Cholesterol is known to play key roles in cardiovascular disorders, obesity, diabetes, and infectious diseases caused by numerous bacterial, viral, and protozoal pathogens. Intracellular pathogens in particular target cholesterol at various stages of infection. For example, Mycobacterium bovis
and Helicobacter pylori
directly target cholesterol as a “docking site” to stabilize interactions with the host cell membrane and initiate internalization (1–3
spp., Brucella suis
, Listeria monocytogenes
, Leishmania donovani
, and Plasmodium falciparum
appear to target cholesterol-rich lipid rafts during entry into both phagocytic and nonphagocytic cells (3–13
). Once inside the cell, cholesterol is often targeted during establishment of the intracellular niche and bacterial growth. For example, Mycobacterium tuberculosis
and Mycobacterium leprae
accumulate cholesterol in the early phagosome as a mechanism to inhibit phagosome-lysosome fusion and promote pathogen survival (14–16
). M. tuberculosis
also utilizes a cholesterol import system to hijack host cell cholesterol as a carbon and energy source (17
). Chlamydia trachomatis
intercepts cholesterol trafficking from the Golgi apparatus and incorporates cholesterol into the Chlamydia
-containing inclusion body as well as the bacterial cell wall (18
). Thus, modulation of cellular cholesterol by diverse microbial pathogens appears to play an important role in promoting pathogen entry, survival, and subsequent disease.
Recent reports have implicated cholesterol as an important factor during infection by the intracellular bacterial pathogen Coxiella burnetii
, a significant cause of culture-negative endocarditis in the United States (19–21
). An obligate intracellular pathogen during natural infection, C. burnetii
forms a unique niche in a modified acidic phagolysosome known as the parasitophorous vacuole (PV). After uptake by the host cell via phagocytosis, the bacterium resides in a tight-fitting nascent phagosome that matures through the default endocytic pathway (22
). Approximately 24 to 48 h postinfection, the C. burnetii
PV expands through fusion with early and late endosomes, lysosomes, and autophagosomes (24
). As a result, the mature PV membrane is a hybrid of host vesicular membranes, and the vacuole displays various characteristics of a phagolysosome, including lysosomal hydrolases (acid phosphatase, cathepsin D, and 5′-nucleotidase) and an acidic pH of ~4.5 to 5 (24
). Establishment of a replication-competent PV requires the C. burnetii
Dot/Icm type 4B secretion system (T4BSS), which manipulates host cell trafficking and signaling pathways via the activity of effector proteins secreted into the host cytoplasm (25
Formation of the C. burnetii
PV is a highly dynamic process involving vesicular trafficking and fusion events, with the PV membrane playing a central role. A distinguishing feature of the C. burnetii
PV membrane, based on staining with the fluorescent sterol-binding compound filipin, is that it is rich in sterols (21
). A role for cholesterol during C. burnetii
infection was suggested by gene expression analysis of infected host cells, which found that genes involved in cholesterol efflux and storage are upregulated during C. burnetii
). Further, a recent screen of a FDA-approved drug library identified 57 drugs that perturb host cell cholesterol homeostasis also block C. burnetii
growth in THP-1 human macrophage-like cells (19
). Intriguingly, these drugs had a more pronounced effect on C. burnetii
than on Legionella pneumophila
, Rickettsia conorii
, or Brucella abortus
, suggesting that C. burnetii
may be uniquely sensitive to altered host cell cholesterol homeostasis. Additionally, when cholesterol transport from endosomes and presumably the C. burnetii
PV was blocked through knockdown of the cholesterol transporter NPC-1, C. burnetii
growth was significantly attenuated (19
). Together, these studies suggest that cholesterol is an important player affecting the C. burnetii
-host cell interaction.
In order to further understand the role of cholesterol during C. burnetii
infection, we developed a novel cholesterol-free host cell tissue culture system using cells lacking DHCR24, the final enzyme in cholesterol biosynthesis (20
). When adapted to serum-free media, DHCR24−/−
mouse embryonic fibroblasts lack both endogenous and exogenous cholesterol sources, and instead, they accumulate desmosterol in cellular membranes. Cholesterol-free cells are an attractive model for deciphering the role of cholesterol in cellular processes, enabling cholesterol manipulation by the addition of exogenous cholesterol to the media. Our prior studies with this model system revealed that C. burnetii
uptake into fibroblast cells was dependent on cholesterol-rich lipid rafts and the integrin αv
). Strikingly, C. burnetii
PV formation and intracellular replication did not require cholesterol. Further, the PV acquired the typical PV markers Rab7, flotillin-2, syntaxin 7, syntaxin 8, and Vamp7 and contained active cathepsin, indicating that the majority of PV maturation events occurred in the absence of cholesterol. However, the lack of the late endosomal marker CD63 in the PV lumen in cholesterol-free cells suggests that cholesterol regulates one or more intracellular trafficking pathways to the PV (20
While studies thus far indicate that cholesterol plays a key role during C. burnetii infection, how cholesterol affects the formation and maintenance of the PV, as well as C. burnetii growth, is not yet known. Here, we utilized cholesterol-free cells to further decipher the role of cholesterol in C. burnetii-host cell interactions. Our studies surprisingly revealed that increasing cholesterol in the C. burnetii PV inhibits fusion between the PV and endosomes, acidifies the PV, and results in C. burnetii degradation. Our data demonstrating a cholesterol-mediated negative effect on an intracellular bacterial pathogen is novel and may have broader implications in the treatment of C. burnetii infection.
Cholesterol is a critical lipid constituent of cellular membranes, regulating membrane dynamics, trafficking, and signaling. Due to its involvement in important host cell processes, an increasing number of pathogens, including Leishmania
spp., Salmonella enterica
, Staphylococcus aureus
spp., and Listeria monocytogenes
, have been reported to exploit host cell cholesterol (3–5
). To understand the role of cholesterol during C. burnetii
-host interaction, we utilized a cholesterol-free tissue culture model system that lacks both endogenous cholesterol (from biosynthesis) and exogenous cholesterol (from serum). In a previous study, we used this system to establish that C. burnetii
entry into fibroblasts occurred through lipid raft-mediated αv
). In addition, with the exception of CD63, endolysosomal markers were associated with the C. burnetii
PV regardless of the presence or absence of cholesterol, indicating that PV maturation was not cholesterol dependent. Here, we made the surprising discovery that increasing cellular cholesterol is detrimental to C. burnetii
survival. C. burnetii
is most sensitive to cholesterol during the early stages of infection, with increasing cholesterol levels leading to altered PV fusion, increased acidity, and bacterial degradation. Cholesterol traffics to the PV and drugs that trap cholesterol in the endolysosomal system are bactericidal, suggesting that PV cholesterol influences PV biology and C. burnetii
pathogenesis. These data strongly support the conclusion that manipulating cholesterol in the bacterium-containing PV kills C. burnetii
growth is sensitive to drugs that target cholesterol biosynthesis and uptake (19
). Further, Czyz et al. reported that treatment of THP-1 cells with FDA-approved drugs that alter cellular cholesterol distribution similar to U18666A also inhibit C. burnetii
intracellular growth (19
). In support of these data, they also showed decreased C. burnetii
growth in THP-1 macrophage-like cells deficient in NPC-1, a cholesterol transporter that facilitates cholesterol export from late endosomes and lysosomes (19
). We found that both plasma membrane cholesterol and the cholesterol-binding exogenous protein LDL travel to the PV. We hypothesize that cholesterol supplementation of cholesterol-free MEFs leads to increased cholesterol in the PV membrane compared to cholesterol-free MEFs. Our model system further revealed that C. burnetii
PV size and bacterial growth are sensitive to cellular cholesterol, further supporting the hypothesis that manipulating host cell cholesterol homeostasis adversely affects C. burnetii
infection. Importantly, our data show that rather than simply blocking bacterial growth or PV formation, increasing PV cholesterol leads to C. burnetii
lysis. Remarkably, treatment for only 3 h with U18666A, which traps cholesterol in the PV, killed 80% of the bacteria. These data, along with data from Czyz et al. (19
), suggest that C. burnetii
is sensitive to altered cholesterol distribution within the cell, particularly accumulation of cholesterol in the endosomal trafficking pathway and the PV. With other bacteria, including Chlamydia trachomatis
, Staphylococcus aureus
, and Mycobacterium
spp., the presence of cholesterol is reported to be beneficial to the bacterium, with cholesterol depletion leading to reduced bacterial growth (11
). The unique sensitivity of C. burnetii
to host cell cholesterol may reflect the distinctive intracellular niche this bacterium occupies.
Cholesterol is a key regulator of endosomal trafficking and fusion (47–49
). We previously found that the endosome marker CD63 was absent in the PV lumen in cholesterol-free MEFs, suggesting that cholesterol is required for late endosomal trafficking to the PV (20
). In this study, we discovered that lytic PVs containing degraded C. burnetii
, which are found only in cells with cholesterol, were no longer fusogenic with the endosomal pathway. Most likely, bacterial degradation leads to a loss of the T4BSS effector proteins required to maintain PV fusogenicity. The C. burnetii
T4BSS is not required for short-term intracellular survival, with a T4BSS mutant persisting for several days in a viable form (25
). Thus, it is unlikely that a cholesterol-dependent loss in PV fusogenicity would lead to bacterial degradation. However, cholesterol is specifically toxic to C. burnetii
during the initial stages of PV biogenesis and expansion, and it is possible that cholesterol plays a role in activating T4BSS secretion early during PV development. Most likely, both the timing and amount of PV cholesterol are tightly regulated by the bacteria to regulate PV-endosome fusion.
In addition to altered fusogenicity, the PVs from MEFs with cholesterol are significantly more acidic than PVs from cholesterol-free cells. Further, U18666A treatment also increased PV acidity in a vATPase-dependent manner. Importantly, blocking acidification by vATPase also rescued bacterial viability, demonstrating that increasing the PV acidity kills C. burnetii
. This is a surprising finding, given that C. burnetii
metabolism is activated by acid (35
) and the PV pH has previously been reported to be approximately 4.8 (37
). Our studies found the PVs in cholesterol-free MEFs and HeLa cells to be slightly more alkaline at pH 5.2. The differences in measured pH between studies might be a result of different pH-sensitive reagents. We utilized the fluorescein derivative Oregon Green 488, given the improved pH sensitivity of Oregon Green 488 in acidic environments (41
). A recent study on the role of lysosome-associated membrane glycoprotein (LAMP) proteins in PV maturation found the pH of the PV to be between 4.0 and 4.5 using LysoSensor Yellow/Blue DND-160 (50
). In our hands, this reagent stained the bacteria and was not free in the PV lumen, prompting us to utilize the dual dextran labeling approach (41
). Regardless, it is clear that the PV is more acidic in MEFs with cholesterol and HeLa cells treated with U18666A, and this increased acidity kills C. burnetii
. However, the mechanism behind C. burnetii
degradation is not known. Host cathepsin D and lysosomal acid phosphatases accumulate in the PV (35
), and the PV is proteolytically active, presumably due to the presence of host proteases (51
). It is possible that increased acidity further activates lysosomal degradative enzymes beyond the threshold the bacteria can survive. Detailed characterization of the PV proteolytic activity is needed to fully understand how C. burnetii
survives in this environment.
Previously, a drug screen revealed that C. burnetii
growth is sensitive to 57 FDA-approved drugs that perturb host cell cholesterol homeostasis (19
). We used several drugs from this screen to further validate our hypothesis that C. burnetii
lysis was due to cholesterol-induced changes in the PV pH. Treatment with six of the eight selected drugs resulted in significant C. burnetii
lysis which could be at least partially rescued by blocking acidification through vATPase. Importantly, these drugs were shown to have little to no effect on C. burnetii
growth in axenic media or on host cell viability (19
). While the mode of action differs between these drugs, we confirmed the results of previous studies that they altered cholesterol distribution within the cell (19
). Treatment with several of these drugs appeared to increase cholesterol within the PV. Together, these findings reveal a potential vulnerability in the C. burnetii
lifestyle which could be targeted with currently available drugs.
The cholesterol-mediated negative effect on intracellular C. burnetii
raises intriguing questions as to how C. burnetii
successfully colonizes cholesterol-containing cells during natural infection, given that cholesterol is an essential lipid for host cells outside the laboratory setting. Accumulating evidence suggests that C. burnetii
possesses multiple mechanisms to manipulate host cholesterol metabolism. For example, Howe and Heinzen reported differential expression of cholesterol biosynthesis-related genes in C. burnetii
-infected Vero cells (21
). Expression profiling of C. burnetii
-infected THP-1 cells suggests that C. burnetii
actively upregulates expression of apoE
, which are involved in cholesterol efflux and storage, respectively (26
). Beyond gene expression, cholesterol storage organelles called lipid droplets have been observed in and around the PVs of infected primary human alveolar macrophages (52
). It is possible that C. burnetii
targets the carefully regulated host cholesterol homeostasis, upregulating storage and efflux while also decreasing biosynthesis. In addition, we recently showed that C. burnetii
recruits the host cell sterol-binding protein ORP1L to the PV, where it participates in membrane contact sites between the PV and endoplasmic reticulum (53
). Finally, C. burnetii
expresses two eukaryote-like sterol reductase enzymes that could modify cholesterol (54
). This intriguing possibility might explain the intense filipin labeling of the PV, with a bacterium-derived β-hydroxysterol other than cholesterol dominating the PV membrane.
In summary, our data suggest that the presence of cholesterol in the PV during the initial phases of PV formation negatively affects PV formation and C. burnetii
survival. While not absolutely required for C. burnetii
growth, some cholesterol is needed for optimal PV development through fusion with late endosomes (20
). However, too much PV membrane cholesterol leads to increased PV acidification, decreased fusion with endosomes, and eventual bacterial degradation. We propose that the amount of cholesterol in the PV membrane regulates key aspects of PV function, and C. burnetii
must maintain a delicate balance of PV membrane cholesterol. This would explain the unique sensitivity of C. burnetii
to drugs that target different aspects of host cholesterol metabolism: any slight shift in host cholesterol homeostasis would impact PV membrane cholesterol levels. Identifying both the bacterial and host pathways involved in this delicate balance may yield novel targets to treat or prevent C. burnetii
MATERIALS AND METHODS
Bacteria and mammalian cells.
Nine Mile Phase II (NMII) (clone 4, RSA439) and mCherry-expressing C. burnetii
) were purified from Vero cells (African green monkey kidney epithelial cells [ATCC CCL-81; American Type Culture Collection, Manassas, VA]) and stored as previously described (56
). Vero cells were maintained in RPMI 1640 medium (Corning, New York, NY) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA) at 37°C and 5% CO2
mouse embryonic fibroblasts (MEFs) were cultured in fibroblast media supplemented with serum-free growth kit (ATCC) and cholesterol (Synthechol; Sigma-Aldrich, St. Louis, MO) as previously described (20
). The multiplicity of infection (MOI) was optimized for each bacterial stock, cell type, and infection condition for a final infection of ca. one internalized bacterium/cell at 37°C and 5% CO2
A total of 5 × 104 MEFs were plated onto ibidi-treated channel µslide VI0.4 (3 × 103 cells per channel; ibidi USA Inc., Verona, WI) and allowed to adhere overnight. After the MEFs were infected with C. burnetii for 1 h, they were washed with phosphate-buffered saline (PBS) to remove extracellular bacteria and incubated in media containing the indicated cholesterol concentrations. At different time points postinfection, cells were fixed with 2.5% paraformaldehyde (PFA) on ice for 15 min and then permeabilized/blocked for 15 min with 0.1% saponin and 1% bovine serum albumin (BSA) in PBS. The cells were incubated with rat anti-LAMP1 (catalog no. 553792; BD Biosciences, San Jose, CA) and rabbit anti-C. burnetii primary antibodies in saponin-BSA-PBS for 1 h, followed by Alexa Fluor secondary antibodies (Invitrogen) for 1 h. Following washing with PBS, ProLong Gold with 4′,6′-diamidino-2-phenylindole (DAPI) (Invitrogen) was added, and the cells on the slides were visualized on a Leica inverted DMI6000B microscope (63× oil immersion objective). Images were captured and processed identically, and a cross-sectional area through the middle of the PV was measured using ImageJ software. Approximately 20 PVs were measured per condition for each of three independent experiments.
C. burnetii growth in MEFs.
MEFs were plated at 1 × 105
cells/well in a six-well plate under different cholesterol conditions and allowed to adhere overnight. After the MEFs were infected with C. burnetii
for 1 h in 500 µl medium, the wells were washed with PBS to remove extracellular bacteria and then gently scraped into 3 ml of medium. For the day 0 sample, 1 ml of the cell suspension was centrifuged at 20,000 × g
for 10 min, and the pellet was frozen at −20°C. The remaining cells were left in the six-well plate in medium supplemented with cholesterol. The medium was changed daily to ensure constant cholesterol concentrations. At 6 days postinfection, the cells were harvested by scraping the cells into the growth medium and centrifuging at 20,000 × g
for 10 min. Bacterial DNA was extracted from the pellets using the UltraClean microbial DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA) according to the manufacturer’s instructions. Quantitative PCR for genome equivalents was performed using a primer set specific for dotA
) and Luminaris Color HiGreen quantitative PCR (qPCR) master mix (Thermo Scientific) with an Applied Biosystems 7500 real-time PCR cycler. Each experiment was done in duplicate.
Quantitation of lytic PVs containing lysed C. burnetii.
DHCR24−/− MEFs were plated under different cholesterol conditions at 5 × 104 cells per well of a six-well plate and infected with mCherry-expressing C. burnetii (mCherry-C. burnetii) for 1 h as described above. Approximately 24 h later, the cells were scraped into fresh medium, resuspended to 1 × 105 cells/ml, and plated onto ibidi-treated channel µslide VI0.4 (3 × 103 cells per channel). The medium was changed daily, and cells were examined live every 24 h on a Leica inverted DMI6000B microscope with a 63× oil immersion objective. PVs with visible mCherry fluorescence in the PV lumen were scored as “lytic PVs” with 50 PVs scored for each condition for three individual experiments.
HeLa cells (5 × 104
) were infected with mCherry-C. burnetii
in 6-well plates for 1 h. At 2 days postinfection, the cells were trypsinized and resuspended to 1 × 105
cells/ml, and plated onto ibidi-treated channel µslide VI0.4
(3 × 103
cells per channel; Ibidi). At 3 days postinfection, dimethyl sulfoxide (DMSO) control, U18666A (1 or 5 µM), or the indicated FDA-approved drugs (see Table S1
in the supplemental material; obtained from Sigma and used at a final concentration of 20 µM) with or without vATPase inhibitor bafilomycin A1 (100 nM) were added to the cells and incubated for the time indicated prior to counting lytic PVs as described above. At least 50 PVs were scored for each condition for three individual experiments.
C. burnetii viability by fluorescent infectious focus-forming unit (FFU) assay.
To test viability of C. burnetii in MEFs under different cholesterol conditions, 1 × 104 cells/well were infected with C. burnetii for 1 h in a 48-well plate, washed extensively with PBS, and incubated with media containing different cholesterol concentrations. At the indicated time points, cells were incubated for 5 min with sterile water, pipetted up and down to lyse cells, and diluted 1:5 in RPMI 1640 with 2% FBS (2% FBS-RPMI). Serial dilutions were added to confluent monolayers of Vero cells in a 24-well plate and incubated for 5 days. Plates were fixed with methanol and stained with rabbit anti-C. burnetii antibody and DAPI to confirm monolayer integrity. Four fields per well were captured on an Evos automated microscope (Thermo Fisher) with a 4X objective, and fluorescent focus units were quantitated using ImageJ. Each experiment was done in duplicate.
To determine bacterial viability in drug-treated cells, HeLa cells were plated at 5 × 104 cells/well in a six-well plate and infected with mCherry-C. burnetii. At 2 days postinfection, the cells were trypsinized and replated in 24-well plates at 5 × 104 cells/well. Approximately 16 h later, the cells were treated with DMSO or drug with or without the vATPase inhibitor bafilomycin A1 (100 nM) for the time indicated, at which point the medium was aspirated from the 24-well plate and the cells were lysed by incubation in sterile water for 5 min. After the cells were pipetted up and down, the released bacteria were diluted 1:5 in 2% FBS-RPMI and plated in 10-fold serial dilutions onto confluent Vero cell monolayers in a 96-well ibidi-treated µplate (ibidi). The plate was fixed with 2.5% PFA 5 days later and stained with DAPI, and the number of fluorescent foci was determined as described above. Each experiment was done in duplicate.
Microscopy for cholesterol trafficking.
To monitor trafficking of plasma membrane cholesterol, fluorescent cholesterol (TopFluor cholesterol; Avanti Polar Lipids) was resuspended at 20 mg/ml in ethanol. Twenty microliters of this solution was added to 1 ml of 10% methyl-beta-cyclodextrin (Sigma) in serum-free RPMI 1640 medium. The solution was sonicated in a water bath sonicator (Avanti) for 30 s, and insoluble material was pelleted by spinning for 2 min at 20,000 × g. MEFs with cholesterol were infected with mCherry-C. burnetii and plated onto an ibidi µslide as described above. At 3 days postinfection, fluorescent cholesterol (final concentration of 30 µg/ml) was added to the cells for 24 h. Live-cell images were taken with a modified PerkinElmer UltraView spinning disk confocal connected to a Nikon Eclipse Ti-E inverted microscope with a 63× oil immersion objective.
For trafficking of BODIPY-LDL, MEFs were infected and plated onto an ibidi µslide as described above. The cells were incubated for 5 min on ice with 25 µg/ml BODIPY-LDL (Invitrogen), washed twice with medium, and visualized after 4 h of incubation at 37°C.
To visualize endogenous free sterols after drug treatment, infected cells were plated onto ibidi µslides and treated as described above. The cells were fixed with 2.5% PFA on ice for 15 min and incubated with 1:100 filipin (Cayman Chemicals, Ann Arbor, MI) in PBS with 1% BSA for 1 h. After the cells were washed with PBS three times, they were incubated with rat anti-LAMP1 for MEFs (catalog no. 553792; BD Biosciences, San Jose, CA) or mouse anti-CD63 for HeLa cells for 1 h, followed by three washes in PBS and a 1 h incubation with Alexa Fluor 488 anti-mouse secondary antibody. After the cells were washed three times with PBS, ProLong Gold was added to the wells, and samples were visualized on a Leica inverted DMI6000B microscope (63× oil immersion objective). Images were captured under identical capture settings and processed identically using ImageJ.
C. burnetii growth in cell-free media.
To complex cholesterol to BSA, 500 µg of cholesterol (10 mg/ml chloroform stock; Avanti) was dried down in a glass tube under a nitrogen stream. The lipid film was resuspended into 2.5 ml of 5% fatty acid-free BSA using a water bath sonicator (Avanti). The resulting 200 µg/ml stock was sterile filtered and added to a final concentration of 5 µg/ml in ACCM-2 (40
). C. burnetii
bacteria were diluted to approximately 1 × 105
genomes/ml in ACCM-2 with BSA or BSA-cholesterol, and 7 ml was transferred to a T25 flask and incubated as previously described (40
). Every 24 h, 50 µl was removed and added to a tube with 150 µl PBS and a half volume of 0.1-mm zirconia-silica beads (BioSpec Products, Bartlesville, OK). Bacteria were lysed by bead beating in a FastPrep FP120 (Thermo Scientific) and analyzed by qPCR as previously described (20
). Each experiment was done in duplicate.
To test bacterial sensitivity to U18666A, ACCM-2 was inoculated at approximately 1 × 105
bacteria/ml with mCherry-C. burnetii
and grown for 5 days as previously described (40
). Bacteria (500 µl) were treated for 6 h with DMSO or U18666A in 24-well plates under normal C. burnetii
culture conditions. The bacteria were diluted 1:10 in 2% FBS-RPMI prior to the FFU assay in 96-well ibidi-treated µplates as described above.
Cells were infected with mCherry-C. burnetii in six-well plates and replated onto ibidi slides at 2 days postinfection as described above. PVs were selected and marked in Elements software on the spinning disk confocal microscope in a live-cell environmental chamber. Individual ibidi channels were pulsed with 1 mg/ml Alexa Fluor 488-dextran (molecular weight [MW] of 10,000) for 10 min in medium, followed by four washes with medium to remove uninternalized dextran and finally replaced with either basal medium or basal medium with cholesterol (5 µg/ml). The PVs were then focused, and confocal images through the entire PV were obtained every 6.33 min for 38 min. The fluorescence intensity of dextran inside the PV was calculated using the average intensity multiplied by the PV volume using ImageJ.
PV pH measurements.
The pH measurement was performed as previously described with slight modifications (57
). Briefly, MEFs were infected with mCherry-C. burnetii
in six-well plates, incubated with and without cholesterol, and replated onto ibidi slides at 2 days postinfection as described above. For measurement of PV pH in U18666A-treated cells, HeLa cells were infected with mCherry-C. burnetii
in six-well plates and replated onto ibidi plates at 2 days postinfection. Under both conditions, at 3 days postinfection, cells were incubated with pH-sensitive Oregon Green 488 dextran (MW, 10,000; Invitrogen) and pH-stable Alexa Fluor 647 dextran (MW, 10,000; Invitrogen) for 4 h at a concentration of 0.5 mg/ml. MEFs were imaged directly with a 63× oil immersion objective under identical capture settings.
To measure the time-dependent change in PV pH, individual PVs were selected and imaged, and then treated with 5 µM U18666A or DMSO as a vehicle control. Starting from 15 min after the treatment, cells were then imaged every 15 min for the next 2 h. The PV fluorescence intensity was measured using ImageJ, and the Oregon Green 488/Alexa Fluor 647 ratio was calculated. To generate a standard curve for MEFs and HeLa cells, the respective infected cells were incubated with the ionophores nigericin (10 µM) and monensin (10 µM) for 5 min at room temperature, followed by buffers with different pHs (pH 4.0 to 7.0) before imaging. At least 20 PVs were imaged at each pH for every experiment, and the ratio of fluorescence intensity at 488/647 nm were plotted against the pH of the respective buffer to obtain a sigmoidal standard curve.
Image processing and analysis were done with ImageJ software (W. S. Rasband, National Institutes of Health, Bethesda, MD) (58
). Statistical analyses were performed using unpaired two-tailed t
test, ordinary one-way or two-way analysis of variance (ANOVA) with Tukey’s or Dunnett’s multiple-comparison test in Prism (GraphPad Software, Inc., La Jolla, CA).