 488-conjugated secondary antibodies (shown in green), and
DsRedm-expressing bacteria appear in red. Arrows indicate BCVs in whole and close-up images. Scale bars are 10 and 2 mm.
compartments of the endocytic pathway, consistent with
the demonstrated accumulation of LAMP-1 on BCV membranes for up to 12 h p.i. (Figure 1B) (2–4). To test this
hypothesis, we decided to further examine vacuolar trafficking of Brucella. Because of the dual intracellular fate
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of Brucella in phagocytes, a macrophage infection model
is not appropriate to study the vacuolar trafficking of
replication-proficient bacteria, which constitute only a small
fraction of the intracellular organisms. To circumvent
this caveat, we used the established model of Brucella
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Brucella Trafficking Through Lysosomes
infection of the HeLa epithelial cell line (3–5,8,13,18), first
examining the bactericidal activities of these cells
toward internalized Brucella. In contrast to BMMs, HeLa
cells did not show any significant bactericidal activities
during the first 12 h p.i. (Figure 2A), indicating that this
model allows for the analysis of only replication-proficient
trafficking events. As in BMMs (Figure 1A), treatment of
HeLa cells with BAF prior to infection prevented Brucella
intracellular growth, while BAF treatment after 8 h p.i. did
not show any significant effect on bacterial replication
(Figure 2B). However, HeLa cells failed to kill bacteria in
BAF-pretreated cells because intracellular viable numbers
remained unchanged over 24 h (Figure 2B). Compared
with untreated cells where BCVs initially acquired and then
excluded LAMP-1, BCVs in BAF-pretreated HeLa cells
remained LAMP-1 positive and bacterial replication was
inhibited (Figure 2C,D). Hence, inhibition of phagosomal
acidification in HeLa cells recapitulated the effects on
Brucella trafficking and replication observed in BMMs,
with the exception of the late bactericidal effect observed
in BMMs pretreated with BAF.
BCVs acquire multiple markers of late endosomes/
lysosomes during maturation
The above data suggested that perturbation of the endocytic pathway affected BCV trafficking and bacterial replication. To determine the level of interaction of BCVs with
the endocytic pathway, particularly late endocytic compartments, we next examined by confocal microscopy the
recruitment of various markers of late endocytic compartments to BCVs containing Brucella constitutively expressing a monomeric red fluorescent protein (DsRedm) at early
(2 h p.i.), intermediate (6 h p.i.) and late (24 h p.i.) stages of
the bacterial intracellular cycle. As previously reported,
BCVs were enriched in LAMP-1 at 2 and 6 h p.i. but then
excluded this marker (Figure 3A,D), concomitant with the
onset of replication (Figure 3D). Interestingly, BCVs also
accumulated, and then lost, the late endosomal/multivesicular body marker CD63, with kinetics similar to
LAMP-1 (Figure 3B,D), demonstrating that late endocytic
markers other than LAMP-1 are transiently recruited to the
BCV. Furthermore, in HeLa cells expressing a green fluorescent protein (GFP)–Rab7 fusion, we clearly detected the
vacuolar recruitment of Rab7 (Figure 3C) during early and
intermediate stages of BCV maturation but not during the
Brucella replication phase (Figure 3D). The kinetics of Rab7
accumulation and loss correlated with that of LAMP-1 and
CD63 (Figure 3D). In non-EGF-treated HeLa cells, LAMP-1
and Rab7 recruitment to BCVs followed the same kinetics
as that in EGF-treated cells (Figure S1), ruling out that
such trafficking events are because of the use of EGF to
promote Brucella entry into non-phagocytic cells. We
conclude that BCVs transiently accumulate numerous late
endocytic proteins during their early and intermediate
stages of maturation, implying that BCVs interact and fuse
with late endocytic compartments.
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BCVs are accessible to late endocytic traffic
during maturation
To further delineate the extent of BCV interactions with
the endocytic pathway, we used high-resolution live cell
confocal microscopy to assess whether BCVs are accessible to incoming endocytic traffic. HeLa cells were
preloaded overnight with Alexa FluorÒ 546-conjugated
dextran, infected with GFP-expressing Brucella in the
absence of dextran and then reincubated with dextran for
the remainder of the experiment. Under these conditions,
the entire endocytic pathway is labeled with dextran. We
found that early (3 h p.i.) and intermediate (6 h p.i.), but not
late (12 h p.i.), BCVs were dextran positive (Figure 4A,B
and data not shown; see also Movies S1 and S2), demonstrating the transfer of luminal content between endocytic
compartments and BCVs. Quantification of such fusion
events revealed that the majority of BCVs were accessible
to fluid-phase markers during early (3 h p.i.) and intermediate (6 h p.i.) trafficking stages, with 69 4.6 and
66 16% of BCVs being dextran positive at 3 and 6 h
p.i., respectively (Figure 4C). Thereafter, the percentage
of dextran-positive BCVs progressively decreased to
20 10% by 12 h p.i. At this stage in the intracellular
cycle, most bacteria were clustered in dextran-negative
replicative BCVs (Figure 4B,C). Hence, BCVs are accessible
to fluid-phase markers delivered from endocytic compartments during the early and intermediate stages of maturation but lose their fusogenicity with endocytic vesicles
concomitant with the biogenesis of the replicative vacuole.
Given our finding that BCVs transiently fuse with endocytic
compartments and recruit late endocytic markers, we next
reassessed whether BCVs also fuse with the lysosomal
compartment during maturation. For this purpose, HeLa
cells were loaded with Alexa Fluor 546-conjugated dextran
the day prior to infection and the loaded dextran was
chased overnight to lysosomes. Cells were then infected
and further incubated in the absence of fluorescent dextran.
We clearly detected dextran-positive BCVs interacting with
dextran-loaded lysosomal vesicles at 3 and 6 h p.i. (Figure
5A, arrowhead and data not shown; see also Movie S3 and
Figure S1). In fact, the majority of BCVs accumulated
dextran at 3 and 6 h p.i. (75 13 and 81 9% respectively; Figure 5D). This clearly demonstrates that Brucella
interacts with lysosomes and that lysosome fusion is not
prevented during early/intermediate BCVs maturation.
However, by 12 h p.i., most of the BCVs were replicative
and no longer dextran positive (27 6%; Figure 5B,D; see
also Movie S4). At this time-point, loaded lysosomes were
also seen to dock to, but not fuse with, BCVs (Figure 5B,
arrowhead). In conclusion, our live cell imaging studies
support a model of Brucella trafficking along the endocytic
pathway to the lysosomal compartment before establishing
a non-fusogenic, replication-permissive niche.
To independently confirm that BCVs interact with lysosomes, we examined whether they acquire lysosomal
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Figure 2: Early acidification of BCVs is required for proper trafficking, survival and replication of Brucella in HeLa cells. A)
Intracellular viability and replication of Brucella in murine BMMs and HeLa cells. BMMs (open circles) or HeLa cells (closed circles) were
infected with Brucella abortus 2308 as described in the Materials and Methods, and the viability of intracellular bacteria was monitored over
24 h by enumeration of CFU. Data are means SD from a representative experiment performed in triplicate. B) Effect of inhibition of BCV
acidification upon bacterial survival and replication. HeLa cells were left untreated (open circles), treated with 100 nM BAF from 1 h prior to
infection to 8 h p.i. (closed squares) or from 8 to 24 h p.i. (open squares), infected with B. abortus 2308 and the viability of intracellular
bacteria was monitored over 24 h by enumeration of CFU. Data are means SD from a representative experiment performed in triplicate.
C) Effect of inhibition of BCV acidification upon BCV maturation. HeLa cells were left untreated (open circles) or treated with 100 nM BAF
from 1 h prior to infection to 8 h p.i. (closed circles), infected with DsRedm-expressing B. abortus 2308 and processed for immunostaining
of LAMP-1. BCV maturation was monitored over 24 h from colocalization of bacteria with LAMP-1 using confocal microscopy. Data are
means SD from three independent experiments. D) Representative confocal micrographs of untreated (control) or BAF-treated HeLa
cells infected with DsRedm-expressing B. abortus 2308 at 4 h p.i. (left-hand panels) and 24 h p.i. (right-hand panels). LAMP-1 was detected
using Alexa Fluor 488-conjugated secondary antibodies (shown in green), and DsRedm-expressing bacteria appear in red. Arrows indicate
BCVs in whole and close-up images. Scale bars are 10 and 2 mm. CFU, colony-forming units.
properties such as proteolytic activity. For this purpose, we
used DQ-Red BSA, a fluorescent probe for proteolytic
activity based on BODIPY fluorescence dequenching upon
proteolysis of conjugated BSA (19). Cells were preloaded
with DQ-Red BSA overnight, infected in the presence of
the fluorescent probe and analyzed by live cell confocal
microscopy. In HeLa cells infected with GFP-expressing
Brucella, the majority of BCVs clearly displayed DQ-Red
fluorescence at 6 h p.i. (79 3.2% of positive BCVs;
Figure 5C, arrowhead and Figure 5D), demonstrating vacuolar proteolytic activities, while replicative BCVs at 12 h
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p.i. did not display significant fluorescence (12 6.5% of
positive BCVs; Figure 5C,D). Consistently, the percentage
of DQ-Red-positive BCVs paralleled that of vacuoles
acquiring dextran from lysosomes during the first 12 h
p.i. (Figure 5D). In addition, BCVs were acidic at 4 and 10 h
p.i. (Figure 9A), but not at 24 h p.i. during extensive
replication, consistent with previous results obtained in
J774A.1 cells (10,15).
To evaluate the extent of BCV fusion with lysosomes, we
quantified dextran accumulation in vacuoles containing
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Brucella Trafficking Through Lysosomes
Figure 3: Maturing BCVs acquire various late endocytic markers. A) Representative confocal micrographs of HeLa cells infected with
DsRedm-expressing Brucella abortus 2308 (red) and immunostained for A) LAMP-1 (green) and B) CD63 (green). HeLa cells were infected,
processed for immunofluorescence labeling at 2, 6 and 24 h p.i. and analyzed by confocal microscopy as described in the Materials and
Methods. C) Representative confocal micrographs of HeLa cells expressing GFP–Rab7 that were infected with DsRedm-expressing
B. abortus 2308 (appears in red) and immunostained for LAMP-1 using Cyanin 5-conjugated secondary antibodies (appears in blue). HeLa
cells were transfected with a derivative of pEGFP-C1 expressing GFP–Rab7, infected, processed for immunostaining at 2, 6 and 24 h p.i.
and analyzed by confocal microscopy as described in the Materials and Methods. Scale bars are 10 and 2 mm. D) Quantification of late
endocytic markers acquired by BCVs. Recruitment of LAMP-1 (open squares), CD63 (open circles) and GFP–Rab7 (closed circles) on BCVs
was scored by confocal microscopy at 2, 6, 12 and 24 h p.i. Arrows indicate the area magnified in the whole images or the positive BCVs in
the insets. Data are means SD from three independent experiments.
either live or heat-killed bacteria. The latter can be considered as a control for the maximal fusion with lysosomes.
At 3 h p.i., the average pixel intensity of dextran fluorescence in BCVs containing live Brucella was reduced by
nearly 70% compared with BCVs containing heat-killed
bacteria (Figure 5E), suggesting reduced delivery. This
indicates that BCV fusion with lysosomes, although evident, is limited and controlled by the bacteria. Altogether,
these results demonstrate that early and intermediate
BCVs (i) acquire various late endosomal/lysosomal proteins, (ii) are accessible to, but limit the delivery of, fluidphase markers from lysosomes and (iii) are acidic and
display proteolytic activities. This provides strong evidence that biogenesis of these bacterial vacuoles involves
transient interactions with the endocytic pathway, including lysosomes, prior to generating an ER-derived replicative niche.
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Perturbation of late endosome/lysosome functions
impairs BCV trafficking and bacterial replication
Having established that BCVs interact with the lysosomal
compartment prior to biogenesis of the ER-derived replicative organelle and bacterial replication, one obvious
question was to determine whether such trafficking
events are essential for biogenesis of the replicative
vacuole and bacterial proliferation. To address this, we
overexpressed mutant forms of Rab7 in HeLa cells and
examined their effects on BCV trafficking and bacterial
replication. In cells expressing the GTP-bound, constitutively active Rab7[Q67L], BCVs recruited both LAMP-1 and
Rab7[Q67L] at early (2 h p.i.) and intermediate (6 h p.i.)
stages of trafficking, and bacteria multiplied in a LAMP-1negative, Rab7[Q67L]-negative compartment at 24 h p.i.
(Figure 6A). This indicates that expression of the constitutively active Rab7 did not affect BCV trafficking or
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Figure 4: Intermediate, but not replicative, BCVs are accessible to fluid-phase markers. HeLa cells were preloaded with Alexa Fluor
546–dextran, infected with GFP-expressing Brucella abortus 2308 in absence of dextran and then incubated with dextran until analysis in
order to label all endocytic compartments. At 3, 6, 9 and 12 h p.i., cells were visualized by live cell confocal microscopy, and approximately
5-min long time-lapse recordings were performed at each time-point. A) Consecutive frames of a representative time-lapse recording of an
Alexa Fluor 546–dextran-loaded HeLa cell (appears in red) infected with GFP-expressing Brucella (appears in green) at 6 h p.i. The arrow
indicates a dextran-positive BCV. The arrowhead indicates a dextran-positive endocytic vesicle docking to and fusing with the BCV. Scale
bar, 1 mm. B) Consecutive frames of a representative time-lapse recording of an Alexa Fluor 546–dextran-loaded HeLa cell (appears in red)
infected with GFP-expressing Brucella (appears in green) at 12 h p.i. The arrow indicates a cluster of replicative bacteria within dextrannegative BCVs. Scale bar, 1 mm. C) Quantification of BCV accessibility to dextran, expressed as the percentage of dextran-positive BCVs
over time. Data are means SD from three independent experiments. The total numbers of BCVs analyzed per time-point were 361 at 3 h
p.i., 257 at 6 h p.i., 389 at 9 h p.i. and 438 at 12 h p.i.
replication and is consistent with previously described
results in NIH3T3 cells where intracellular growth was
not affected by Rab7[Q67L] expression (13). The kinetics
of Rab7[Q67L] recruitment to BCVs was indistinguishable
from that of wild-type Rab7 and LAMP-1 over time
(Figures 6B and 3D), confirming the recruitment of Rab7
to LAMP-1 BCVs during maturation. By contrast, the GDPbound, dominant-negative allele, Rab7[T22N], whose
expression affects Rab7-dependent membrane trafficking,
lysosome positioning and functions (14), was not recruited
to BCVs (Figure 6C,D), indicating that Rab7 recruitment to
BCVs is dependent upon its GTP-binding activity. Expression of Rab7[T22N] did not prevent LAMP-1 accumulation
on BCVs (Figure 6C,D), suggesting that LAMP-1 recruit684
ment to BCVs is Rab7 independent or that the residual
activity of endogenous Rab7 in the cell is sufficient for
LAMP-1 recruitment to BCVs. To extend these results and
confirm the relevance of Rab7 recruitment to BCVs, we
examined whether the Rab7 effector RILP (20,21) was
also recruited. In cells overexpressing a GFP–RILP fusion,
GFP–RILP clearly labeled maturing BCVs (Figure 6E and
Figure S2). Because RILP is only recruited to membranes
by GTP-bound Rab7 (20,21), this indicates the presence of
activated, endogenous active Rab7 on BCVs.
Interestingly, at 24 h p.i., Brucella remained enclosed
within LAMP-1-positive BCVs in a small but significant
fraction of cells expressing GFP–Rab7[T22N] and did not
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Figure 5: Intermediate, but not replicative, BCVs fuse with the lysosomal compartment. HeLa cells were preloaded with Alexa Fluor
546–dextran that was chased for 16 h to lysosomes before infection with GFP-expressing Brucella abortus 2308 was performed in
absence of dextran. At 3, 6, 9 and 12 h p.i., cells were visualized by live cell confocal microscopy, and approximately 5-min long time-lapse
recordings were performed at each time-point. A) Consecutive frames of a representative time-lapse recording of an Alexa Fluor 546–
dextran-loaded HeLa cell (appears in red) infected with GFP-expressing Brucella (appears in green) at 6 h p.i. The arrow indicates a dextranpositive BCV. The arrowhead indicates a dextran-positive lysosome docked to the BCV. Scale bar, 1 mm. B) Consecutive frames of
a representative time-lapse recording of an Alexa Fluor 546–dextran-loaded HeLa cell (appears in red) infected with GFP-expressing
Brucella (appears in green) at 12 h p.i. The arrow indicates a cluster of replicative bacteria within dextran-negative BCVs. The arrowhead
indicates a dextran-positive lysosome that docks to a BCV without delivering its content. Scale bar, 1 mm. C) Representative live cell
confocal micrographs of HeLa cells loaded with DQ-Red BSA and infected for 6 h (upper panels) or 12 h (lower panels) with GFPexpressing B. abortus 2308. Intermediate BCVs at 6 h p.i. accumulated DQ-Red fluorescence (arrowhead), while replicative BCVs at 12 h
p.i. did not. D) Quantification of BCV accessibility to lysosomal dextran and intravacuolar proteolytic activity, expressed as the percentage
of dextran- or DQ-Red-positive BCVs over time. Data are means SD from three independent experiments. The numbers of dextranpositive BCVs analyzed per time-point were 295 at 3 h p.i., 365 at 6 h p.i., 540 at 9 h p.i. and 838 at 12 h p.i. The numbers of DQ-Redpositive BCVs analyzed per time-point were 267 at 3 h p.i., 254 at 6 h p.i., 352 at 9 h p.i. and 374 at 12 h p.i. E) Quantification of
intravacuolar dextran accumulated in BCVs containing live or heat-killed Brucella. Values are normalized fluorescence pixel intensities and
are means SD from three independent experiments. Asterisk indicates a statistically significant difference (Student’s t-test, p < 0.01).
replicate (Figure 6C,D). This correlated with a significant
decrease in bacterial replication because 37 5.1% of
GFP–Rab7[T22N]-expressing cells were replication permissive compared with 50 3.4, 59 5.2 and 58 6.1% of
GFP-, GFP–Rab7- and GFP–Rab7[Q67L]-expressing cells,
respectively (Figure 6G; p < 0.01). Hence, perturbation of
Rab7-dependent functions affects BCV maturation and
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subsequent bacterial replication. Overexpression of GFP–
RILP induced the collapse of the late endosomal/lysosomal
compartment to a juxtanuclear location close to the microtubule-organizing center [MTOC; (20,21); Figure 6E]. This is
because of the increased recruitment of the dynein–
dynactin motor complexes to late endosomes/lysosomes
and their subsequent enhanced centripetal movements
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(21). In these cells, Brucella remained enclosed in LAMP-1positive, RILP-positive, clustered BCVs at 24 h p.i. and did
not display significant signs of replication (Figure 6E,F).
Only 25 5.3% of RILP-overexpressing cells supported
bacterial growth compared with 50 3.4% in GFPexpressing cells (Figure 6G; p < 0.01). It remains possible
that the dramatic displacement and aggregation of late
endosomal/lysosomal compartments because of RILP
overexpression prevents BCV conversion into a replicative
organelle. These results nonetheless indicate that perturbation of Rab7-dependent functions prevents proper trafficking of BCVs and subsequent bacterial replication.
Therefore, Brucella replication requires trafficking through
a functional late endosomal/lysosomal compartment to
successfully generate a replicative organelle.
To confirm these findings, we examined whether BCVs in
cells expressing dominant alleles of Rab7 or overexpressing RILP acquired ER membrane markers, a readout of
replicative organelle biogenesis (2,4,5). In untransfected or
Rab7[Q67L]-expressing cells, BCVs acquired calreticulin
from 12 h p.i. and onwards (Figure 7A,B), concomitant
with bacterial replication (Figure 7A), indicating that BCVs
had fully matured into an replication-permissive, ERderived vacuole. By contrast, BCVs in Rab7[T22N]- or
RILP-expressing cells never acquired calreticulin (Figure
7A,B), demonstrating that they did not convert into ERderived, replicative organelles. Therefore, perturbation of
Rab7-dependent functions prevents biogenesis of the
Brucella replicative organelle.
Rab7-dependent functions are required for BCV
maturation in RAW264.7 cells
To confirm in phagocytic cells the observations made in
HeLa cells, we examined Rab7 recruitment to BCVs and
the effect of expression of Rab7 dominant alleles upon
Brucella intracellular growth in RAW264.7 macrophagelike cells. RAW264.7 cells were transiently transfected to
express GFP–Rab7, infected with DsRedm-expressing
Brucella and Rab7 and LAMP-1 recruitment was quantified. By 6 h p.i., most BCVs were replication permissive
(data not shown), and Rab7 clearly decorated the majority
of these BCVs (82 1.0%; Figure 8A,B), indicating that
replication-proficient intermediate BCVs interact with late
endocytic compartments during maturation in phagocytic cells. Moreover, expression of GFP–Rab7[T22N] in
RAW264.7 cells significantly decreased bacterial replication at 24 h p.i. because only 8.9 3.3% of GFP–
Rab7[T22N]-transfected cells supported bacterial growth
compared with 47 13% in untransfected cells (p < 0.05;
Figure 8C,D). By contrast, expression of GFP–Rab7 (34 11%) or GFP–Rab7[Q67L] (33 5.5%) did not significantly
affect bacterial replication compared with control cells
(p > 0.2; Figure 8C,D). Taken together, these results
demonstrate a role for Rab7-dependent functions in BCV
maturation and bacterial replication in phagocytic cells,
consistent with our data from epithelial cells.
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Decreased BCV–lysosome fusion correlates with
defective vacuolar trafficking
Because of the essential role of acidification in BCV
maturation and the similar defective BCV trafficking
observed in epithelial cells expressing Rab7[T22N] or
treated with BAF (Figures 1B,C, 2C,D and 6C,D), we
postulated that BCV acidification in Rab7[T22N]-expressing
cells was impaired, which consequently affects vacuolar
trafficking of bacteria. To test this hypothesis, BCV acidity
was assessed by treating GFP-expressing Brucellainfected HeLa cells at 4, 10 and 24 h p.i. with 5 mg/mL
acridine orange for 10 min followed by live cell imaging
analysis for red fluorescent BCVs. Acridine orange red
fluorescence emission was specific for acidic compartments in uninfected cells as it was completely abolished
by BAF pretreatment for 1 h and progressively restored
upon BAF washout (data not shown). In both untransfected and Rab7[T22N]-transfected cells, the majority of
BCVs displayed red fluorescence at 4 and 10 h p.i.
(Figure 9A), indicating that these BCVs remain acidic
during the early and intermediate maturation stages. BCV
acidity was completely abolished in cells pretreated with
BAF and analyzed at 4 h p.i. (data not shown). At 24 h p.i.,
replicative BCVs in control cells were not acidic, while
BCVs in cells expressing Rab7[T22N] remained acidic,
consistent with their defect in maturation (Figure 6C,D).
Thus, the defect in BCV maturation observed in cells
expressing GFP–Rab7[T22N] cannot solely be explained
by a lack of acidification.
Given that Rab7 controls fusion with the late endosomal/
lysosomal compartments, we next examined whether
stalled BCVs were defective for accessibility to fluid-phase
markers delivered from lysosomes by measuring dextran
accumulation in BCVs by quantitative live cell imaging.
Dextran accumulation in BCVs was significantly reduced in
cells expressing GFP–Rab7[T22N] or cells treated with
BAF (65 10 and 42 3.5% of the control, respectively;
Figure 9B,C) compared with untreated cells, indicating that
failed BCV maturation correlates with decreased intermediate BCV–lysosome fusion.
Discussion
Intravacuolar, intracellular pathogens have devised various
strategies to avoid degradation along the endocytic pathway (22). Whether they avoid interactions with the endocytic pathway, like Legionella pneumophila and Chlamydia
species (23), or arrest the maturation of their phagosome
at various stages, such as Mycobacterium tuberculosis
(22), they ultimately avoid fusion with degradative lysosomes. Such is also the case for Brucella, whose intracellular survival is commonly considered to rely upon
inhibition of fusion with lysosomes (2,3,9,11,18,24). This
view of Brucella intracellular trafficking is based upon
cumulative data from immunofluorescence and electron microscopy of fixed samples. In these experiments,
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Brucella Trafficking Through Lysosomes
Figure 6: Perturbation of the late endosomal/lysosomal compartment functions and positioning impairs BCV maturation and
bacterial replication. A, C and E) Representative confocal micrographs of HeLa cells expressing GFP–Rab7[Q67L] (A), GFP–Rab7[T22N]
(C) and GFP–RILP (E) that were infected with DsRedm-expressing Brucella abortus 2308 (appears in red) and immunostained for LAMP-1
using Cyanin 5-conjugated secondary antibodies (appears in blue). Transfected, infected HeLa cells were processed for immunostaining at
2, 6 and 24 h p.i. and analyzed by confocal microscopy as described in the Materials and Methods. Arrows indicate the area magnified in
the whole images or the positive BCVs in the insets. Scale bars, 10 or 2 mm. B, D and F) Effect of overexpression of GFP–Rab7[Q67L] (B),
GFP–Rab7[T22N] (D) or GFP–RILP (F) on BCV maturation. Colocalization of bacteria with the Rab7 dominant alleles or RILP (closed circles)
and LAMP-1 (open circles) was scored by confocal microscopy at 2, 6, 12 and 24 h p.i. Data are means SD from four independent
experiments. G) Quantification of bacterial replication in HeLa cells overexpressing Rab7 alleles or RILP. HeLa cells expressing GFP only
(control), GFP–Rab7, GFP–Rab7[Q67L], GFP–Rab7[T22N] or GFP–RILP were infected with DsRedm-expressing B. abortus 2308 for 24 h,
and intracellular replication was scored by fluorescence microscopy. Infected cells were considered to support bacterial replication when at
least 10 bacteria were present in replicative clusters. Data are means SD from five independent experiments. Asterisks indicate
a statistically significant difference compared with control conditions (Student’s t-test, p < 0.01).
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Figure 7: Perturbation of the late endosomal/lysosomal
compartment functions and positioning impairs biogenesis
of replicative BCVs. A) Representative confocal micrographs of
untransfected HeLa cells (control) or HeLa cells expressing GFP–
Rab7[Q67L], GFP–Rab7[T22N] or GFP–RILP that were infected
with DsRedm-expressing Brucella abortus 2308 and immunostained for calreticulin using Cyanin 5-conjugated secondary antibodies. Left-hand panels show GFP fluorescence in transfected
cells. Right-hand panels show overlays of Brucella (appears red)
and calreticulin (pseudocolored in white) fluorescence. Boxes on
micrographs indicate the area magnified in the insets. Arrows
indicate intracellular bacteria. Scale bars, 10 or 2 mm. B) Quantification of calreticulin recruitment to BCVs. Infected HeLa cells
were processed for immunostaining at 6, 12 and 24 h p.i. and
analyzed for colocalization between bacteria and ER by confocal
microscopy as described in the Materials and Methods. Data are
means SD from three independent experiments.
detected because of the limited sensitivity of the methodologies used. For example, permeabilization steps associated with fixation and immunostaining procedures can lead
to extraction of fluid-phase markers or luminal antigens
from their compartments and loss of corresponding signals, an artifact that was recently quantified by Drecktrah
et al., where fixation and immunofluorescence processing
of HeLa cells preloaded with fluorescent dextran led to a
>90% decrease of the dextran-associated fluorescence (12).
conclusions were drawn from the failure to deliver lysosomal luminal content to the BCV in the form of cathepsin
D (2,3,18,24) and preloaded fluid-phase markers such as
gold-labeled BSA (11) or fluorescent dextran (9). Yet, there
remained the possibility that fusion events were not
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Here, we have reassessed the trafficking of replicationproficient BCVs using a model of B. abortus infection of
epithelial HeLa cells and high-resolution live cell confocal
microscopy. Unlike what was previously believed, we
show that intermediate BCVs traffic along the endocytic
pathway and interact with late endocytic compartments,
including lysosomes. Such interactions are transient and
occur prior to BCV interaction and fusion with the ER and
subsequent conversion into replicative vacuoles. Our
results are consistent with the transient accumulation of
the late endosomal/lysosomal marker LAMP-1 on maturing BCVs (2,18), a hallmark of BCV trafficking that has
remained difficult to explain in the context of the current
model of rapid BCV segregation from the endocytic pathway. We also clearly show BCV recruitment of other late
endosomal/lysosomal markers such as CD63, Rab7 and
RILP. These findings also solve a long-lasting controversy
on Brucella intracellular trafficking, whereby the model of
rapid segregation from the endocytic pathway and inhibition of fusion with lysosomes did not accommodate the
fact that early BCV acidification is essential to bacterial
survival (15). Our findings on the nature of the intermediate
BCV are also consistent with the unusual trafficking of
opsonized B. abortus observed in human monocytes
where the bacteria remain and eventually replicate within
an ER-negative, LAMP-1-positive compartment (25). By
analogy with our results, such a compartment resembles
an intermediate BCV that would eventually become replication permissive. It remains unclear why these intracellular bacteria do not reach the ER, although opsonization of
bacteria may alter their intracellular trafficking.
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Brucella Trafficking Through Lysosomes
Figure 8: Rab7-dependent
functions
are required for Brucella replication in
RAW264.7 cells. A) Representative confocal micrograph of a GFP–Rab7-transfected RAW264.7 cell that was infected
with DsRedm-expressing Brucella abortus
2308 (appears in red) for 6 h and immunostained for LAMP-1 using Cyanin 5conjugated secondary antibodies (appears
in blue). Arrows indicate the area magnified in the whole image or the positive
BCVs in the insets. Scale bars, 10 or 2 mm.
B) Quantification of Rab7- and LAMP-1positive BCVs in GFP–Rab7-transfected
RAW264.7 cells that were infected with
DsRedm-expressing B. abortus 2308 for
6 h. Data are means SD from three independent experiments. C) Representative
confocal micrographs of either GFP–
Rab7[T22N]- or GFP–Rab7[Q67L]-transfected RAW264.7 cells that were infected
with DsRedm-expressing B. abortus 2308
(appears in red) for 24 h and immunostained for LAMP-1 using Cyanin 5conjugated secondary antibodies (appears
in blue). Arrows indicate the area magnified in the whole image or the positive
BCVs in the insets. Scale bars, 10 or 2 mm.
D) Quantification of bacterial replication in RAW264.7 cells overexpressing
Rab7 alleles. RAW264.7 cells expressing
no transgene (control), GFP–Rab7, GFP–
Rab7[Q67L] or GFP–Rab7[T22N] were infected with DsRedm-expressing B. abortus
2308 for 24 h, and intracellular replication
was scored by fluorescence microscopy.
Infected cells were considered to support
bacterial replication when at least 10 bacteria were present in replicative clusters.
Data are means SD from three independent experiments. The asterisk indicates a
statistically significant difference between
control and Rab7[T22N]-expressing cells
(Student’s t-test, p ¼ 0.03).
Although BCV fusion with lysosomes was obvious in live cells,
it was not as extensive as fusion events that occurred with
vacuoles containing heat-killed Brucella. This indicates that
live Brucella limit fusion of their vacuole with lysosomes,
possibly to avoid accumulation of lysosomal content to
bactericidal levels. The potential loss of luminal antigens
associated with immunostaining, the tight luminal space of
BCVs and the limited fusion of BCVs with lysosomes
together explain why previous studies have failed to detect
significant amounts of luminal lysosomal enzymes, such as
cathepsin D, inside BCVs (2,18). In support of our findings,
intermediate BCVs also displayed proteolytic activities, as
judged by the intravacuolar detection of DQ-Red BSA
fluorescence, which indicated delivery of proteolytic enzymes into maturing BCVs. Recruitment of active Rab7 to
BCVs strongly argues for the interaction of intermediate
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BCVs with the late endosomal/lysosomal compartment as
this small GTPase controls traffic to, fusion with and positioning of this compartment (14). These results contradict
previous observations that Rab7 is not recruited to BCVs in
macrophages (2). However, this previous study assessed
Rab7 recruitment to BCVs using immunostaining of endogenous Rab7. The absence of detectable antibody signal on
BCVs may be explained by the difficulties detecting low
steady-state levels of endogenous antigens that rapidly cycle
on and off intracellular compartments, such as Rab GTPases.
Not only Rab7 was recruited to BCVs in both HeLa and
RAW264.7 cells but also its activity was required for the
proper maturation of the BCV into a replication-permissive,
ER-derived organelle, indicating that Brucella traffic
through late endocytic compartments is requisite for
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Figure 9: Perturbation of late endosomal/lysosomal compartments decreases BCV fusion with lysosomes. A) Quantification of
BCV acidity in HeLa or GFP–Rab7[T22N]-overexpressing HeLa cells. Cells were infected with GFP-expressing Brucella abortus 2308, and BCV
acidity was evaluated after loading cells with acridine orange using live cell imaging analysis at 4, 10 and 24 h p.i. Acidic BCVs displayed
acridine orange red fluorescence. Data are means SD from three independent experiments. B) Single frames of representative time-lapse
recordings of untreated, GFP–Rab7[T22N]-expressing or BAF-treated HeLa cells that were preloaded with Alexa Fluor 546–dextran to label
lysosomes and infected with GFP-expressing Brucella for 6 h. Left-hand panels show both GFP and Alexa Fluor 546–dextran fluorescence,
while right-hand panels show dextran fluorescence only. Note that the bacterial GFP signal was stronger than the GFP–Rab7[T22N] signal,
easily allowing the detection of bacteria in transfected cells. Arrows indicate dextran-positive BCVs. C) Quantification of intravacuolar dextran
accumulated in BCVs in cells expressing GFP–Rab7[T22N] or treated with BAF compared with untransfected or untreated cells, respectively.
The fluorescence pixel intensities measured were normalized to controls in each independent experiment, and data are means SD from
three independent experiments. Asterisks indicate a statistically significant difference compared with control conditions (Student’s t-test,
p < 0.05).
intracellular replication. These results clearly establish late
endosomal BCVs as the intermediate niche for replicationproficient bacteria and rule out the possibility that, in
macrophages, LAMP-1-positive BCVs only contain bacteria
that are routed to a degradative phagolysosome. ChavesOlarte et al. have reported that overexpression of the
constitutively active Rab7[Q67L] allele in stably transfected NIH3T3 fibroblasts did not affect Brucella intracellular growth or trafficking, which led the authors to
conclude that Rab7 is not required for Brucella intracellular
trafficking (13). However, the effect of the dominantnegative allele of Rab7, Rab7[T22N], on Brucella intracellular fate was not examined in this study. Our results using
transient transfection of HeLa or RAW264.7 cells with
GFP–Rab7[Q67L] concur with this previous study. Yet, our
experiments with Rab7[T22N] in HeLa and RAW264.7
cells clearly demonstrate a significant role for this GTPase
in BCV trafficking and bacterial replication. This is in
agreement with previous work showing that overexpression of Rab7[Q67L] does not significantly affect functionality of late endocytic compartments, while that of
Rab7[T22N] decreases endocytic functions by about 50%
(14). Overexpression of the Rab7 effector GFP–RILP,
which induces a dramatic collapse of late endosomes
and lysosomes around the MTOC because of enhanced
recruitment of dynein–dynactin motor complexes (21), also
affected BCV trafficking and prevented bacterial replication, confirming that intermediate BCVs traffic through late
endocytic compartments. The effect of RILP overexpression suggests that proper positioning, in addition to
functioning, of this compartment might be necessary for
efficient BCV trafficking. However, overexpression of
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Rab7[T22N] did not significantly affect BCV positioning
with respect to the MTOC (data not shown), even though it
is known to increase the retrograde movement of late
endosomes in uninfected cells (14). This suggests that
BCV positioning in the cell does not only rely upon Rab7dependent mechanisms.
In cells expressing Rab7[T22N], most BCVs failed to traffic
to the ER and generate replication-permissive organelles.
Such a trafficking defect was also observed in cells
pretreated with the v-ATPase inhibitor BAF, which blocks
acidification and subsequent maturation of phagosomes
along the endocytic pathway. BAF treatment prevented
bacterial replication in both macrophages and epithelial
cells. As reported by Porte et al. (15), early, but not late,
inhibition of vacuolar acidification affected Brucella intracellular fate, a timing consistent with the kinetics of BCV
interaction with late endocytic compartments. This suggests that proper BCV maturation requires a specific
sequence of timed intracellular events. The similar effects
of BAF treatment and Rab7[T22N] expression on Brucella
trafficking suggest a role for intravacuolar pH in both cases
because both reportedly affect intraluminal pH of endocytic compartments (14,16). However, defects in endosome maturation resulting from these treatments may
also be invoked. Indeed, late BCVs remained acidic in cells
expressing Rab7[T22N], ruling out the theory that phagosomal pH is solely responsible for BCV trafficking defects. Instead, both BAF treatment and overexpression of
Rab7[T22N] decreased BCV fusion with lysosomes in
HeLa cells. It is thus possible that environmental cues
other than acidification resulting from BCV interaction with
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Brucella Trafficking Through Lysosomes
late endosomes/lysosomes are necessary for further trafficking toward the ER. Further work detailing the physiochemical environment within the intermediate BCV is
needed to address these questions.
Our work provides evidence that intermediate BCVs
interact with the endocytic pathway and experience limited fusion with lysosomes to generate a transient, intravacuolar environment conducive to further trafficking of
the BCV toward the ER (Figure 10). Because the VirB type
IV secretion system is required for subsequent trafficking
events, such as the biogenesis of the ER-derived replicative organelle (2,3,8), it is tempting to speculate that
intermediate BCVs provide environmental cues required
for VirB expression and/or activation and that treatments
affecting proper maturation of the intermediate BCV affect
VirB functions. In support of this hypothesis, intracellular
B. abortus virB gene induction is maximal at 4 h p.i. in
Figure 10: Revised model of Brucella intracellular trafficking.
A) Upon entry, newly formed BCVs traffic along the endocytic
pathway, sequentially interacting with early compartments of the
endocytic pathway (shown in yellow) and late endosomes and
multivesicular bodies (MVB; shown in green) before fusing with
lysosomes (shown in red). Such interactions are required for
further trafficking (B), whereby intermediate BCVs interact with
ER exit sites (ERES) and subsequently fuse with the ER (shown in
blue) (C) to generate ER-derived, replicative BCVs (D). BCVs
containing VirB-defective mutants that cannot sustain interactions
with the ER (2) and/or limit fusion with the lysosomal compartment and mature into a bactericidal vacuole (E).
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J774A.1 cells (26) when bacteria are within intermediate
BCVs. Moreover, expression of the VirB type IV secretion
system encoding genes in B. suis can be induced in vitro at
acidic pH, and BAF reduces intracellular virB promoter
activity in J774A.1 cells (17). Consistent with this hypothesis, Brucella mutants in the VirB type IV secretion system
in untreated cells exhibit trafficking defects (2,3,8) similar
to those we observed for wild-type bacteria in BAF-treated
cells or cells expressing Rab7[T22N]. This suggests that
the trafficking defects because of perturbation of late
endosomal/lysosomal compartments may be because of
an impaired expression, or activation, of VirB. Although we
could reproduce the inhibitory effect of BAF on B. abortus
virB gene induction (17) in both BMMs and HeLa cells
using quantitative reverse transcriptase–polymerase chain
reaction of virB4 (Figure S2, see also Supplementary
Materials and Methods) or virB11 messenger RNAs (data
not shown), our attempts at measuring VirB expression in
cells expressing dominant alleles of Rab7 were unsuccessful. Despite this, our data do support a model whereby
transient fusion of BCVs with lysosomes provides the
intravacuolar cues required for proper expression of VirBassociated functions, which in turn allows for trafficking to
the ER and replication.
The demonstrated interaction of intermediate BCVs with
the late endosomal/lysosomal compartment suggests that
Brucella is capable of resisting a potentially degradative
environment, at least transiently. The fact that VirBdeficient mutants of Brucella do not traffic further than
intermediate BCVs and are progressively killed (2) indicates that such an organelle can acquire bactericidal
functions over time. It also raises the possibility that, once
expressed and assembled, the VirB apparatus translocates
effector molecules that support bacterial survival in the
intermediate BCV, possibly by limiting fusion with lysosomes. Such a process would allow bacteria to redirect the
trafficking of their vacuole to the ER in a VirB-dependent
manner. Cyclic b-1,2-glucan synthesized by Brucella has
also been proposed to control avoidance of lysosome
fusion based on experiments using delivery of cathepsin
D as evidence for BCV–lysosome (24). It will be interesting
to assess the relative contributions of cyclic b-1,2-glucan
and the type IV secretion system using our live cell assays,
which are extremely sensitive in measuring the extent of
BCV–lysosome fusion. Ultimately, the identification of
substrates of the VirB apparatus and their characterization
will be required to clarify if, and how, the VirB apparatus
participates in the trafficking transition of the BCV from the
endocytic pathway to the ER.
Materials and Methods
Bacterial strains and plasmids
The bacterial strains used in this study were the smooth virulent B. abortus
strain 2308 and two derivatives expressing either the GFP from plasmid
pJC43 (5) or the DsRedm from plasmid pJC44. To construct pJC44, a fusion
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between the aphA3 gene promoter driving GFP expression on pJC43 and
the DsRedm gene from pDsRedm (Clontech) was generated using the primers
pairs JC213 (50 -TGCAGGAATTCCCAGCGAACC-30 )/JC214 (50 -TTAATAAACCTCCTTTCGGATCCG-30 ) and JC215 (50 -AAGGAGGTTTATTAAATGGACAACACCGAGGACGTC-30 )/JC216 (50 -GAATTCTAGAGTCGCGGCCGCTC-30 ),
respectively. Both fragments were subsequently fused by overlap extension
polymerase chain reaction amplification using primers JC213 and JC216. The
resulting fragment was cloned into pCR2.1TOPO (Invitrogen), and its
sequence was confirmed. The fusion fragment was then excised using
EcoRI and XbaI sites present on primers JC213 and JC216 and subcloned into
the same restriction sites on pBBR1-MCS2 (27) to give pJC44. This plasmid
was then introduced by electroporation into the wild-type strain 2308. All
bacteria were grown in tryptic soy broth (TSB; Sigma) or on tryptic soy (TS)
agar plates (TSA; Sigma), supplemented with kanamycin (50 mg/mL) to select
the pBBR1-MCS2 derivatives pJC43 and pJC44. For infection of eukaryotic
cells, 2 mL of TSB was inoculated with a few bacterial colonies from a freshly
streaked TSA plate and grown at 378C for 16 h to early stationary phase. Heatkilled bacteria were prepared by incubating an aliquot of an early stationary
phase culture at 808C for 30 min.
Cell culture and infections
To obtain BMMs, bone marrow cells were isolated from femurs of
6–10 week old, C57BL/6J female mice (Jackson Laboratories) and differentiated into macrophages for 5 days at 378C and 7% CO2 in 1 g/L glucose DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen), 10%
L-conditioned medium and 2 mM L-glutamine in non-tissue culture-treated
Petri dishes. 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 replated in 24-well cell culturetreated plates (1 105 BMMs/well). BMMs were further incubated at 378C
under 7% CO2 atmosphere for 48 h, replenishing with complete medium
24 h before infection. HeLa cells [American Type Culture Collection (ATCC)
clone CCL-2] were cultured at 378C under 7% CO2 atmosphere in DMEM
supplemented with 10% fetal calf serum (FCS) and 2 mM L-glutamine and
seeded 24 h before infection either on 12-mm glass coverslips in 24-well
plates (immunofluorescence; 1 105/well) or on WillCo-dishÒ glass bottom 35-mm dishes (live cell imaging; 1 105/dish; WillCo Wells BV).
RAW264.7 cells (ATCC number TIB-71) were cultured at 378C under 7%
CO2 atmosphere in DMEM supplemented with 10% FCS and 2 mM
L-glutamine and seeded 48 h before infection on 12-mm glass coverslips
in 24-well plates (immunofluorescence; 3 105/well). For transfections,
HeLa or RAW264.7 cells were transfected 1 day prior to infection using
either the FuGene 6ä transfection reagent (HeLa; Roche) or the FuGene
HDä transfection reagent (RAW264.7; Roche) according to the manufacturer’s instructions.
For infections, bacterial cultures were diluted in complete medium and
added to chilled cells at a theoretical multiplicity of infection of 25 (BMMs),
100 (RAW264.7 cells) or 1000 (HeLa cells). To improve Brucella uptake by
HeLa cells and allow statistically significant analyses, EGF at a final
concentration of 50 ng/mL (recombinant human EGF; Calbiochem) was
added to the cells at the time of infection, which induced transient
membrane ruffling. EGF-mediated uptake did not alter trafficking of
internalized bacteria compared with untreated HeLa cells (Figure S1).
Bacteria were centrifuged onto cells at 400 g for 10 min at 48C, and
infected cells were incubated for either 20 min (BMMs and RAW264.7
cells) or 30 min (HeLa cells) at 378C under 7% CO2 atmosphere following
a rapid warm up in a 378C water bath to synchronize bacterial uptake.
Infected cells were then washed five times with DMEM to remove
extracellular bacteria, incubated for either an additional 30 (BMMs and
RAW264.7 cells) or 60 min (HeLa cells) in complete medium before
medium containing 100 mg/mL gentamicin was added for 60 min (BMMs
and RAW264.7 cells) or 90 min (HeLa cells) to kill extracellular bacteria.
Thereafter, infected cells were maintained in gentamicin-free medium.
When required, BAF (AG Scientific) was added to cells at a final concentration of 100 nM and replenished every 6 h.
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Intracellular replication assays
To evaluate intracellular replication of Brucella, BMMs or HeLa cells
seeded in 24-well plates were infected as described above, and the
number of viable intracellular bacteria per well was determined in triplicate
for each time-point. Infected cells were washed three times with sterile
PBS and then lysed with 1 mL of 0.1% Triton-X-100 in water for 2 min at
room temperature. Serial dilutions were rapidly plated onto TSA plates, and
plates were incubated for 3 days at 378C before enumeration of colonyforming units.
Immunofluorescence microscopy
Infected cells grown on 12-mm glass coverslips in 24-well plates were
washed three times with PBS, fixed with 3% paraformaldehyde, pH 7.4, at
378C for 20 min, washed three times with PBS and then incubated for
10 min in 50 mM NH4Cl in PBS in order to quench free aldehyde groups.
Samples were blocked and permeabilized in 10% horse serum and 0.1%
saponin in PBS for 30 min at room temperature. Cells were labeled by
inverting coverslips onto drops of primary antibodies diluted in 10% horse
serum and 0.1% saponin in PBS and incubating for 45 min at room
temperature. Primary antibodies used were rat anti-mouse LAMP-1 clone
1D4B, mouse anti-human LAMP-1 clone H4A3, mouse anti-human CD63
clone H5C6 (developed by J. T. August and 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, The University of Iowa, Iowa
City, IA, USA) and rabbit polyclonal anti-calreticulin antibodies (Affinity
BioReagents). Bound antibodies were detected by incubation with 1:500
dilutions of either Alexa Fluor 488 donkey anti-mouse, anti-rat antibodies or
Cyanin 5-conjugated goat anti-rabbit or donkey anti-rat antibodies (Jackson
ImmunoResearch Laboratories) for 45 min at room temperature. Cells
were washed twice with 0.1% saponin in PBS, once in PBS, once in H2O
and then mounted in Mowiol 4-88 mounting medium (Calbiochem).
Samples were observed on a Nikon Eclipse E800 epifluorescence microscope equipped with a Plan Apo 60/1.4 objective for quantitative analysis
or a Carl Zeiss LSM 510 confocal laser scanning microscope for image
acquisition (Carl Zeiss Micro Imaging). Confocal images of 1024 1024
pixels were acquired as projections of three consecutive slices with a
0.38-mm step and assembled using ADOBE PHOTOSHOP CS (Adobe Systems).
Live cell imaging
To assess BCV accessibility to fluid-phase markers, HeLa cells were
preincubated for 16 h in complete medium containing 100 mg/mL Alexa
Fluor 546-conjugated dextran (10 000 MW; Molecular Probes) and then
infected with GFP-expressing bacteria in the absence of dextran until uptake
was completed. Thereafter, fluorescent dextran was added to the medium
until sample analysis. To assess BCV fusion with lysosomes, HeLa cells were
preincubated for 12 h in complete medium containing 200 mg/mL Alexa Fluor
546-conjugated dextran (10 000 MW; Molecular Probes), which was subsequently chased for 12 h in complete medium before infection to ensure
all dextran had trafficked to lysosomes. Cells were then infected with GFPexpressing bacteria in absence of dextran. Because GFP fluorescence was
destroyed by heat treatment, heat-killed bacteria were stained using SYTO-9
dye according to the manufacturer’s instructions (Invitrogen). To evaluate
proteolytic activities in BCVs, HeLa cells were preincubated for 16 h in
complete medium containing 50 mg/mL DQ-Red BSA (Molecular Probes),
which was maintained all throughout the subsequent infection process.
At the time of analysis, cells were washed with CO2-independent medium
(Invitrogen) and incubated with CO2-independent medium supplemented
with 10% FBS, 4 mM L-glutamine and 1 mM Trolox (Calbiochem) during live
cell imaging. Time-lapse recording was performed immediately on a Carl
Zeiss LSM 5 live confocal live cell imaging microscope fitted with a Pecon
heated stage insert and LCI Plan 63/1.45 NA objective using 488 and
532 nm solid-state lasers for sequential excitation. Images (1024 1024
pixels) were acquired using the Carl Zeiss LSM 5 LIVE 4.0 SP2 software.
Individual frames were assembled using ADOBE PHOTOSHOP CS, and
Traffic 2008; 9: 678–694
Brucella Trafficking Through Lysosomes
time-lapse movies were assembled as Quick Time movies using
PREMIERE PRO software (Adobe Systems).
ADOBE
To quantify dextran acquisition by BCVs, randomly chosen BCVs were
imaged, regions of interest (ROI) corresponding to BCVs were defined and
the associated dextran fluorescence measured using the Carl Zeiss LSM 5
LIVE 4.0 SP2 software. Because of differences in the planes of focus between
the BCVs analyzed, the total fluorescence intensity measured was normalized to the ROI area (in pixels) to generate average fluorescence intensity
values per pixel that were comparable between vacuoles. Data were
expressed as the means standard deviations of average pixel intensities
from three independent experiments. A standard Student’s two-tailed t-test
was used to assess significance between conditions (p < 0.05).
Acknowledgments
We are grateful to Cecilia Bucci for the generous gift of Rab7 alleles and
RILP, Olivia Steele-Mortimer, Jessica Edwards and Audrey Chong for critical
reading of the manuscript, Steve Porcella and Kimmo Virtaneva for their help
with quantitative reverse transcriptase–polymerase chain reaction experiments, Anita Mora and Austin Athman for assistance with graphics and the
Rocky Mountain Laboratories Genomics Unit for DNA sequencing. This work
was supported by the Intramural Research Program of the National Institutes
of Health, National Institute of Allergy and Infectious Diseases.
Supplementary Materials
Supplementary Materials and Methods
Quantitative real-time polymerase chain reaction.
Figure S1: BCV interaction with late endosomal/lysosomal compartments in non-EGF-treated HeLa cells. A) Representative confocal micrographs of either untransfected, GFP–Rab7- or GFP–RILP-transfected HeLa
cells that were infected for 6 h with DsRedm-expressing Brucella abortus
2308 in the absence of EGF and immunostained for LAMP-1 using either
Alexa Fluor 488- (untransfected cells) or Cyanin 5-conjugated secondary
antibodies. Arrows indicate the area magnified in the whole images or the
positive BCVs in the insets. Scale bars, 10 or 2 mm. B) Quantification of
LAMP-1 Rab7 and RILP on BCVs at 2, 6, 12 and 24 h p.i. in non-EGF-treated
HeLa cells. The kinetics of acquisition and loss of these markers are
indistinguishable from those in EGF-treated HeLa cells (compare with
Figure 3D). Data are means SD of three independent experiments. C)
Single frames of representative time-lapse recordings of Alexa Fluor 546–
dextran-loaded HeLa cells (appears in red) infected in the absence of EGF
with GFP-expressing Brucella (appears in green) at 6 or 12 h p.i.. The arrows
indicate either dextran-positive (6 h p.i.) or -negative (12 h p.i.) BCVs.
Intermediate, but not replicative, BCVs acquire dextran through fusion with
lysosomes. Scale bar, 10 mm.
Figure S2: Inhibition of intracellular induction of virB genes by BAF.
A) The time–course of intracellular expression of virB4 inside BMMs.
Untreated BMMs (closed circles) or BMMs treated with BAF from 1 h
prior to infection to 8 h p.i. (open circles) were infected with Brucella
abortus 2308 and processed for TaqMan analysis of virB4-specific mRNAs
at 1, 4, 8 and 24 h p.i. Values are expressed as relative mRNA fold changes
compared with levels measured at 1 h p.i. and are means SEM of
a representative experiment out of three. B) Effect of BAF upon virB4
induction inside BMMs or HeLa cells. BAF-treated or untreated BMMs or
HeLa cells were infected with B. abortus 2308 and processed for TaqMan
analysis of virB4-specific mRNAs at 0 and 4 h p.i. Values are expressed as
relative mRNA fold changes between 0 and 4 h p.i. and are means SEM
of a representative experiment out of two. mRNA, messenger RNA.
Traffic 2008; 9: 678–694
Movie S1: Time-lapse confocal images of a HeLa cell loaded with Alexa
Fluor 546-conjugated dextran and infected with GFP-expressing Brucella
abortus 2308 for 6 h, showing an intermediate BCV accessible to dextran
delivered from endosomes. The left-hand image shows an overlay of both
dextran- (red) and bacteria- (green) associated fluorescence, while the righthand image shows dextran-associated fluorescence only. Seventy-five
consecutive frames were acquired through sequential excitation of the
sample using 488 nm and then 543 nm laser lines, with no interval
between acquisitions.
Movie S2: Time-lapse confocal images of a HeLa cell loaded with Alexa
Fluor 546-conjugated dextran and infected with GFP-expressing Brucella
abortus 2308 for 12 h, showing replicative BCVs that are not accessible to
dextran delivered from endosomes. The left-hand image shows an overlay
of both dextran- (red) and bacteria- (green) associated fluorescence, while
the right-hand image shows dextran-associated fluorescence only.
Seventy-five consecutive frames were acquired through sequential excitation of the sample using 488 nm and then 543 nm laser lines, with no
interval between acquisitions.
Movie S3: Time-lapse confocal images of a HeLa cell whose lysosomes
were preloaded with Alexa Fluor 546-conjugated dextran before infection
with GFP-expressing Brucella abortus 2308 for 6 h. An intermediate BCV is
accessible to dextran delivered from lysosomes. The left-hand image
shows an overlay of both dextran- (red) and bacteria- (green) associated
fluorescence, while the right-hand image shows dextran-associated fluorescence only. Seventy-five consecutive frames were acquired through
sequential excitation of the sample using 488 nm and then 543 nm laser
lines, with no interval between acquisitions.
Movie S4: Time-lapse confocal images of a HeLa cell whose lysosomes
were preloaded with Alexa Fluor 546-conjugated dextran before infection
with GFP-expressing Brucella abortus 2308 for 12 h. Replicative BCVs are
not accessible to dextran delivered from lysosomes. The left-hand image
shows an overlay of both dextran- (red) and bacteria- (green) associated
fluorescence, while the right-hand image shows dextran-associated fluorescence only. Seventy-five consecutive frames were acquired through
sequential excitation of the sample using 488 nm and then 543 nm laser
lines, with no interval between acquisitions.
Supplemental materials are available as part of the online article at http://
www.blackwell-synergy.com
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