It has been previously demonstrated that C. burnetii transits the endocytic and autophagic pathways generating an acidic and replicative niche with phagolysosomal characteristics which develops in a large CRV at 48 h of infection (Akporiaye et al., 1983; Maurin et al., 1992; Heinzen et al., 1999; Grieshaber et al., 2002).

We and others have used LysoTracker to label the CRV indicating that these vacuoles have an acidic internal environment. To assess whether the entire population of CRVs posesses the same internal acidic characteristics, infected CHO cells were incubated with LysoSensor green. This membrane permeant dye increases its fluorescence in acidic environments. Living cells labeled with this compound were analyzed by confocal microscopy. To our surprise, we observed that several CRVs were indeed positive for LysoSensor but most of them displayed less or almost no fluorescence (Figures 1A,B), suggesting that the CRVs did not present homogeneous acidic properties at the time of observation.

Given these striking observations, a different experimental approach was used to assess the acidic pH inside the CRV. pHrodo is a compound that is almost non-fluorescent at neutral pH, and fluoresces brightly in an acidic environment. Thus, this pH marker allows detecting acidification or neutralization events by monitoring variations in its fluorescence intensity. Therefore, we first verified that the dextran/pHrodo beads that we were using actually responded to the changes in pH, as expected. As shown in the Figures S1A,B, the beads displayed a clear fluorescence intensity dependency according to the pH of the media. It is well-known that the Coxiella vacuole is highly fusogenic and readily fuses with other endosomes or phagosomes (e.g., bacteria-containing compartments). Therefore, we allowed the cells to endocytose pHrodo dextran or to phagocytose pHrodo-conjugated heat-killed Staphylococcus aureus (SA/pHrodo). CHO cells were infected with C. burnetii and incubated with pHrodo dextran or SA/pHrodo. The acidification of the CRV was visualized by fluorescence live cell imaging. As depicted in Figures 1C–F, pHrodo fluorescence displays heterogeneity between CRVs, suggesting different populations of these vacuoles depending on the pH. Our results indicate that a large percentage (more than 50%) of the vacuoles have a pH 7 or above because their low fluorescence intensity (Figures S1D–E). Interestingly, these differences were not dependent on the vacuole diameter (Figure S1C), suggesting that the fluorescence variability was not due to a dilution of the fluorochrome in the large CRV. Therefore, we can infer that there are different populations of CRVs with respect to their pH and that the possible transient loss of acidity in some of them seems to be independent on the size of the vacuole.

In order to assure that the large majority of the vacuoles has incorporated the pHrodo dye, we did some additional controls to the experiments described above. CHO cells were incubated with SA/pHrodo. The S. aureus particles were previously incubated with Hoechst to label the bacteria. Cells were analyzed by confocal live imaging (Figure S1D). The percentage of S. aureus positive vacuoles (labeled with Hoechst) as well as vacuoles pHrodo positive or negative was determined. As shown in Figure S1E, approximately 90% of the C. burnetii vacuoles contained S. aureus particles. However, only 40% of them were labeled with pHrodo, indicating that the pH of the rest of the vacuoles was not acidic. We also observed that the pHrodo fluorescence was dispersed throughout the CCV and not associated with the staph particle. We think this phenomenon is likely due to the release of the dye from the bacterial particles inside the internal environment of the CCV through a mechanism that remains to be determined.

On the other hand, CHO cells were infected with C. burnetii for 48 h. Then, the cells were co-incubated with Texas red dextran (red), as a control for dextran particles incorporation, and pHrodo dextran (green). Samples were analyzed by confocal microscopy (Figures S1F–G). The percentage of vacuoles Texas red dextran positive (red) or both Texas red dextran and pHrodo dextran positive (red and green) was determined. As indicated in Figure S1G whereas 100% of the Coxiella vacuoles have incorporated Texas red dextran, only 35–40% of the vacuoles were also labeled with pHrodo green. This result clearly indicates that the dextran particles were able to reach all the Coxiella vacuoles but only a proportion of those were labeled by pHrodo, indicating that a fraction of the vacuoles were not acidic.

To further verify the pH of the C. burnetii vacuole, we used ratiometric techniques. The C. burnetii vacuole was loaded with Oregon green dextran via fluid-phase endocytosis which has spectral properties that respond to low pH. C. burnetii infected CHO cells were loaded for 20 h with Oregon green dextran at a concentration of 1 mg/ml. The pH for the vacuole was calculated to be 5.68 ± 0.3 (n = 22; Figure 1G). We found that there are two different populations in Coxiella vacuoles pH, as represented in Figure 1H, supporting the existence of intact vacuoles with acidic pH around 5.2 and transient damaged ones with less acidic pH. This experiment was performed in CHO cells whereas in Akporiaye et al. (1983) and Maurin et al. (1992), macrophages were used. In Akporiaye et al. (1983), the experiment was performed with C. burnetii phase I in J774 cells and authors determined that the pH was 5.21 ± 0.07, whereas in Maurin et al. (1992), the pH in infected P388DI cells was 4.85 ± 0.3. On the other hand, in Vero cells, the pH of C. burnetii's vacuole was 4.88 ± 0.14 as shown in the study by Grieshaber et al. (2002). Thus, it is possible that the differences observed in the measurement of the pH in the mentioned papers can be due to the cell type and/or the Coxiella burnetii type used.

To assess the degradative properties of the CRV, CHO cells were infected with C. burnetii for 48 h and then incubated with DQ-BSA green, a fluorescent marker for degradative compartments. Cells were visualized by fluorescence live cell imaging. As depicted in Figures 1I,J populations of CRVs labeled or not with DQ-BSA green were observed, in agreement with the existence of heterogeneous populations of the Coxiella replicative niche.

In addition, we performed a staining of infected C. burnetii CHO cells with LysoTracker (red). Consistently with the non-homogeneous distribution of the LysoTracker labeling, the presence of small vesicles inside the CRV marked by both probes was clearly observed (Figure 1K), confirming the multivesicular nature of the Coxiella-vacuole.

All together these results clearly indicate that at a given time point during infection, the CRVs are highly heterogeneous not only regarding to their acidic pH but also degradative properties.

We reasoned that the presence of different populations of C. burnetii's vacuole with respect to their pH indicated that these vacuoles were damaged allowing passive movements of molecules across the CRV membrane. We hypothesized that the observed drop in acidity was a consequence of the leaking of protons through a damaged limiting CRV membrane.

To ascertain whether C. burnetii containing vacuoles were permeable to cytoplasmic markers in a live cell system, soluble dextran of 3 kDa coupled to the Texas Red fluorochrome was microinjected into the cytoplasm of CHO cells previously infected with C. burnetii for 48 h. Vacuolar membrane permeability can be measured through the access of dextrans loaded into the cytoplasm to the CRV. The presence of dextran inside the vacuole was assessed by determining the fluorescence signal inside the CRV, using a spinning disk microscope. For each image of microinjected cells, a sequential optical series (z series) and time lapse images were recorded to determine the signal inside the CRV. Tagged dextrans of 3 kDa in size were able to gain access to vacuoles containing live C. burnetii. Figure 2A shows two images of one optical slice of the z serie and time lapse taken in each microinjection experiment. Figure 2A shows that Texas red dextran entered the CRVs labeled as CRV1 and CRV2. In contrast, the fluorescent marker was excluded from the CRV3 depicted in Figure 2B. The quantification of the fluorescence intensity inside each vacuole is depicted in Figure 2B. The differences among the three selected CRVs are clearly depicted in the insets shown in Figure 2C. The percentage of vacuoles accessed by the microinjected dextran is shown in Figure 2D. Thus, 20% of the vacuoles presents a relatively substantial damage of the limiting membrane. As a control of microinjection, a 200 kDa dextran was microinjected in the cytoplasm of infected CHO cells and the cells were visualized by confocal imaging as before. As depicted in the Figure S2A, this dextran could not get into de CRV, which means that vacuoles were not permeable to this size of molecules and that the microinjection procedure itself was not damaging the CRV.

In order to assure that the vacuoles are permeable, we had to adapt a FRET based reporter assay that was previously used to monitor the intracellular localization of Salmonella and Shigella species (Keller et al., 2013). In the host cell, membrane permeable CCF4-AM molecules diffuse freely across the cellular plasma membrane, gain access to the cytoplasm and are excluded from endosomes and other organelles by anion conversion into CCF4-AM upon cytosolic esterase action. The use of this probe relies on the bacterial expression and exposure of beta-lactamase. When the beta-lactamase gets in contact with the cytosolic substrate there is a switch in the FRET signal from 535 nm (green) to 450 nm (blue) upon 405 nm excitation, indicating cleavage of CCF4-AM. The change in the FRET signal indicates rupture of the phagosome/vacuole membrane. Ray et al. (2010). For our assay a modified approach was used. CHO cells were infected with Coxiella for 48 h and allowed to internalize 1 μm latex beads coupled with beta-lactamase. Afterwards, cells were incubated with the CCF4 substrate which is loaded in the cytoplasm. As previously demonstrated for M. marinum (Simeone et al., 2012) and other bacteria (Ray et al., 2010), when the beta-lactamase gets in contact with the cytosolic substrate there is a switch in the FRET signal from 535 nm (green) to 450 nm (blue), indicating rupture of the phagosome/vacuole membrane. As shown in the Figure 2E almost all the Coxiella infected cells (INF) turned blue whereas the uninfected (N-INF) cells remained green. The quantification of these results, using an algorithm segmenting individual cells and measuring both fluorescent channels, is shown in Figure 2F. High ratios (above 0.5) indicate the damage of the phagosome membrane as clearly depicted in the panel. As shown in the Figures S2B,C some of the incorporated beta-lactamase beads (red) can be visualized in a Coxiella-containing compartment (green).

In addition, we have also performed transmission electron microscopy of CHO cells infected with C. burnetii at early times of infection (24 h). In Figure 2G, we can observe several disruptions in both a Coxiella-containing phagosome membrane as indicated by yellow arrowheads.

Altogether these results noticeably indicate that the CRV surrounding membrane integrity is affected and permeable to cytoplasmic loaded compounds.

It has been reported that C. burnetii transits through the endocytic pathway, which delivers the bacteria to the low pH environment of a lysosome. It is known that CRV maturation to a phagolysosome takes approximately 2 h. Since galectins are considered useful molecules to detect damage of intracellular membranous compartments, we were interested in determining whether, at some time point during infection, the CRV membrane integrity was altered as suggested by the experiments described above. It has been reported that Gal3 and Gal8 detect membrane damage in bacteria's vacuoles like Shigella and Salmonella (Dupont et al., 2009; Paz et al., 2010; Thurston et al., 2012). Thus, we analyzed the association of these galectins with Coxiella phagosomes at early infection times (i.e., 6 h) and later infection times once the large vacuole is generated (i.e., 24, 48 h). To this aim, we overexpressed these galectins in CHO cells and infected cells with C. burnetii. At different times of infection (6, 24, and 48 h), cells were fixed and analyzed by confocal microscopy as shown in Figure 3A. Figure 3B shows the fluorescence intensity diagram along the yellow line across the CRV depicted in Figure 3A inset. We could observe galectins distributed at the limiting membrane and in the interior of the CRV as is shown in the Figure S3A, using Structured Illumination Microscopy (SIM) image of a transfected GFP-Gal3 CHO cell infected with C. burnetii. The quantification along the time of infection is depicted in Figure 3C, indicating that YFP-Gal8 and -Gal3 were clearly recruited to a population of the CRVs (30–40%) at all times of infection analyzed. We also screened a panel of other human galectins for their presence at the C. burnetii-containing vacuole and we determined whether the interaction of these galectins with the CRV was dependent on the time of infection. For this purpose, CHO cells were transfected for 24 h with YFP-Gal4, YFP-Gal10, YFP-Gal1 and YFP-Gal9 and cells were infected for different periods of time. Figure S3C shows confocal images of the kinetic analysis for the Gal1, Gal4, Gal9, and Gal10. The population of C. burnetii phagosomes that recruited the tested galectins during the course of infection was also quantified (Figure S3D). As depicted, the colocalization between C. burnetii and YFP-Gal1 increased at 24 h of infection and it remained constant at 48 h. In contrast, in the case of YFP-Gal9 and -Gal4 the proportion decreased at later infection times whereas no colocalization along infection was observed in the case of YFP-Gal10.

Since overexpressed Gal3, Gal8, and Gal1 interact with C. burnetii at different stages of vacuole development and persist along the infection process (48 h), we were interested in determining whether the endogenous proteins were also recruited at the CRV. Cells were infected with C. burnetii for 48 h and both bacteria and endogenous galectins were detected using specific antibodies. As shown in Figure 3D, we observed the accumulation of endogenous Gal3, Gal1, and Gal 8 at the CRV surrounding membrane (insets). The quantification of the percentage of CRVs positive for endogenous Gal3, Gal1, or Gal8 is depicted in Figure 3F. Our results clearly indicate that 25–30% of the CRVs are decorated by these galectins. Also, there is a large amount of colocalization between Gal3 and Gal8 at early times of infection (6 h; Figure 4A). CHO cells were transfected with mCherry-Gal8 and infected with C. burnetii. Then, the cells were fixed and subjected to immunofluorescence with specific antibodies against Gal3. The quantification of colocalization is depicted in Figure 4B, indicating that both galectins are recruited to the same CRV.

In addition, we were interested in determining the presence of galectins at earlier times of infection. For this purpose, CHO cells transfected with YFP-Gal8 or YFP-Gal3 were infected for 2 h with mCherry-C. burnetii. The cells were analyzed by fluorescence confocal videomicroscopy and a multidimensional image was constructed from a z-interval (Figure 4C). The image clearly shows the recruitment of YFP-Gal8 to a small phagosome containing just a bacterium, confirming that Gal8 associates to the Coxiella phagosome at very early times after infection. Figure 4D, shows the quantification of the recruitment of these galectins at 2 h of infection.

In order to dynamically image the recruitment of galectins to the Coxiella phagosome, CHO cells were transiently transfected with GFP-Gal3 and infected with C. burnetii incubated with Hoechst. The cells were analyzed by fluorescence confocal videomicroscopy. Figure 4E shows an infected cell with an early recruitment of Gal3 around a group of bacteria. The sequence shows images taken every 5 min in a sequence of the total time lapse analyzed from 0 to 50 min. Hence, there is a recruitment of Gal3 around bacteria starting at the time point 25 min of infection and remaining during the whole period of time analyzed while the phagosomes become larger.

Moreover, in order to assess whether some galectin-positive compartments have lost their acidity, CHO cells were transiently transfected with RFP-Gal8. Then, the cells were infected with C. burnetii for 24 h. Afterwards, the cells were incubated with pHrodo green dextran. The acidification of the CRV was visualized by fluorescence live cell imaging (Figure 4F). As depicted in the Figure 4G, the large majority of the mCherry Gal8-positive vacuoles are pHrodo negative. Nevertheless, Coxiella-containing vacuoles positive for Gal8 and pHrodo are also clearly observed. This can be interpreted as a transient membrane damage, hence neutralizing the vacuolar pH, before vacuoles recover acidification as membrane is repaired. In other words, at some point the vacuole was pierced and this damage was detected by galectins but since the damage is transient they recover the acidity but the CRV still remain positive for galectin. Thus, healing the membrane damage may contribute to the recovering of acidity necessary for the Coxiella metabolic activity.

In order to determine whether the galectin recruitment is specifically dependent on bacterial infection, a control experiment with an inert particle was performed. CHO cells were transfected with GFP-Gal3 and incubated with rhodamine beads for different times (2, 6, 24, 48 h), fixed and analyzed by confocal microscopy (Figure S4A). In addition, in a parallel experiment, endogenous Gal3 was determined in CHO cells incubated with FITC beads at the same time points (Figure S4C). As depicted in the graphs of fluorescence intensity along the yellow line across beads phagosomes, no recruitment of galectins to the beads-containing phagosomes was observed. Figures S4B,D show the quantification of the association of Gal3 to beads phagosomes.

The damage of the vacuole exposes host glycans which may cause Gal8 and Gal3 accumulation at the CRV. As indicated in the introduction, Gal3 has only one carbohydrate recognition domain (CRD) whereas Gal8 belongs to the group of “tandem repeat type” galectins that exhibit two structurally distinct CRDs, being able to interact with dissimilar carbohydrates. The requirement for carbohydrate binding by Gal8 and Gal3 was tested using point mutations in their respective CRDs. CHO cells were transfected with the corresponding carbohydrate-binding mutants YFP-Gal8 (R275H), YFP-Gal8 (R69H) and GFP-Gal3 (R186S). All these mutants carry a mutation in one critical aminoacid at the CRD so that they are not able to recognize carbohydrates. Afterwards, the cells were infected with C. burnetii and, at 48 h of infection they were fixed and analyzed by confocal microscopy (Figures 5A,B). As shown in the quantification depicted in Figure 5C, in contrast to the wt YFP-Gal8 or GFP-Gal3, none of the mutants did accumulate at the CRD, indicating that either the Gal3 CRD or both Gal8 CRD domains are absolutely required for recruitment to the Coxiella vacuoles.

Furthermore, to demonstrate that host glycans are indeed important for the recruitment of galectins to the CRV, we transfected CHO and CHO-Lec3.2.8.1 cells, which lack mature glycans, with mCherry-Gal8. Transfected cells were infected with GFP-C. burnetii and at 48 h of infection, cells were fixed and analyzed by confocal microscopy (Figure 5D). As shown in Figure 5E, recruitment of Gal8 to CRVs was severely impaired in the CHO-Lec3.2.8.1 cells. This indicates that the presence of mature glycans of the host cell is important for the recruitment of Gal8 to the CRV.

To assess whether Gal3 inside C. burnetii-containing vacuole may arrive from the plasma membrane when C. burnetii is phagocytosed, we utilized the reagent N-Acetyl-D-lactosamine, which blocks the interactions between Gal3 and its ligands at the cell surface. CHO cells were incubated with (N-Acetyl-D-lac) or without (Control) the inhibitor and after 2 h, the cells were infected with C. burnetii (red) for 48 h in the continuous presence of the inhibitor. Afterwards, the cells were fixed and endogenous Gal3 (green) was detected by immunofluorescence and analyzed by confocal microscopy (Figures 5F,G). The same experiment was performed with YFP-Gal3 transfected CHO cells (Figures 5H,I). We could observe the presence of Gal3 recruited to the vacuole in both control conditions and when incubated with the inhibitor (N-Acetyl-D-lac). This result indicates that the Gal3 present at the CRV does not come from the plasma membrane but from the cytoplasm.

It has been shown that C. burnetii possesses a type 4 secretion system (T4SS) termed Dot and Icm. Recent studies have demonstrated that Dot/Icm function is required for cytosolic delivery of several substrates which have been identified as proteins effectors important for intracellular replication, development of the large CRV and apoptosis protection of infected THP-1 cells. Nevertheless, it was found that translocation of Dot/Icm substrates is not required for axenic growth or uptake by host cells (Carey et al., 2011; Newton and Roy, 2011; Beare et al., 2012).

In previous reports, it has been demonstrated that T4SS function can be inhibited by chloramphenicol treatment. In order to assess whether the association between C. burnetii and galectins is an active phenomenon that depends on bacterial protein synthesis, C. burnetii was pretreated with 100 μg/ml of chloramphenicol for 1 h and CHO cells were then infected for 48 h, with the continuous presence of the antibiotic in the culture medium. Cells were fixed and subjected to a double immunofluorescence, using specific antibodies against C. burnetii and Gal3 (Figure 5J). Consistently with the results presented above, we observed in the control condition that a colocalization of about 30–40% of the CRV recruited this galectin, whereas with the chloramphenicol-treated bacteria the percentage of colocalization was significant smaller (around 10%; Figure 5K). This result clearly indicates that the recruitment of galectins depends on an active protein synthesis by C. burnetii. To actually demonstrate that galectin recruitment is dependent on T4SS, CHO cells were infected with GFP-wt C. burnetii or a DotA mutant GFP- C. burnetii (Tn292). This mutant carries an independent transposon insertion in the gene CBU_1648, which encodes DotA, an essential component of the Coxiella Dot/Icm secretion system. At 48 h of infection, cells were fixed and subjected to immunofluorescence against endogenous Gal3 (red). Similar to chloramphenicol treatment, cells infected with DotA mutant did not recruit Gal3 compared to control wt Coxiella (Figures 5L,M). These results show that C. burnetii's Dot/Icm secretion system is important for the recruitment of galectins.

It has been shown in previous publications that Salmonella enterica serotype Typhimurium and Streptococcus pyogenes are recognized by NDP52 and recruits polyubiquitinated proteins once in the cytosol (Thurston et al., 2009). NDP52 bridges LC3 and ubiquitin molecules around bacteria allowing the transport of cytosolic bacteria to the autophagic pathway. Based on these evidences, we next assessed the association of overexpressed RFP-NDP52 at the CRV. As shown in Figures S5A,B, the results indicate that the protein was found in 20–30% of the vacuoles generated after 48 h. In order to determine whether galectins also colocalized with NDP52 in the same Coxiella vacuole, YFP-Gal8 or YFP-Gal3 and RFP-NDP52 were coexpressed in CHO cells which were subsequently infected with C. burnetii for 48 h. As shown in Figures S5C,D, we found that around 80% of the galectin-positive CRVs accumulated NDP52. Furthermore, NDP52 also markedly colocalized with LC3 in the large vacuoles generated at 48 h infection (Figures S5E,F). The results suggest that galectin-dependent recruitment of NDP52 to the CRV is likely to participate to the further association of LC3.

The protein p62 (also known as SQSTM1) targets intracellular pathogens for degradation in autophagolysosomes. P62 is a receptor for cargo with multiple protein-protein interaction domains that binds ubiquitin and also LC3 (Komatsu and Ichimura, 2010). Because NDP52 was recruited to the C. burnetii's CRV, we also evaluated the presence of p62 at the vacuole membrane. CHO cells were infected with mCherry-C. burnetii for 48 h and the cells were subjected to immunofluorescence with a specific antibody against p62. In the Figure S6A the association of p62 to the CRV membrane is depicted. Figure S6B shows the quantification of the percentage of vacuoles that recruit p62. Thus, we were next interested to also determine the presence of ubiquitin to the vacuoles. CHO cells were infected with C. burnetii for 48 h and then the cells were fixed. As depicted in Figure S6C, the vacuoles were decorated by ubiquitin (see quantification in Figure S6D) suggesting that C. burnetii recruits in an ubiquitin-dependent manner adaptors of the autophagy machinery.

In several previous publications it has been shown the association of LC3 to the large CRV (Berón et al., 2002; Gutierrez et al., 2005; Romano et al., 2007). Therefore, we next examined whether both Gal3 or Gal8 and LC3 were recruited to the same vacuole. To examine in details the respective distribution of galectins and LC3 during C. burnetii infection, cells were co-transfected with YFP-Gal3 or YFP-Gal8 and RFP-LC3 and they were infected with C. burnetii for 48 h. Figure S5G clearly shows the presence of either YFP-Gal3 or YFP-Gal8 and RFP-LC3 decorating the large CRV. The image shows the accumulation of LC3 (red) and galectins (green) at the surrounding CRV membrane but also in small structures inside the CRV that may represent small internal vesicles (Figure S5G insets). Figure S5H shows the quantification of LC3 positive vacuoles (from a total around of 30%) that colocalize with Gal8 or Gal3. We can conclude that in 48 h vacuoles, galectins and LC3 are both present suggesting that similar to other pathogens galectins may mediate the recruitment of LC3 to the CRV.

In order to assess whether the recruitment of Gal3 was modulated by the autophagic pathway, CHO cells overexpressing Gal3 were infected with C. burnetii duting 48 h. Then, the cells were subsequently incubated in full nutrient media (Control) or under starvation conditions (Stv) or with Rapamycin (Rap) to induce autophagy. As shown in Figure S5J, no major differences were observed in the recruitment of Gal3 in all the conditions tested.

To directly assess the role of autophagy in galectins recruitment, we used a mouse embryonic fibroblast (MEF) cell line harboring a knock-out for the atg5 locus (atg5 −/−). These cells lacking Atg5 are deficient in macroautophagy because this protein is essential for the early steps of autophagosome formation. To demonstrate the significance of the membrane damaged caused by Coxiella and the role of autophagy in this process, MEF wt and Atg5−/− cells were infected with C. burnetii and following 48 h of infection, the cells were fixed, subjected to immunofluorescence against Gal3 (red) and analyzed by confocal microscopy. As expected, in control cells, endogenous Gal3 was recruited to a population of the CRVs. However, in MEF Atg5−/− cells, a higher percentage of vacuoles decorated by endogenous Gal3 were observed, indicating that a larger number of CRVs were damaged and that autophagy is likely required for membrane repair (Figures 6A,B). Similar results were also observed when cells MEF wt or Atg5−/− overexpressing YFP-Gal3 were infected with C. burnetii confirming that this galectin was recruited to the vacuole in an autophagy-independent manner (Figures 6C,D). However, the number of CRV positive for galectin was dependent on a functional autophagy pathway.

As indicated above pHrodo is a fluorogenic dye that becomes highly fluorescent as the pH decreases, therefore, it is a useful probe to assess acidic compartments. To corroborate that in autophagy incompetent cells more vacuoles were actually damaged and, as a consequence, the acidity of the CRV was lost due to the leaking of protons, we used pHrodo as a marker of acidic vacuoles. For this purpose, MEF wt and Atg5−/− infected cells were incubated with pHrodo-conjugated to heat-killed Staphylococcus aureus to be internalized and visualized by fluorescence live cell imaging. As depicted in Figure 6E, in Atg5−/− cells, there was a decrease in the number of vacuoles labeled with the fluorogenic compound, suggesting that there were less acidic vacuoles in autophagy-deficient cells. This result supports the participation of autophagy in keeping the integrity of the Coxiella vacuole limiting membrane.

We next determined the importance of a functional autophagy pathway in Coxiella growth. To assess the role of autophagy in bacteria replication, a focus-forming unit (FFU) assay was performed as follows: MEF wt and Atg5−/− cells were infected with C. burnetii and after 24 h of infection, cells were scraped and lysed by sonication. Sonicates were diluted in infection medium (i.e., DMEM) and a monolayer of Vero cells were infected with a dilution of the obtained supernatant (1:100). After 72 h of incubation (chase), cells were fixed and examined by fluorescence microscopy (please see Experimental Procedures for more details). The FFU assay showed a marked decrease in the replicative capacity of C. burnetii in MEF Atg5−/− in comparison with wt cells, indicating an active role of autophagy in Coxiella growth (Figure 6F).

We have previously demonstrated that the autophagic protein Beclin 1 was recruited to the Coxiella replicative vacuole and that overexpression of this protein favors the development of the CRV (Vázquez and Colombo, 2010). In order to confirm the significance of the membrane damaged caused by Coxiella and the role of autophagy in this process based in the participation of Beclin1, we performed the silencing of this protein using a specific siRNA plasmid (Vázquez and Colombo, 2010). Control and Beclin 1-KD cells were infected with C. burnetii and following 48 h of infection, the cells were fixed, subjected to immunofluorescence against Gal3 and analyzed by confocal microscopy. As expected, in control cells, endogenous Gal3 was recruited to a population (20%) of the CRVs. However, in Beclin 1-KD cells, a much higher percentage of vacuoles decorated by endogenous Gal3 were observed (Figure 6G).

In order to determine the acidity of the vacuoles, control, and Beclin 1-KD infected cells were incubated with pHrodo green dextran and visualized by fluorescence live cell imaging. As depicted in Figure 6H, in Beclin1 KD cells, there is a decrease in the number of vacuoles labeled with the fluorogenic compound. To verify whether Beclin 1 was also involved in the replicative capacity of Coxiella, HeLa cells were transfected with a pEGFP plasmid as a control or co-transfected with a plasmid encoding a small interference RNA against Beclin 1 (pSuper Beclin 1-KD), in order to knockdown the endogenous Beclin in transfected cells (Figure 6J). After 24 h cells were infected with C. burnetii for additional 24 h. The replication capacity of the bacteria was determined by the FFU assay in cells subjected to Beclin 1 knockdown. As shown in Figure 6I, left panel, a significant decrease in bacteria replication was observed when Beclin 1 was silenced which indicates that this autophagic protein is involved in the generation of an appropriate environment for bacteria growth. Taken together, these results suggest that autophagy has an important role in keeping the integrity of the membrane vacuole favoring also bacteria replication.

Minimum essential Medium Alpha Medium (alfa-MEM) and Dulbecco's Modified Eagle Medium (D-MEM) were obtained from Gibco Laboratories (Invitrogen, Argentina); fetal bovine serum (FBS) was obtained from GIBCO BRL/Life Technologies (Buenos Aires, Argentina). Rabbit polyclonal anti-Coxiella antibody and mCherry-Coxiella burnetii were generously provided by Dr. Robert Heinzen (Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA). Plasmids encoding YFP-Gal4, YFP-Gal9, YFP-Gal3, YFP-Gal10, YFP-Gal1, YFP-Gal8, and RFP-NDP52 were kindly provided by Dr. Felix Randow (MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, U.K.). Rabbit and mouse polyclonal antibodies anti-Gal3, -Gal1, -Gal8 and a plasmid encoding Gal3 were generously provided by Dr. Gabriel Rabinovich (Instituto de Biología y Medicina Experimental, INYME-CONICET, Buenos Aires, Argentina). The Beclin 1 KD plasmid was a generous gift of Dr. William Maltese (Medical University of Ohio, Toledo, Ohio, USA). Tandem fluorescent-tagged Galectin-3, tfGal3 was kindly provided by Dr. Tamotsu Yoshimori (Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan). mCherry-Gal8 was kindly provided by Dr. Mauricio Terebiznik (Department of Biological Sciences, University of Toronto, Toronto, Canada). Coxiella-GFP (Tn1832) and DotA mutant GFP (Tn292) were kindly provided by Dr. Matteo Bonazzi (Cell Biology of Bacterial Infections, UMR 5236 CPBS, Montpellier, France). CHO-Lec3.2.8.1 cells were kindly provided by Dr Pamela Stanley (Albert Einstein College of Medicine, New York, USA). Ubiquitin antibody was from Abcam.

Texas Red-tagged dextran (3 kDa), pHrodo™ Succinimidyl Ester, pHrodo™ Green dextran, 10,000 MW, LysoTracker, LysoSensor green DND-189 and DQ-BSA were purchased from Molecular Probes.

Vero (ATCC, CCL-81), MEF (ATCC, SCRC-1040), and Chinese hamster ovary cells (CHO; ATCC, CCL-61) were grown on coverslips in D-MEM or α-MEM supplemented with 15% FBS, at 37°C in an atmosphere of 95% air and 5% CO2, in 24-well-plates to 80% confluence.

MEF and CHO cells were transfected with the plasmids (1 μg/μl) using LipofectAMINE 2000 reagent (Invitrogen, Argentina) as previously described in Campoy et al. (2013). Transfected cells were incubated for 24 h in DMEM and were infected with C. burnetii as described above.

Obtaining C. burnetii was performed as previously described in Romano et al. (2007). Briefly, C. burnetii clone 4, phase II, strain Nine Mile bacteria were provided by Ted Hackstadt (Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT) and handled in a biosafety level II facility. For initial infection, 80% confluent Vero cells were infected with C. burnetii for 2 weeks at 37°C under 5% CO2. C. burnetii were obtained from infected cells by lysis of the host cells followed a differential centrifugation of the supernatants. Purified C. burnetii in the resulting pellet were resuspended in PBS and frozen at −70°C.

Cells were plated on coverslips distributed in 6 or 24 well-plates and 0.5–1 ml of a dilution of C. burnetii suspension was added per well. Afterwards the cells were incubated at 37°C in an atmosphere of 95% air and 5% CO2 for the indicated time periods as described (Campoy et al., 2013).

GFP-LC3, YFP-Gal8, YFP-Gal3, YFP-Gal9, YFP-Gal10, YFP-Gal4, YFP-Gal, YFP-Gal8 (R275H), YFP-Gal8 (R69H), and GFP-Gal3 (R186S) or RFP-NDP52 transfected CHO cells were analyzed by confocal microscopy using an Olympus FluoViewTM FV1000 confocal microscope (Olympus, Argentina), with the FV10-ASW (version 01.07.00.16) software. Images were processed using Adobe CS3 (Adobe Systems). Confocal images (0.39-μm sections) were collected.

High-resolution images were acquired on a ElyraPS1 microscope system (Zeiss, Carl Zeiss-Strasse 22 73447 Oberkochen, Germany) using a 100 oil-immersion lens (NA 1.46). This SIM system can achieve a resolution of 100 nm along the x–y axis and 300 nm along the z-axis. Laser lines at 488 and 561 nm were used for excitation. SIM images (15 images with five different phases for 3 different angular orientations of illumination for each SIM image) were acquired with an EMCCD camera (Andor Technology Ltd., Millennium WaySpringvale Business Park, Belfast BT12 7AL, UK; 1,002 1,004 pixels) and processed with Zen (Zeiss) software.

Some in vivo experiments were recorded using an inverted AxioObserver Z1 microscope fitted with a Zeiss Axiocam® MRm camera (Carl Zeiss, Oberkochen, Germany) and operated by Axiovision® software. Images were acquired with a Plan-Apo 100 × /1.46 oil immersion objective (Carl Zeiss, Oberkochen, Germany). For video microscopy, the fluorophore excitation system was composed of a Colibri system (Zeiss, Carl Zeiss, Oberkochen, Germany) with 365, 470, 555, and 590 nm LEDs. For pH measurements, images were adquired in a LEICA fluorescence microscope spinning disk (AF6000-X; Campus CNRS CCHB, Bio Imaging Center Lille—Campus Lille 1, Université de Lille).

The cells grown on coverslips were fixed with 3% paraformaldehyde solution in PBS for 10 min at room temperature, washed with PBS followed by quenching with 50 mM NH4Cl in PBS. Subsequently, cells were permeabilized with 1% saponin in PBS containing 1% BSA, and further incubated with the primary antibody dilution in PBS. Afterwards, the coverslips were incubated with a conjugated secondary antibody (Jackson immune Research Laboratories, EE. UU). After 3x washing with PBS cells were mounted with Mowiol (plus Hoechst) and examined by confocal microscopy as described previously (Campoy et al., 2013).

Infected CHO cells were injected as previously described (Teitelbaum et al., 1999) with 3 kDa Texas red dextran, using a Micromanipulator 5,171 and a Transjector 5,426 plus (Eppendorf). Needles were pulled on a Sutter pipette puller, model P97. Infected CHO cells were microinjected with an initial pressure of 600 k Pa. Successive optical sections were collected and analyzed in a spin disk microscope (Olympus). The z series collection was initiated once diffusion of the injected marker was determined by imaging fluorescence. Injected cells were imaged for 20 min immediately after microinjection.

Preparations of Staphylococcus aureus particles conjugated to pHrodo were performed as previously described (Kobayashi et al., 2010). Heat-dead bacteria were washed twice and resuspended in PBS at an appropriate concentration (1:10 or 1:20 dilution). pHrodo-succinimidyl ester (Invitrogen, P36600) was added to the bacteria suspension and the samples were incubated in buffer bicarbonate for 45 min at room temperature. After conjugation, bacteria were washed and finally suspended in PBS. The resulting suspension (~10 μl) was mixed with 90 μl of culture medium and added to cells by replacing the medium. After incubation with the mixture for 1 h at 37°C, cells were washed with fresh medium to remove particles or not phagocytosed bacteria, and cells were subsequently incubated for 2 h.

A double-hit protocol was performed as previously described in Vázquez and Colombo (2010). HeLa cells were plated in six-well-plates at 40% confluence and then transfected with GFP (control) or GFP and Beclin 1-KD (plasmid) After 24 h, cells were transfected with 24 h later, cells were transfected again with the GFP (control) or GFP and Beclin 1-KD (plasmid) again. Twenty four hours later cells were infected with C. burnetii during 48 h.

A fluorescent infectious FFU assay was used to quantify the replication and viability of C. burnetii in MEF wt or Atg5−/− cells or in Beclin1 KD HeLa cells. In brief, the infected cells were lysed and samples were serially diluted. Vero cells were infected with these lysates in a 24 well-plate. After 72 h of infection, Vero cells were fixed and processed for fluorescent microscopy. Approximately 1,000 cells were scored per coverslip to determine an average number of FFU for each sample.

For standard curves, C. burnetii infected cells were incubated with 10 mM nigericin and 10 mM valinomycin in the pH 7.0 calibration Buffer. The coverslips were subsequently incubated for 10 min in pH calibration buffers adjusted to pH of 4.0, 5.0, 6.0, or 7.0. Then, they were incubated with Oregon green dextran 10,000 MW at a concentration of 1 mg/ml in culture medium overnight. Images for ratiometric calculations were acquired in a Leica spinning disk at excitation wavelengths of 440 nm and 480 nm with a fixed emission wavelength of 520 nm. Ratio values for the standard curves were determined by measuring the average 480/440 ratio value. To determine the pH of C. burnetii's vacuoles, infected cells were incubated with Oregon green dextran in culture medium overnight. Ratio values were plotted against the standard curves to determine the pH of C. burnetii replicative vacuoles.

Amine functionalized beads were incubated for 4 h in 8% glutaraldehyde in PBS on a rolling wheel at RT. Activated beads were washed and resuspended in lactamase containing solutions in saturating conditions (estimated following the manufacturer's instructions). They were left overnight on a rolling wheel at 4°C. Beads were then washed and stocked at 4°C in PBS.

CHO cells were seeded on coverslips in 24-well plates at 2 × 10⁴ cells per well for experiments with fixed samples. CHO cells were infected with C. burnetii and after 48 h, they were loaded with 0.1 mM CCF4/AM substrate (Invitrogen) in EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM, 0.8 mM MgCl2, 5 mM glucose and 25 mM Hepes, pH 7.3) containing 6 mM probenecid, an inhibitor of anions transporters, for 2 h at RT, washed twice with EM and transferred to fresh medium containing 6 mM probenecid for a further 30 min. CCF4-loaded CHO cells were directly incubated with beta-lactamase coated beads (see Coating of beads with lactamase) for 8 h at 37°C. The adquisition was performed with a spinning-disk confocal microscope is composed of two diode laser sources emitting at wavelength of 488 and 440 nm (Omicron, Germany) injected in a spinning-disk confocal system (Yokogawa CSU-X1, Tokyo, Japan) adapted on a Leica (Lognes, FR) DMI6000B inverted microscope. Cells were imaged using a 63 × water-immersion objective (Leica HCX Plan Apo NA 1.20 W Corr CS). Fluorescence emitted was then successively routed by a dichroic mirror (Semrock Di01-T405/488/568/647 and 405/440/514 respectivlely), spectrally filtered (Semrock, FF01-520/35 nm) and detected with a Quatem 512SC EMCCD coupled to an optical zoom (×2). Every 512 × 512 image was acquired using 300 ms exposure time. The acquisition was performed by separated spectra to avoid potential overlap between emissions. Results were analyzed using ImageJ software (live cell imaging).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.