ABSTRACT
Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to the pathogenesis of this intracellular pathogen is the secretion of bacterial effectors into host cells by a Type 4b Secretion System (T4SS), to subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole allowing intracellular replication. The characterization of prokaryotic Serine/Threonine Protein Kinases (STPKs) in bacterial pathogens is emerging as an important strategy to better understand host-pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a bacterial protein kinase identified in C. burnetii by in silico analysis. Here, we demonstrated that this putative protein kinase undergoes autophosphorylation on Ser, Thr, and Tyr residues, and phosphorylates a classical eukaryotic protein kinase substrate in vitro. This dual Ser/Thr and Tyr kinase activity is similarly observed for eukaryotic dual specificity Tyr phosphorylation-regulated kinase class. CstK is translocated during infections and localizes at Coxiella-containing vacuoles (CCVs). Moreover, a C. burnetii mutant strain overexpressing CstK displays a severe CCVs development phenotype, suggesting a finely tuned regulation by the bacterial kinase during infection. Protein-protein interaction studies identified the Rab7-GTPase activating protein (GAP) TBC1D5 as a candidate CstK-specific host target, suggesting a role for this eukaryotic GAP in Coxiella infections. Indeed, CstK colocalizes with TBC1D5 in non-infected cells, and TBC1D5 is recruited at CCVs during infection. Accordingly, depletion of TBC1D5 from infected cells significantly affects CCVs development. Our results indicate that CstK has a critical role during infection as a bacterial effector protein that interacts with host proteins to facilitate vacuole biogenesis and intracellular replication.
Signal transduction is an essential and universal function that allows all cells, from prokaryotes to eukaryotes, to translate environmental signals to adaptive changes. By this mechanism, extracellular inputs propagate through complex signaling networks whose activity is often regulated by reversible protein phosphorylation. Signaling mediated by Serine/Threonine/Tyrosine protein phosphorylation has been extensively studied in eukaryotes, however, its relevance in prokaryotes has only begun to be appreciated. The recent discovery that bacteria also use Ser/Thr/Tyr kinase-based signaling pathways has opened new perspectives to study environmental adaptation, especially in the case of bacterial pathogens, with respect to host infection. Thus, advances in genetic strategies and genome sequencing have revealed the existence of “eukaryotic-like” serine/threonine protein kinases (STPKs) and phosphatases in a number of prokaryotic organisms, including pathogens such as, Streptococcus spp. (1–4), Mycobacteria (5–10), Yersinia spp. (11,12), Listeria monocytogenes (13,14), Pseudomonas aeruginosa (15), Enterococcus faecalis (16) or Staphylococcus aureus (17–20). Consequently, the study of STPKs in human bacterial pathogens is emerging as an important strategy to better understand host-pathogen interactions and develop new, targeted antimicrobial therapies. However, if on one hand it is clear that STPKs and phosphatases regulate important functions in bacterial pathogens, their signal transduction mechanism remains ill-defined and restricted to a limited number of microbes.
Importantly, STPKs expressed by pathogenic bacteria can either act as key regulators of important microbial processes, or be translocated by secretion systems to interact with host substrates, thereby subverting essential host functions including the immune response, cell shape and integrity (21). Phosphorylation of host substrates has been demonstrated for some bacterial STPKs, whereas others seem to require their kinase activity but their phosphorylated substrates remain to be identified (21). Therefore, biochemical mechanisms of these pathogen-directed targeted perturbations in the host cell-signaling network are being actively investigated and STPKs are proving to be molecular switches that play key roles in host-pathogen interactions (21).
Among emerging human pathogens, Coxiella burnetii is a highly infectious bacterium, responsible of the zoonosis Q fever, a debilitating flu-like disease leading to large outbreaks with a severe health and economic burden (22–24). The efficiency of infections by C. burnetii is likely associated with the remarkable capacity of this bacterium to adapt to environmental as well as intracellular stress. Indeed, outside the host, C. burnetii generates pseudo-spores that facilitate its airborne dissemination. During infection, C. burnetii has developed a unique adaptation to the host, being the only bacterium that thrives in an acidic compartment containing active lysosomal enzymes. Similarly to other intracellular bacterial pathogens, C. burnetii adaptation depends on the translocation of bacterial effectors by a Dot/Icm Type 4b Secretion System (T4SS). Some of these effectors modulate important signaling pathways of infected cells, including apoptosis and inflammasome activation (25–27), whereas others localize at Coxiella-containing vacuoles (CCVs) and manipulate host membrane traffic to facilitate the development of the intracellular replicative niche (28,29). C. burnetii genome analysis revealed a close homology to the facultative intracellular pathogen Legionella pneumophila, in particular at the level of Dot/Icm core genes (30). In silico analysis identified over 100 candidate effector proteins encoded in the C. burnetii genome, some of which have been validated for secretion using either C. burnetii or L. pneumophila as a surrogate model (31–33). In this study, we investigated the candidate effector CBU_0175, which encodes the only putative Coxiella Ser/Thr kinase (CstK). We demonstrated CstK translocation by C. burnetii during infection and we reported its localization at CCVs. In vitro kinase assays revealed that CstK undergoes autophosphorylation on Ser, Thr, and Tyr residues, and CstK displays kinase activity towards a test substrate of eukaryotic protein kinases. Furthermore, the identification of the Rab7 GTP-activating protein (GAP) TBC1D5 as a CstK interactor suggests that this protein might be phosphorylated during infections to facilitate CCVs biogenesis. Indeed, TBC1D5 is actively recruited at CCVs during Coxiella infections and TBC1D5-targeting siRNAs significantly affect CCVs development. Our data provide the first evidence that a C. burnetii secreted kinase might control host cell infection.
RESULTS
Coxiella burnetii genome encodes a single putative protein kinase
In silico analysis of the epidemic C. burnetii strain RSA493 NMI genome revealed only one gene encoding a putative Serine/Threonine Protein Kinase (STPK). To date, no STPKs have been characterized in this organism. This gene was named cstK for C. burnetii serine threonine Kinase and encodes a 246 amino acids protein with an estimated molecular mass of 31 kDa. ClustalW and BLAST analysis revealed the presence of most of the essential amino acids and sequence subdomains characterizing the Hanks family of eukaryotic-like protein kinases (34). CstK shares a common eukaryotic protein kinases superfamily fold with two lobes and a Gly-rich loop. These include the central core of the catalytic domain, and the invariant lysine residue in the consensus motif within subdomain II, which is usually involved in the phosphotransfer reaction and required for the autophosphorylating activity of eukaryotic STPKs (Fig. 1A) (34,35). The activation loop in the catalytic domain is particularly short in CstK, and the DFG motif is substituted by a GLG motif. Interestingly, the transmembrane domain usually presents in classical prokaryotic STPKs is lacking in CstK, thus it is a so-called cytoplasmic STPK.
CstK is a Dot/Icm effector protein
Bioinformatics analysis using the prediction software S4TE2 (36) indicated that CstK harbors features corresponding to secreted effector proteins, including a promoter motif typically found in effector proteins from intra-vacuolar bacterial pathogens and a putative C-terminal E-block domain (37), suggesting that CstK is indeed a Coxiella effector protein (Fig. 1A). Consistently, previous studies by Chen and colleagues have shown that CstK is secreted in a T4SS-dependent manner by the surrogate host L. pneumophila, albeit with low efficiency. In order to validate CstK secretion in C. burnetii, we engineered plasmids encoding, either CstK or CvpB (a known C. burnetii effector protein) (38), fused to Beta-Lactamase (BLAM) and expressed in Coxiella Tn1832 (a C. burnetii transposon mutant that phenocopies wild-type C. burnetii) or dotA::Tn (a dotA transposon mutant, defective for Dot/Icm secretion). By means of a BLAM secretion assay, we could observe that BLAM-CstK was secreted by Coxiella at 48 and 72 hours, but not at 24 hours post-infection (Fig. 1B). Secretion of BLAM-CvpB or BLAM-CstK was not detectable in cells infected with the dotA::Tn strain, indicating that both CvpB and CstK are C. burnetii Dot/Icm substrates (Fig. 1B). Next, we ectopically expressed HA-tagged CstK in non-infected and C. burnetii infected U2OS cells, to investigate its intracellular localization. CstK mainly localized at vesicular compartments in non-infected cells, and accumulated at CCVs upon C. burnetii infection, suggesting an active role in the biogenesis of this compartment (Figure 1C).
CstK displays autokinase and protein kinase activities
To determine whether CstK is a functional protein kinase, this protein was overproduced in E. coli and purified as a recombinant protein fused to glutathione S-transferase (GST) tag. The purified tagged CstK protein (Fig. 2A, upper panel) was then assayed for autokinase activity in presence of the phosphate donor [γ-33P]ATP. As shown in Fig. 2A (lower panel), CstK incorporated radioactive phosphate from [γ-33P]ATP, generating a radioactive signal corresponding to the expected size of the protein isoform, strongly suggesting that this kinase undergoes autophosphorylation.
To confirm CstK autophosphorylation and exclude that contaminant kinase activities from E. coli extracts might phosphorylate CstK, we mutated the conserved Lys55 residue present in subdomain II into CstK by site-directed mutagenesis. Indeed protein sequence analysis revealed that Lys55 in CstK is similarly positioned as a conserved Lys residue usually involved in the phosphotransfer reaction and also required for the autophosphorylating activity of eukaryotic-like STPKs (34,35). Thus, Lys55 was substituted by a Met residue, and the mutated form of CstK, CstK_K55M, was purified as described above (Fig. 2A, upper panel) and then tested for autophosphorylation in presence of [γ-33P]ATP. As expected, no radioactive signal could be detected (Fig. 2A, lower panel), thus establishing that CstK displayed autophosphorylation activity.
A kinetic analysis of CstK phosphorylation was next performed to determine the initial CstK phosphorylation rate (Fig. 2B). Incorporation of γ-phosphate occurred rapidly, reaching about 50% of its maximum rate within 5 min of reaction. This autokinase activity was dependent on bivalent cations such as Mg2+ and Mn2+ as shown in Fig. 2C, and abolished by addition of 20 mM EDTA (data not shown).
The recombinant CstK protein was further characterized by studying its ability to phosphorylate exogenous proteins and was thus assayed for in vitro phosphorylation of the general eukaryotic protein kinase substrate, myelin basic protein (MBP), in the presence of [γ-33P]ATP. MBP is a commonly used substrate for both Ser/Thr and Tyr kinases. A radiolabeled signal at the expected 18-kDa molecular mass of MBP was detected, thus demonstrating that CstK phosphorylates protein substrates such as MBP (Fig. 2A). Altogether, these data indicate that in vitro, CstK possesses intrinsic autophosphorylation activity and displays kinase functions for exogenous substrates.
Identification of CstK autophosphorylation sites
To determine the specificity of this kinase, we next identified its autophosphorylation sites. A mass spectrometry approach was used since this technique allows precise characterization of post-translational modifications including phosphorylation (39,40). NanoLC/nanospray/ tandem mass spectrometry (LC-ESI/MS/MS) was applied for the identification of phosphorylated peptides and for localisation of phosphorylation sites in CstK. This approach led to 97% of sequence coverage, while the remaining residues uncovered did not include Ser, Thr or Tyr residues able to be phosphorylated.
As detailed in Table 3, analysis of tryptic digests allowed the characterization of three phosphorylation sites in CstK. Surprisingly, unlike classical Ser/Thr or Tyr kinases, CstK was phosphorylated on two Tyr residues (Y14 and Y209) in addition to one Thr site (T232). Since protein sequence analysis did not reveal a classical activation loop in this kinase, the contribution of T232, Y14 and Y209 to CstK kinase activity was individually assessed. Hence, these residues were mutated either to phenylalanine to replace tyrosine residues or alanine to replace threonine residue, thus generating the single mutants CstkK_Y14F, CstK_Y209F, and CstK_T232A as well as the CstK_Y14F/Y209F/T232A triple mutant. Next, in vitro kinase assays with [γ-33P]ATP were carried out and revealed that maximum loss in CstK autophosphorylation activity was observed in the CstK_Y14F mutant (Fig. 2D), suggesting that this site is central for CstK activation. In contrast, the CstK_Y209F mutant exhibited a slight hyperphosphorylation, which might indicate that Y209 only plays an accessory role in controlling CstK autophosphorylation (Fig. 2D). Finally, the CstK_T232A mutant showed a reduced CstK phosphorylation and displayed diminished kinase activity towards the exogenous substrate MBP (Fig. 2D). Note that mutating all three autophosphorylation sites fully abrogated CstK kinase activity (Fig. 2D). These results indicate that Y14 and T232 are the major phosphorylation sites in CstK and strongly suggest that CstK might be a dual specificity (Thr/Tyr) kinase.
CstK regulates vacuole development and C. burnetii replication within infected cells
As a first step towards the understanding of CstK functions in the course of infection, and to appreciate the extent to which this kinase is required for growth and viability of C. burnetii, we attempted to inactivate the corresponding chromosomal gene. Unfortunately, after several attempts we were unable to generate a null mutant suggesting that cstK might be essential. However, we had previously isolated a C. burnetii mutant (Tn2496) carrying a transposon insertion at position 156783, 32 bp upstream of the starting codon of cstK (41) (Fig. 1A). To determine the effect of this transposon insertion for cstK gene expression, we assessed the expression level of cstK mRNA from the C. burnetii wild type and Tn2496 strains. Surprisingly, cstK expression was significantly upregulated in the mutant strain, suggesting that the transposon insertion may have released a transcriptional negative regulation (Fig. 3A). This suggested that a putative transcriptional regulator might bind the cstK promoter and control its activity during host invasion.
We next examined the phenotypic effects of CstK overexpression in C. burnetii. This mutant was tested for its capacity of invading and replicating within host cells. Tn2496 maintained the capacity of invading host cells (Fig. 3B), however, intracellular growth was significantly reduced over 7 days of infection with an intermediate phenotype between WT and the non-secreting dotA::Tn mutant (Fig. 3B&3C). Accordingly, this was reflected in a strong reduction in the size of intracellular C. burnetii colonies and CCVs (Fig. 3D). Therefore, we could conclude that CstK might participate in the formation of the C. burnetii replicative vacuole, and that its expression must be finely tuned for optimal intracellular replication.
CstK specifically interacts with host cell proteins
Since CstK is a secreted protein, we assume that this kinase would interfere with host cell signal transduction pathways to subvert host cell defenses to the benefit of the bacteria. To identify host cell proteins that could interact with CstK, we made use of the model amoeba Dictyostelium discoideum. D. discoideum is a eukaryotic professional phagocyte amenable to genetic and biochemical studies. Lysate from cells overexpressing CstK tagged with a C-terminal FLAG epitope (CstK-FLAG) was incubated with beads coupled to an anti-FLAG antibody. Beads were extensively washed and bound proteins were separated by SDS-PAGE before mass-spectrometry analysis. The putative interactants of CstK identified by this approach are mainly involved in the endocytic pathway (data not shown).
CstK interacts with TBC1D5 in vitro and in cells
Among several identified proteins, we focused on a RabGAP/TBC domain-containing protein, DDB_G0280253 (UniProtKB - Q54VM3). This 136.4 kDa protein is homologous to mammalian TBC1D5 (http://dictybase.org), a GTPase-activating protein (GAP) for Rab7a (42), thus an interesting putative target regarding the role of Rab7a in C. burnetii vacuole formation (43). Therefore, to verify the interaction between CstK and TBC1D5, we assayed the interaction in vivo of FLAG-CstK with human TBC1D5 (Hs-TBC1D5) by using a GFP Trap assay (Fig. 4A). HEK cells expressing FLAG-CstK, Hs_TBC1D5-GFP or both proteins were used for immunoprecipitation using GFP-trap beads and CstK was only detected as co-immunoprecipitated in presence of Hs_TBC1D5-GFP thus confirming that Hs-TBC1D5 is a bona fide CstK interactant in vivo, though we cannot formally exclude that other host cell proteins contribute to this interaction. This interaction was also observed in confocal microscopy studies. Ectopic expression of HA-CstK and GFP-TBC1D5 in non-infected U2OS cells, resulted in the recruitment of TBC1D5 at CstK-positive compartments, as opposed to cells overexpressing GFP-TBC1D5 alone, where the TBC1D5 was found diffused in the cytoplasm (Fig. 4B).
TBC1D5 is recruited at CCVs and regulates their biogenesis
Given its recently reported role in the development of L. pneumophila-containing vacuoles, we investigated the localization of TBC1D5 in U2OS cells infected either with C. burnetii control transposon mutant Tn1832 or the Dot/Icm-defective mutant dotA::Tn. TBC1D5 seems to accumulate at CCVs in a Dot/Icm-dependent manner as the eukaryotic protein failed to accumulate around intracellular C. burnetii dotA::Tn (Fig. 4C). Next, we used siRNA to deplete cells of TBC1D5 prior to C. burnetii infection, to investigate a possible role in CCVs development and intracellular bacterial replication. Indeed, vacuole development was significantly reduced in cells exposed to TBC1D5-targeted siRNAs as opposed to cells treated with non-targeting siRNA oligonucleotides (Fig. 4D).
TBC1D5 is not phosphorylated in vitro by recombinant CstK
Next, we assessed whether CstK might phosphorylate the recombinant Hs-TBC1D5. Despite the in silico prediction of several Ser/Thr and Tyr phosphorylatable residues in Hs-TBC1D5, we failed to detect Hs-TBC1D5 phosphorylation using several in vitro kinases assays (data not shown). In addition, Hs-TBC1D5 phosphorylation status was also investigated upon transfection with CstK or its inactive derivative (K55M) followed by Hs-TBC1D5 immunoprecipitation. No phosphorylation could be detected in our conditions, though a sensitivity issue regarding the detection cannot be ruled out.
DISCUSSION
Our results provide the first biochemical analysis of the secreted kinase CstK and its involvement in the process of infection and CCVs development. Our study also delineates important differences with the classical Ser/Thr kinases. In particular, we provided evidence that CstK is a dual kinase able to autophosphorylate on Thr and Tyr residues. Our overexpressing mutant suggested the presence of a negative transcriptional regulation of cstK expression and its phenotype highlighted the importance of regulating cstK expression during C. burnetii infections. Moreover, the identification of candidate host interactors of CstK further corroborated an important role of the bacterial kinase during infection. We were not able to detect TBC1D5 phosphorylation by CstK in our conditions. However, lack of phosphorylation of host interactors of bacterial STPKs is not uncommon (21) as interaction between STPKs and host proteins might as well perturb protein interaction networks at play in host cells. The biochemical mechanisms of these pathogen-directed targeted perturbations of host cell-signaling networks are being actively investigated. Regardless, siRNA depletion of TBC1D5 in C. burnetii-infected cells points at a role of the eukaryotic protein in CCVs development. In mammals, TBC1D5 was suggested to function as a molecular switch between endosomal and autophagy pathways. Indeed TBC1D5 associates the retromer VPS29 subunit involved in endosomal trafficking, and upon autophagy induction, the autophagy ubiquitin-like protein LC3 can displace VPS29, thus orienting TBC1D5 functions towards autophagy instead of endosomal functions (44). It is thus tempting to propose that CstK might interfere with this tight regulation between TBC1D5, LC3 and VPS29, and redirect TBC1D5 functions to support efficient C. burnetii intracellular replication. Further work needs to be carried out to understand how CstK recognize these host substrates and how they participate in the establishment of C. burnetii parasitophorous vacuoles. Another perspective of this work is the opening of a new field of investigation for future drug development to fight this pathogen. Because CstK seems to be essential, specific inhibitors capable of preventing C. burnetii growth would be extremely useful for the development of new therapies.
EXPERIMENTAL PROCEDURES
Bacterial strains and growth conditions
Bacterial strains and plasmids are described in Table 1. Strains used for cloning and expression of recombinant proteins were Escherichia coli TOP10 (Invitrogen) and E. coli BL21(DE3)Star (Stratagene), respectively. E. coli cells were grown and maintained at 25 °C in LB medium supplemented with 100 µg/ml ampicillin when required. Coxiella burnetii RSA439 NMII and transposon mutants Tn1832, Tn2496, and dotA::Tn were grown in ACCM-2 (45) supplemented with kanamycin (340 mg/ml) or chloramphenicol (3 mg/ml) as appropriate in a humidified atmosphere of 5% CO2 and 2.5% O2 at 37°C.
Cloning, expression and purification of CstK derivatives
The cstK (CBU_0175) gene was amplified by PCR using Coxiella burnetii RSA439 NMII chromosomal DNA as a template with the primers listed in Table 2 containing a BamHI and HindIII restriction site, respectively. The corresponding amplified product was digested with BamHI and HindIII, and ligated into the bacterial pGEX(M) plasmid, that includes a N-terminal GST-tag, thus generating pGEX(M)_cstK. pGEX(M)_cstK derivatives harboring different mutations were generated by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA), and resulted in the construction of plasmids detailed in Table 2. All constructs were verified by DNA sequencing. Purifications of the GST-tagged recombinants were performed as described by the manufacturer (GE Healthcare). For expression in mammalian cells, the cstK gene was amplified by PCR using pGEX(M)_cstK as a template with primers listed in Table 2 containing a BamHI and HindIII restriction site, respectively. The corresponding amplified product was digested with BamHI and HindIII, and ligated into the eukaryotic pcDNA3.1(-) plasmid, thus generating pcDNA3.1(-)_Flag_cstK. cstK coding sequence was also optimized for mammalian cell expression (GenScript), amplified by PCR and cloned into pDXA-3C (46) containing a FLAG-tag for C-terminal fusion. After sequencing, the plasmid was linearized by the restriction enzyme ScaI and transfected in D. discoideum as described (47). Clone selection was made with 10 mg/ml of G418, and protein expression was assayed by Western blot analysis of D. discoideum crude extract with an anti-FLAG rabbit polyclonal antibody (GenScript, USA). For ectopic expression assays, cstK with optimized codons was cloned in pRK5-HA using the primer pair CstKopt-BamHI-Fw/CstKopt-EcoRI-Rv.
Cloning, expression and purification of TBC1D5 derivatives
The D. discoideum GFP-tagged TBC1D5 was previously generated (48). Cells were grown at 22°C in HL5 medium as previously described (47). Human TBC1D5 coding sequence was obtained from the hORFeome v8.1 (ORF 2659, Q92609, fully-sequenced cloned human ORFs in Gateway Entry clones ready for transfer to Gateway-compatible expression vectors). HsTBC1D5 coding sequence has been recombined into pEGFP-N1 RfC Destination vector by GateWay reaction (MGC Paltform Montpellier) thus generating pEGFP-N1_HsTBC1D5 coding for HsTBC1D5 with a C-terminal GFP Tag.
RNA extraction and quantitative RT-PCR (qRT-PCR)
Fifty ml of C. burnetii culture was harvested, resuspended in 600 µL of RNA protect reagent (Qiagen) and incubated 5 min at room temperature. Bacteria were centrifuged and resuspended in 200 µl TE (10 mM Tris-HCl, 1 mM EDTA, pH 8) containing 1 mg/ml lysozyme. Bacterial suspension was incubated at room temperature for 5 min and bacteria were disrupted by vigorous vortexing for 10 sec every 2 min. 700 µl of RLT buffer from RNA easy kit (Qiagen), were added to the bacterial suspension and disruption was completed by vortexing vigorously. RNA was purified with the RNA easy kit according to manufacturer’s instructions. DNA was further removed using DNAse I (Invitrogen). cDNA was produced using Superscript III reverse transcriptase (Invitrogen). Controls without reverse transcriptase were done on each RNA sample to rule out possible DNA contamination. Quantitative real-time PCR was performed using LightCycler 480 SYBR Green I Master (Roche) and a 480 light cycler instrument (Roche). PCR conditions were as follows: 3 min denaturation at 98°C, 45 cycles of 98°C for 5 sec, 60°C for 10 sec and 72°C for 10 sec. The dotA gene was used as internal control. The sequences of primers used for qRT-PCR are listed in Table 2. The expression level of cstK in the wild type strain was set at 100 and the expression levels of cstK in the Tn2496 mutant were normalized to the wild type levels.
In vitro kinase assays
In vitro phosphorylation was performed with 4 μg of wild-type CstK or CstK derivatives in 20 µl of buffer P (25 mM Tris-HCl, pH 7.0; 1 mM DTT; 5 mM MnCl2; 1 mM EDTA; 50μM ATP) with 200 µCi ml−1 (65 nM) [γ-33P]ATP (PerkinElmer, 3000 Ci mmol−1) for 30 min at 37 °C. For substrate phosphorylation, 4 µg of MBP (Myelin Basic Protein) (Sigma) and 4 µg of CstK were used. Each reaction mixture was stopped by addition of an equal volume of Laemmli buffer and the mixture was heated at 100 °C for 5 min. After electrophoresis, gels were soaked in 16% TCA for 10 min at 90 °C, and dried. Radioactive proteins were visualized by autoradiography using direct exposure to films.
Mass spectrometry analysis
For mass spectrometry analysis, CstK was phosphorylated as described above, excepted that [γ-33P]ATP was replaced with 5 mM cold ATP. Subsequent mass spectrometry analyses were performed as previously reported (31–34). Spectra were analyzed with the paragon algorithm from the ProteinPilot® 2.0 database-searching software (Applied Biosystems) using the phosphorylation emphasis criterion against a homemade database that included the sequence of CstK.
C. burnetii infections
U2OS epithelial cells were challenged either with Coxiella burnetii RSA439 NMII, the transposon mutants Tn1832, dotA::Tn or Tn2496 as previously described (38,49). For gene silencing, U2OS cells were seeded at 2,000 cells per well in black, clear-bottomed, 96-well plates in triplicate and transfected with siRNA oligonucleotides 24 h later by using the RNAiMAX transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s recommendations. At 24 h post transfection, cells were challenged with C. burnetii (MOI of 100) and further incubated for 5 d. Cells are then fixed and processed for immunofluorescence. Samples were imaged with an ArrayScan VTI Live epifluorescence automated microscope (Cellomics) equipped with an ORCA-ER CCD camera (Hamamatsu). 25 fields per well were acquired for image analysis. ImageJ, ICY, and CellProfiler software were used for image analysis and quantifications. Phenotypic profiles (expressed as z-scores) were calculated following median based normalization of 96-well plates. Plates effects were corrected by the median value across wells that are annotated as control.
Beta-lactamase translocation assay
Effector proteins translocation assays were performed as previously described (38). Briefly, Coxiella burnetii Tn1832 (wt) and dotA::Tn were transformed either with pXDC-Blam (negative control), pXDC-Blam-CvpB (positive control) or pXDC-Blam-CstK. Each strain was used to infect U2OS epithelial cells. After 24, 48 or 72 hours of infection, cells were loaded with the fluorescent substrate CCF4/AM (LiveBLAzer-FRET B/G loading kit; Invitrogen) in HBSS (20 mM HEPES pH 7.3) containing 15 mM probenecid (Sigma). Cells were incubated in the dark for 1 h at room temperature and imaged using an EVOS inverted fluorescence microscope. Images were acquired using DAPI and GFP filter cubes. The image analysis software CellProfiler was used to segment and identify all cells in the sample (GFP), positive cells (DAPI) and to calculate the intensity of fluorescence in each channel. The percentage of positive cells versus the total number of infected cells was then calculated and used to evaluate effector translocation.
Immunoprecipitation from D. discoideum lysates
For immunoprecipitation assays, 2×107 cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.5% NP40, protease inhibitors (Roche)) and cleared by centrifugation for 15 min at 14 000 rpm in a microfuge. Lysate supernatants were incubated overnight at 4°C with anti-flag monoclonal antibody coated on agarose beads (Genscript). The beads were then washed five times in wash buffer (50mM Tris-HCl pH 7.5, 300 mM NaCl, 0,1% NP40) and once in PBS. Bound proteins were migrated on SDS-PAGE and analysed by LC-MS/MS.
GST pull-down
GST fusion proteins were produced as described above and bound to Glutathione Sepharose 4B beads according to manufacturer’s instructions (GE Heathcare). To prepare cell lysates, D. discoideum cells (2×107) were incubated 15 min in lysis buffer (20 mM HEPES buffer, pH 7.0; 100 mM NaCl; 5 mM MgCl2; 1% Triton X100; protease inhibitors (Roche)) and centrifuged for 15 min at 14 000 rpm in a refrigerated microfuge. GST beads were then incubated with cell lysates (800 µg) overnight at 4°C on a wheel. After three washes in lysis buffer, beads were heated at 95°C for 10 min. Bound proteins were separated by SDS-PAGE and transferred to nitrocellulose before incubation with an anti-myc antibody (9E10; Sigma). Blots were revealed with the Odyssey Western Detection System.
Cell culture, heterologous expression and GFP-Trap immunoprecipitation
HEK cells were grown in a DMEM (Gibco) containing 10% (vol/vol) FBS, 1% glutamax (Gibco, 200 mM stock), 0,5% Penicillin/Streptomycin (Gibco, 10000 U/mL stock) and maintained under standard conditions at 37 °C in a humidified atmosphere containing 5% CO2. Cells were transiently transfected using the jetPEI Transfection Reagent (Polyplus-Transfection Inc.) to express either Hs-TBC1D5_GFP, CstK_FLAG or both proteins. Cells were used 48 h after transfection for immunoprecipitation assay. Transfected cells were washed two times in cold PBS and lysed in lysis buffer (20 mM Hepes, 100 mM NaCl, 5 mM MgCl2, 0,2 % Triton X100, 10 % Glycerol, protease inhibitors (Roche)). Cleared lysate (700 µL, approximately 0.8 mg total proteins) were incubated with GFP-Trap (Chromotek) for 30 min at room temperature under gentle rotation. The beads were washed three times in lysis buffer, boiled in 2X Laemmli sample buffer and loaded on ExpressPlus™ PAGE Gels (GenScript). The eluted proteins were visualized by Western Blotting with the following antibodies: anti-FLAG M2 from Sigma Aldrich, anti-GFP from Torrey Pines, donkey anti-mouse or anti-rabbit from Jackson ImmunoResearch.
CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest with the contents of this article.
ACKNOWLEDGMENTS
The wish to thank the Montpellier RIO imaging facility at the University of Montpellier 2. This work was supported by grants from the ATIP/AVENIR Program for V.M. and M. B., the Region Languedoc-Roussillon for S. B., a Marie Curie CIG to E.M. (grant n. 293731), the Agence Nationale de la Recherche (ANR) Grant ANR-14-CE14-0012-01 (project AttaQ) to M.B.