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Mediates its Restoration and Bacterial Killing1
* Unité des Rickettsies, Faculté de Médecine, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6020, and
Centre d’Immunologie de Marseille-Luminy, Marseille, France; and
Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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restored C.
burnetii killing and phagosome maturation as assessed by
colocalization of C. burnetii with active cathepsin D.
In addition, when IFN- was added before cell infection, it was able
to stimulate C. burnetii killing but it also induced
vacuolar alkalinization. These findings suggest that IFN- mediates
C. burnetii killing via two distinct mechanisms,
phagosome maturation, and phagosome alkalinization. Thus, the tuning of
vacuole biogenesis is likely a key part of C. burnetii
survival and the pathophysiology of Q fever. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subdivision of proteobacteria, is the agent of Q
fever (1). Whereas acute Q fever is controlled by
cell-mediated immunity, this latter is defective in chronic Q fever
(2). The establishment of C. burnetii infection
is based on a specific strategy of invasion of monocytes/macrophages.
Virulent organisms are poorly internalized by macrophages and their
uptake requires the engagement of
v
3 integrin;
avirulent variants are efficiently internalized through
v3 integrin and
complement receptor type 3. The selective use of phagocytic
receptors is an active process based on interference with complement
receptor type 3-mediated phagocytosis (3). Once
internalized, C. burnetii survives and replicates in an
acidic environment (4, 5), which is needed for bacterial
metabolism (6, 7). This low pH also accounts for the
relative inefficiency of antibiotics toward C. burnetii
(1). The association of acidic pH and phagolysosomal
features has led most authors to consider the intracellular life of
C. burnetii as a paradigm of intracellular survival without
alteration of intracellular traffic (5). However, most of
these studies were performed with avirulent C. burnetii
(8, 9). In addition, these studies used fibroblasts and
murine macrophage-like cells, in which virulent and avirulent C.
burnetii replicate (5), whereas only virulent
C. burnetii survives in human monocytes/macrophages
(3).
The fate of intracellular microorganisms including C.
burnetii depends on the microbicidal properties of macrophages and
their regulation by cytokines. A defective killing of C.
burnetii was found in monocytes from patients with chronic Q fever
(10), which partly results from IL-10-mediated impairment
of macrophage microbicidal activity. Indeed, IL-10 elicits the
replication of C. burnetii in resting monocytes, and
neutralizing anti-IL-10 Abs restore microbicidal activity against
C. burnetii in patients with chronic Q fever
(11). In contrast, IFN-, known to stimulate the
microbicidal activity of macrophages, triggers C. burnetii
killing in THP-1 monocytes (12). The ability of IFN- to
stimulate the microbicidal activity of macrophages has been related to
oxygen-dependent mechanisms (13), but reactive oxygen
intermediates are not involved in the killing of C. burnetii
(12). As IFN--induced killing of Listeria
monocytogenes and Mycobacterium avium has been
associated with the modulation of phagosome maturation
(14, 15, 16), we hypothesized that such mechanisms may be
involved in C. burnetii killing.
We show in this study that the survival of C. burnetii
in THP-1 monocytes is associated with altered phagosome maturation.
C. burnetii organisms are present in phagosomes that acquire
markers of late endosomes and late endosomes-early lysosomes but not
the lysosomal enzyme cathepsin D. The survival of C.
burnetii depends on the activation of THP-1 cells. Indeed, IFN-
induces C. burnetii killing and restores phagolysosomal
fusion. This study also provides evidence that IFN--induced killing
of C. burnetii involves two distinct mechanisms, phagosome
maturation and late phagosome alkalinization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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THP-1 monocytic cells were cultured as previously described (12). Cells (5 x 104 cells/assay) were seeded on 12-mm round coverslips in flat-bottom 24-well plates (Nunc, Roskilde, Denmark) and were treated with 10 ng/ml PMA (Sigma-Aldrich, St. Louis, MO) to become adherent. After 24 h at 37°C, cells were washed three times in antibiotic-free RPMI 1640 supplemented with 10% FCS and 2 mM L-glutamine (Invitrogen, Eragny France). PBMC were isolated from healthy volunteers on Ficoll gradient (MSL, Eurobio, Les Ullis, France), and monocytes were purified by adherence on glass Labtek chamber/slides (Miles, Naperville IL), as previously described (11). Nonadherent cells were removed by washing, and remaining cells were cultured for 3 days in RPMI 1640 supplemented with 10% FCS and 2 mM L-glutamine. Virulent and avirulent C. burnetii organisms (Nine Mile strain, ATCC VR-615; American Type Culture Collection, Manassas, VA) were obtained as previously described (3). In brief, virulent organisms were isolated from infected mice and cultured in L929 cells for two passages whereas avirulent variants were cultured in L929 cells by repeated passages. Two other virulent strains of C. burnetii, Priscilla and Q212, isolated from an infected goat and a patient with acute Q fever, respectively, were cultured like the virulent Nine Mile strain. Bacteria were layered on 25–45% linear Renograffin gradient, and the gradients were spun down. Purified bacteria were then collected, washed, and suspended in HBSS before being stored at -80°C. The number of bacteria was determined by Gimenez staining. The viability of C. burnetii was assessed using the LIVE/DEAD BacLight bacterial viability kit (Molecular Probes, Eugene, OR) as recommended by the manufacturer. Briefly, the C. burnetii suspension was incubated with SYTO 9 stain and propidium iodide, and examined with a fluorescence microscope. Results are expressed as the ratio of viable bacteria and the total number of bacteria. Only C. burnetii preparations containing >90% of viable organisms were used. Heat-killed virulent organisms were obtained by heating the bacterial suspension at 100°C for 1 h, and were stored at -80°C.
Intracellular fate of C. burnetii
THP-1 cells and monocytes from healthy donors were incubated
with C. burnetii in antibiotic-free RPMI 1640 containing
10% FCS. After 24 h at 37°C, cells were washed to remove free
bacteria (this time was designated as day 0): this
procedure was sufficient to remove noninternalized and loosely attached
organisms (3). Monocytic cells were again cultured for
different periods. In some experiments, THP-1 cells were incubated with
human rIFN- (R&D Systems, Abingdon, U.K.) for 16 h, and then
infected with C. burnetii. After 24 h, cells were
washed to remove free bacteria (corresponding to day 0), and were again
incubated with IFN-. Alternatively, IFN- was added to infected
cells and the same procedure was used. Intracellular bacteria were
revealed by indirect immunofluorescence. Briefly, cell preparations
were fixed with 1% formaldehyde, incubated with human Abs to C.
burnetii (purified IgG from patients with Q fever endocarditis,
1/4000 dilution) in the presence or the absence of 0.1 mg/ml
lysophosphatidylcholine, washed, and incubated with a 1/200 dilution of
FITC-conjugated F(ab')2 anti-human IgG Abs
(Beckman Coulter, Roissy, France). Results are expressed as an
infection index, which is the product of the mean number of bacteria
per infected cell and the percentage of infected cells x 100
(3). The viability of intracellular bacteria was assessed
using the bacterial viability kit. The infected cells were homogenized
in water and vigorously mixed. The cell lysate was centrifuged at
8000 x g for 10 min, and pelleted bacteria were
collected. The combination of SYTO 9 stain and propidium iodide was
added to the bacterial suspension and the fluorescence of organisms was
observed. Results are expressed in percentage of live bacteria.
Phagosome acidification
The phagosome acidification was analyzed using DM-NERF dextran (molecular mass, 10 kDa; Molecular Probes), a fluorescent probe of phagosomal pH (17). THP-1 cells were incubated with 20 µg/ml DM-NERF dextran and C. burnetii, or latex beads (0.8 µm; Sigma-Aldrich) as control, for 24 h. In some experiments, infected cells loaded with DM-NERF dextran were incubated with 10 nM bafilomycin A1 (Sigma-Aldrich), a specific inhibitor of vacuolar proton ATPase (V-H+-ATPase)3 (18) for 2 h. Bacteria were revealed by human Abs to C. burnetii and Texas Red-conjugated F(ab')2 anti-human IgG Abs (Beckman Coulter) used at a 1/100 dilution. The intraphagosomal pH was measured by ratiometric analysis of fluorescence intensities of DM-NERF dextran (excitation, 490/440 nm; emission, 530 nm). Infected cells were incubated with buffer solutions with graded pH (4.0, 5.0, 6.0, 7.0, and 7.4) in the absence or the presence of 10 µM monensin (Sigma-Aldrich), which equilibrated the intravacuolar pH with extracellular pH (19). After 1 h, fluorescence was recovered using a microplate fluorescent reader (Fisher Scientific, Elancourt, France). The mean pH value of the samples was calculated using a reference pH curve.
Bacterial trafficking
Bacterial trafficking was studied by immunofluorescence as follows (20). THP-1 cells and monocytes were infected by C. burnetii (200 virulent bacteria or 25 avirulent bacteria per cell) for 4 h (considered as h 0), washed to remove free organisms, and incubated for additional periods. Cell preparations were then fixed in 3% paraformaldehyde for 20 min. After washing, cells were incubated with ammonium chloride to neutralize free aldehydes and were permeabilized by PBS containing 0.1% saponin and 10% horse serum for 30 min. Human Abs specific for C. burnetii were used at a 1/4000 dilution. The Abs to intracellular markers were: rabbit anti-lysosome-associated membrane protein-1 (Lamp-1) Abs (a gift from Dr. M. Fukuda, The Burnham Institute, La Jolla, CA) used at a 1/1000 dilution, anti-cation-independent mannose-6-phosphate R (M6PR) Abs (a gift from Dr. B. Hoflack, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany) used at a 1/500 dilution, anti-Rab7 Abs (a gift from Dr. M. Zerial, Max Planck Institute of Molecular Cell Biology and Genetics) used at a 1/200 dilution, anti-cathepsin D Abs (a gift from Dr. S. Kornfeld, Washington University School of Medicine, St. Louis, MO) used at a 1/1000 dilution; mouse anti-CD63 mAbs (BD Biosciences, le Pont de Claix, France) used at a 1/1000 dilution, and anti-V-H+-ATPase mAbs (Chemicon International, Temecula, CA) used at a 1:500 dilution. Primary Abs were added to cell preparations in PBS containing 0.1% saponin and 5% horse serum for 30 min. After being washed, monocytic cells were incubated with fluorescent secondary Abs in 0.1% saponin. Bacteria were revealed by Texas Red-conjugated F(ab')2 anti-human IgG Abs and intracellular markers by FITC-conjugated F(ab')2 anti-rabbit or anti-mouse IgG Abs (Beckman Coulter), both Abs being used at a 1:100 dilution. The colocalization of bacteria with intracellular markers was examined with a laser scanning confocal fluorescence microscope (Leica TCS 4D; Heidelberg, Germany). Optical sections of images were collected at 0.5-µm intervals and analyzed using Adobe Photoshop V5.5 software (Mountain View, CA). C. burnetii phagosomes were scored as positive for soluble markers when fluorescence was observed in the phagosome lumen; for membrane markers, phagosomes were scored as positive when a fluorescence ring surrounded organisms. About 30 C. burnetii-containing vacuoles were scored per coverslip, and at least three distinct experiments were performed per condition. Results are expressed as the percentage of phagosomes expressing intracellular markers.
Lysosomal tracker and in situ measurement of cathepsin D activity
THP-1 cells were infected with C. burnetii for 4 h. The lysosomotropic probe neutral red (Molecular Probes) was added at 5 µg/ml to infected cells 2 h before the end of the infection time. After being washed, cells were fixed in 3% paraformaldehyde. Bacteria were revealed by indirect immunofluorescence with FITC-conjugated secondary Abs, and neutral red was observed with excitation and emission filters for Texas Red. The cathepsin D-sensitive near-infrared fluorescence (NIRF) probe was prepared as previously described (21, 22). It was conjugated with FITC to monitor probe internalization and with a Cy5.5 marker that became fluorescent in the near-infrared spectrum after cathepsin D activation. NIRF at 0.1 µM was added to cells 1 h before the end of the infection time. THP-1 cells were then washed to remove noninternalized organisms and the free NIRF probe and were fixed with methanol at -20°C for 5 min. Bacteria were revealed by indirect immunofluorescence with Texas Red-conjugated secondary Abs. The colocalization of bacteria with the NIRF probe was examined with the laser scanning confocal fluorescence microscope equipped with appropriate excitation and emission filters for FITC, Texas Red, and Cy5.5. Images were analyzed using Adobe Photoshop version 5.5 software.
Statistical analysis
Results, given as mean ± SE, were compared with Student’s t test. Differences were considered significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As suspended THP-1 cells are not suitable tools for studying
intracellular traffic of C. burnetii, they were treated by
PMA to induce their spreading and adhesion, and then incubated with
C. burnetii. Adding virulent organisms (200:1 bacterium-cell
ratio) to adherent THP-1 cells for 24 h led to the infection of
85 ± 6% of cells with 1–2 bacteria per cell. Cellular infection
slightly decreased from days 0 to 3 by 17% and then steadily reached
the initial value of infection after 5 days (Fig. 1
A). Beyond this time, a
decrease in viability of THP-1 cells impaired the determination of
cellular infection. The changes in bacterial number correlated with
bacterial viability, which was assessed under the same experimental
conditions. The percentage of live virulent organisms did not vary over
5 days (Fig. 1B). In contrast, avirulent variants of
C. burnetii were eliminated by adherent THP-1 cells. As they
were more efficiently internalized than virulent organisms, they were
added to THP-1 cells at a bacterium-cell ratio of 25:1. At day 0,
84 ± 8% of cells were infected with 1 or 2 bacteria, and
cellular infection decreased by 42% at day 1 and slowly went down to
reach 84% inhibition at day 5 (Fig. 1A). The viability of
avirulent organisms rapidly decreased during the course of experiments
because it was diminished by 35% at day 0 and 84% at day 5 (Fig. 1B). These results indicate that only virulent C.
burnetii survived in PMA-treated THP-1 cells.
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As previous reports suggested that C. burnetii
replicates in acidic compartments (5), we investigated the
relationship between acidic pH and C. burnetii survival in
adherent THP-1 cells. The intravacuolar pH was determined by
ratiometric analysis of fluorescence intensities of pH-sensitive
DM-NERF dextran. In uninfected THP-1 cells, the pH of phagosomes
containing latex beads was acidic. After 24 h of incubation of
THP-1 cells with C. burnetii, intravacuolar pH was 5.0
± 0.1 for virulent organisms and 5.2 ± 0.1 for avirulent
organisms (Table I). The acidic pH of
C. burnetii vacuoles results from the acquisition of
V-H+-ATPase, known to acidify phagosomes. The
percentage of vacuoles that accumulated
V-H+-ATPase was 58 ± 11% at h 0, and it
progressively increased to 80 ± 8% at h 72.
V-H+-ATPase also colocalized with avirulent
C. burnetii: 72 ± 10 and 88 ± 8% of vacuoles
containing avirulent organisms colocalized with
V-H+-ATPase at hours 0 and 72, respectively (Fig. 2). The V-H+-ATPase
was functional as demonstrated by using bafilomycin A1, a specific
inhibitor of V-H+-ATPase. In the presence of
bafilomycin A1, the pH of vacuoles containing virulent or avirulent
C. burnetii was significantly (p <
0.02) higher than that in the absence of bafilomycin A1 (Table I).
Hence, C. burnetii is present in vacuoles that are acidified
by V-H+-ATPase, independently of organism
virulence.
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As the acidic pH of C. burnetii vacuoles cannot account
for the survival of virulent organisms in THP-1 cells, we suggested
that the dynamics of vacuoles containing virulent organisms is distinct
from that of avirulent organisms. The intracellular traffic of C.
burnetii vacuoles was studied by measurement of organism
colocalization with the lysosomal protease cathepsin D. Cathepsin D did
not accumulate in vacuoles containing virulent C. burnetii
(Fig. 3A). At h 0, only
10 ± 5% of virulent C. burnetii colocalized with
cathepsin D, but cathepsin D appeared in the lumen of 42 ± 6% of
vacuoles containing avirulent organisms (Fig. 3A). It is
noteworthy that the amount of infection with avirulent C.
burnetii remained higher than in cells infected with virulent
organisms as a consequence of distinct phagocytosis efficiency. The
lack of cathepsin D colocalization with C. burnetii was not
due to its delayed acquisition. Indeed, the percentage of vacuoles
containing virulent C. burnetii that colocalized with
cathepsin D did not exceed 20% whatever the postinfection
time, but it steadily increased in cells infected with avirulent
variants, reaching 88 ± 7% after 96 h (Fig. 3B).
To confirm that defective acquisition of cathepsin D corresponds to
impaired phagosome-lysosome fusion, two probes that accumulated in the
lysosomal compartment were used. First, whereas the NIRF probe did not
colocalize with virulent organisms, it accumulated in phagosomes
containing avirulent organisms (Fig. 4A). Second, the
lysosomotropic probe neutral red colocalized only with phagosomes
containing avirulent variants of C. burnetii (Fig. 4B). Defective phagosome-lysosome fusion was not
strain-dependent. Indeed, the percentage of phagosomes containing
organisms from Priscilla and Q212 strains that colocalized with
cathepsin D was 10 ± 3% and 15 ± 4%, respectively (data
not shown). In addition, when virulent C. burnetii organisms
were heat-killed, they regained the ability to colocalize with
cathepsin D (33 ± 5% of positive vacuoles at day 0 and 80
± 9% of positive vacuoles after 96 h). Hence, defective
phagosome-lysosome fusion was related to C. burnetii
virulence.
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). The
colocalization of avirulent C. burnetii with Lamp-1 was
similar to that of virulent organisms. In addition, C.
burnetii colocalized with CD63: the colocalization was high at h 0
(55 ± 13% for virulent organisms and 62 ± 15% for
avirulent organisms), and it remained elevated during the incubation
time (Fig. 2). M6PR is a marker of late endosomes that is transiently
acquired by phagosomes containing inert particles. After 2 h,
24 ± 17% of phagosomes containing virulent C.
burnetii and 27 ± 10% of phagosomes containing avirulent
variants expressed M6PR. Although the percentage of vacuoles containing
C. burnetii organisms that expressed M6PR remained low, it
was similar in cells infected with virulent or avirulent organisms
(Fig. 2). Hence, the acquisition of Lamp-1, CD63, and M6PR by C.
burnetii phagosomes was not related to bacterial virulence.
Distinct results were obtained with Rab7, a small GTPase involved in
phagosome maturation (23). Rab7 was diffusively stained
within the cytoplasm and its fluorescence was concentrated around
bacteria (Fig. 5A). Only
26 ± 9% of vacuoles containing virulent organisms colocalized
with Rab7 at h 0; the percentage remained constant until 96 h
despite a moderate increase (38 ± 5%) at h 2. In contrast,
65 ± 7% of vacuoles containing avirulent organisms were
colocalized with Rab7 at h 0, and this percentage steadily decreased to
values similar to those of virulent organisms after 96 h (Fig. 5B). Hence, vacuoles containing virulent C.
burnetii partially acquired Rab7. Taken together, these data
suggest that vacuoles containing virulent C. burnetii follow
the endosomal pathway, as revealed by the acquisition of
V-H+-ATPase, Lamp-1, and CD63, but partially
acquire M6PR and Rab7.
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stimulates bacterial killing, maturation of C.
burnetii vacuoles, and changes of vacuolar pH
Because IFN- induces the killing of virulent C.
burnetii in THP-1 cells (12), we wondered whether
IFN- also affects the maturation of C. burnetii vacuoles.
The addition of IFN- (at 200 U/ml) to adherent THP-1 cells before
their infection decreased the viability of virulent C.
burnetii from 92 ± 8% at day 0 to 16 ± 5% after 2
days (Fig. 6). The induction of C.
burnetii killing was dose-dependent: a concentration of 50 U/ml
IFN- was sufficient to reduce C. burnetii viability (30
and 40% inhibition at days 1 and 2, respectively), and maximum killing
(85% inhibition) was obtained with 200 U/ml IFN-. We investigated
the effect of IFN- on intravacuolar pH and colocalization of
C. burnetii with endosome/lysosome markers. First, IFN-
significantly (p < 0.05) raised the pH of
vacuoles containing virulent C. burnetii to 6.2 ± 0.3,
equivalent to pH values obtained by treating monocytes with bafilomycin
A1 (Table I). IFN- exerted the same effect on the pH of vacuoles
containing avirulent organisms. The alkalinization of bacterial
vacuoles occurred after 24 h, suggesting that it was a relatively
late event. Second, in the presence of 200 U/ml IFN-, the percentage
of vacuoles containing virulent C. burnetii that colocalized
with cathepsin D was 62 ± 6% at h 0 and 80 ± 7% after h
24, whereas it never exceeded 20% in the absence of IFN- (Fig. 6).
IFN--mediated restoration of bacterial colocalization with cathepsin
D was observed with concentrations of IFN- similar to those required
for bacterial killing (data not shown). However, the cathepsin D that
colocalized with virulent organisms was not active. Indeed, the NIRF
probe colocalized with virulent organisms as demonstrated by FITC
fluorescence, but cathepsin D was inactive as shown by the lack of
Cy5.5 fluorescence (Fig. 7). In contrast,
the ability of IFN- to stimulate the maturation of C.
burnetii phagosomes involves Rab7. Indeed, IFN- increased the
colocalization of virulent C. burnetii with Rab7 as compared
with untreated cells (Fig. 6). The percentage of phagosomes that
expressed Rab7 was 53 ± 8% at h 0, and it increased to 83
± 9% at h 2. After 24 h, 45 ± 10% of phagosomes still
expressed Rab7. Taken together, these results show that the
pretreatment of THP-1 cells by IFN-, which induces C.
burnetii killing, improves the access of C. burnetii
phagosomes to some endosomal markers without leading to complete
maturation in phagolysosomes.
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to infected cells promotes C.
burnetii killing and phagosome maturation without changing the
pH
We wondered whether IFN--induced C. burnetii killing
results from the acquisition of endosomal/lysosomal markers or the
alkalinization of bacterial vacuoles. To discriminate between these two
hypotheses, THP-1 cells were infected with C. burnetii, thus
providing vacuoles containing live organisms, and then were treated
with 200 U/ml IFN-. This treatment reduced C. burnetii
viability after 24 and 48 h (72 ± 9 and 38 ± 3% of
viable bacteria, respectively), thus confirming the microbicidal effect
of IFN- administered before C. burnetii infection (Fig. 6). Adding IFN- to infected cells increased the colocalization of
C. burnetii with cathepsin D. Indeed, 42 ± 14 and
55 ± 12% of C. burnetii vacuoles colocalized with
cathepsin D after 8 and 24 h, respectively (Fig. 6). Vacuolar
cathepsin D was active as demonstrated by the Cy5.5 fluorescence of the
NIRF probe (Fig. 7). In addition, IFN- treatment of infected cells
restored the colocalization of Rab7 with C. burnetii
phagosomes as did IFN- pretreatment (Fig. 6). In contrast to the
effect of IFN- pretreatment of THP-1 cells, the addition of IFN-
to C. burnetii-infected cells did not affect the vacuolar pH
(Table I). Thus, the effects of IFN- on cathepsin D acquisition and
vacuolar pH are likely distinct.
Virulent C. burnetii survives and impairs phagosome-lysosome fusion in circulating monocytes
To extend the findings we reported in THP-1 cells to circulating
monocytes, isolated monocytes were cultured for 3 days to increase
their spreading without inducing their maturation into macrophages.
This procedure was required to visualize bacterial phagosomes with
confocal microscopy. First, we measured the viability of C.
burnetii in monocytes. The viability of avirulent organisms
decreased by 85% after 3 days of culture (Table II) and was residual after 5 days (data
not shown). In contrast, the viability of virulent organisms remained
constant during the 5 days of culture. Hence, only virulent C.
burnetii survived in circulating monocytes, confirming previous
results (3, 11). Second, we assessed the colocalization of
C. burnetii organisms with two markers of phagosome
maturation, Lamp-1 and cathepsin D. The percentage of vacuoles that
colocalized with Lamp-1 was high in monocytes infected with virulent or
avirulent C. burnetii at day 0, and all phagosomes had
acquired Lamp-1 at day 3 (Table II). In contrast, the pattern of
cathepsin D colocalization with C. burnetii was different.
At day 0, one-third of vacuoles acquired cathepsin D in monocytes
infected with avirulent C. burnetii, and almost all vacuoles
were positive for cathepsin D at day 3. In monocytes infected with
virulent organisms, 26 ± 7% of vacuoles colocalized with
cathepsin D at day 0, and this percentage remained low even at day 3
(Table II). Taken together, these results show that the survival of
virulent C. burnetii in circulating monocytes is associated
with impaired acquisition of cathepsin D.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IFN- stimulated the killing of C. burnetii by THP-1 cells
and affected the maturation of C. burnetii vacuoles as
assessed by the acquisition of cathepsin D. IFN- likely affects
cathepsin D acquisition by distinct mechanisms. IFN- pretreatment of
THP-1 cells induced the accumulation of inactive cathepsin D by
C. burnetii vacuoles, which is reminiscent of the results of
Ullrich et al. (33), who found that M. avium
phagosomes acquire an inactive form of cathepsin D. IFN- also
stimulated the alkalinization of C. burnetii vacuoles.
Vacuole alkalinization did not result from the exclusion of
V-H+-ATPase because the colocalization of
V-H+-ATPase and C. burnetii was
similar in THP-1 cells treated or not with IFN-. This finding is
surprising because IFN- has been reported to lower the pH of
M. avium vacuoles through the accumulation of
V-H+-ATPase (14) and to impair the
interaction of phagosomes with late endosomes and lysosomes without
interfering with acidification (16). In contrast, adding
IFN- to infected THP-1 cells stimulated the acquisition of active
cathepsin D but it had no effect on vacuolar pH. Thus, it is likely
that IFN--induced phagolysosomal fusion and vacuolar alkalinization
play different roles in C. burnetii killing. These results
have pathophysiological consequences. Resting monocytic cells are
unable to kill virulent C. burnetii but cannot support
bacterial replication. This latter is only achieved when monocytes are
specifically deactivated by IL-10 (11). IL-10 does not
modify the traffic of C. burnetii vacuoles in monocytes (our
unpublished data). In contrast, the activation of monocytic
cells by IFN- reprogrammed them to be microbicidal against C.
burnetii through phagosomal maturation. As IFN- is associated
with the cure of C. burnetii infections (2), it
is likely that the restoration of phagosome-lysosome fusion is critical
for the control of Q fever.
The survival of C. burnetii into THP-1 cells and monocytes
is associated with altered phagosome maturation. The activation of
these professional phagocytes by IFN- leads to C.
burnetii killing and restores phagosomal maturation. We propose
two potential mechanisms for IFN--induced killing of C.
burnetii: an early mechanism based on phagosome maturation and a
late mechanism involving modulation of vacuolar pH. Therapeutic
elimination of C. burnetii in Q fever might benefit from
exploring these two parameters of bacterial killing.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jean-Louis Mege, Unité des Rickettsies, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6020, Institut Fédératif de Recherche 48, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. E-mail address: Jean-Louis.Mege{at}medecine.univ-mrs.fr
3 Abbreviations used in this paper: V-H+-ATPase, vacuolar proton ATPase; Lamp, lysosome-associated membrane protein; M6PR, cation-independent mannose-6-phosphate R; NIRF, near-infrared fluorescence.
Received for publication April 22, 2002. Accepted for publication August 5, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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v3 integrin and CR3. J. Immunol. 163:6078.-mediated control of Coxiella burnetii survival in monocytes: the role of cell apoptosis and TNF. J. Immunol. 162:2259.. Annu. Rev. Immunol. 15:749.[Medline]
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 J. Ochoa-Reparaz, J. Sentissi, T. Trunkle, C. Riccardi, and D. W. Pascual Attenuated Coxiella burnetii Phase II Causes a Febrile Response in Gamma Interferon Knockout and Toll-Like Receptor 2 Knockout Mice and Protects against Reinfection Infect. Immun., December 1, 2007; 75(12): 5845 - 5858. [Abstract] [Full Text] [PDF]
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 K. Lubick, M. Radke, and M. Jutila Securinine, a GABAA receptor antagonist, enhances macrophage clearance of phase II C. burnetii: comparison with TLR agonists J. Leukoc. Biol., November 1, 2007; 82(5): 1062 - 1069. [Abstract] [Full Text] [PDF]
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M. Andoh, G. Zhang, K. E. Russell-Lodrigue, H. R. Shive, B. R. Weeks, and J. E. Samuel T Cells Are Essential for Bacterial Clearance, and Gamma Interferon, Tumor Necrosis Factor Alpha, and B Cells Are Crucial for Disease Development in Coxiella burnetii Infection in Mice Infect. Immun., July 1, 2007; 75(7): 3245 - 3255. [Abstract] [Full Text] [PDF]
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 P. A. Beare, D. Howe, D. C. Cockrell, and R. A. Heinzen Efficient Method of Cloning the Obligate Intracellular Bacterium Coxiella burnetii Appl. Envir. Microbiol., June 15, 2007; 73(12): 4048 - 4054. [Abstract] [Full Text] [PDF]
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A. Honstettre, S. Meghari, J. A. Nunes, H. Lepidi, D. Raoult, D. Olive, and J.-L. Mege Role for the CD28 Molecule in the Control of Coxiella burnetii Infection Infect. Immun., March 1, 2006; 74(3): 1800 - 1808. [Abstract] [Full Text] [PDF]
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 N. Martin-Orozco, N. Touret, M. L. Zaharik, E. Park, R. Kopelman, S. Miller, B. B. Finlay, P. Gros, and S. Grinstein Visualization of Vacuolar Acidification-induced Transcription of Genes of Pathogens inside Macrophages Mol. Biol. Cell, January 1, 2006; 17(1): 498 - 510. [Abstract] [Full Text] [PDF]
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J.-D. Sauer, J. G. Shannon, D. Howe, S. F. Hayes, M. S. Swanson, and R. A. Heinzen Specificity of Legionella pneumophila and Coxiella burnetii Vacuoles and Versatility of Legionella pneumophila Revealed by Coinfection Infect. Immun., August 1, 2005; 73(8): 4494 - 4504. [Abstract] [Full Text] [PDF]
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 Z. Hmama, K. Sendide, A. Talal, R. Garcia, K. Dobos, and N. E. Reiner Quantitative analysis of phagolysosome fusion in intact cells: inhibition by mycobacterial lipoarabinomannan and rescue by an 1{alpha},25-dihydroxyvitamin D3-phosphoinositide 3-kinase pathway J. Cell Sci., April 15, 2004; 117(10): 2131 - 2140. [Abstract] [Full Text] [PDF]
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 D. A. Drevets, P. J. M. Leenen, and R. A. Greenfield Invasion of the Central Nervous System by Intracellular Bacteria Clin. Microbiol. Rev., April 1, 2004; 17(2): 323 - 347. [Abstract] [Full Text] [PDF]
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D. S. Zamboni and M. Rabinovitch Phagocytosis of Apoptotic Cells Increases the Susceptibility of Macrophages to Infection with Coxiella burnetii Phase II through Down-Modulation of Nitric Oxide Production Infect. Immun., April 1, 2004; 72(4): 2075 - 2080. [Abstract] [Full Text] [PDF]
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D. S. Zamboni Genetic Control of Natural Resistance of Mouse Macrophages to Coxiella burnetii Infection In Vitro: Macrophages from Restrictive Strains Control Parasitophorous Vacuole Maturation Infect. Immun., April 1, 2004; 72(4): 2395 - 2399. [Abstract] [Full Text] [PDF]
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 A. Honstettre, E. Ghigo, A. Moynault, C. Capo, R. Toman, S. Akira, O. Takeuchi, H. Lepidi, D. Raoult, and J.-L. Mege Lipopolysaccharide from Coxiella burnetii Is Involved in Bacterial Phagocytosis, Filamentous Actin Reorganization, and Inflammatory Responses through Toll-Like Receptor 4 J. Immunol., March 15, 2004; 172(6): 3695 - 3703. [Abstract] [Full Text] [PDF]
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A. M. Woolsey, L. Sunwoo, C. A. Petersen, S. M. Brachmann, L. C. Cantley, and B. A. Burleigh Novel PI 3-kinase-dependent mechanisms of trypanosome invasion and vacuole maturation J. Cell Sci., September 1, 2003; 116(17): 3611 - 3622. [Abstract] [Full Text] [PDF]
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C. Capo, A. Moynault, Y. Collette, D. Olive, E. J. Brown, D. Raoult, and J.-L. Mege Coxiella burnetii Avoids Macrophage Phagocytosis by Interfering with Spatial Distribution of Complement Receptor 3 J. Immunol., April 15, 2003; 170(8): 4217 - 4225. [Abstract] [Full Text] [PDF]
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Untreated cells; 
, IFN-