The Coxiella burnetii type IV secretion system substrate CaeB inhibits intrinsic apoptosis at the mitochondrial level

Manipulation of host cell apoptosis is a virulence property shared by many intracellular pathogens to ensure productive replication. For the obligate intracellular pathogen Coxiella burnetii anti‐apoptotic activity, which depends on a functional type IV secretion system (T4SS), has been demonstrated. Accordingly, the C. burnetii T4SS effector protein AnkG was identified to inhibit pathogen‐induced apoptosis, possibly by binding to the host cell mitochondrial protein p32 (gC1qR). However, it was unknown whether AnkG alone is sufficient for apoptosis inhibition or if additional effector proteins are required. Here, we identified two T4SS effector proteins CaeA and CaeB (C . burnetii a nti‐apoptotic e ffector) that inhibit the intrinsic apoptotic pathway. CaeB blocks apoptosis very efficiently, while the anti‐apoptotic activity of CaeA is weaker. Our data suggest that CaeB inhibits apoptosis at the mitochondrial level, but does not bind to p32. Taken together, our results demonstrate that C. burnetii harbours several anti‐apoptotic effector proteins and suggest that these effector proteins use different mechanism(s) to inhibit apoptosis.     

Probing formation of cargo/importin‐α transport complexes in plant cells using a pathogen effector

Importin‐αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin‐α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin‐α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin‐α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co‐opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin‐α paralogs from Arabidopsis thaliana. A crystal structure of the importin‐α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin‐αs expressed in rosette leaves have an almost identical NLS‐binding site. Comparison of the importin‐α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin‐α, sequence variation at the importin‐α NLS‐binding sites and tissue‐specific expression levels of importin‐αs determine formation of cargo/importin‐α transport complexes in plant cells.     

Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L

Coxiella burnetii is a gram‐negative intracellular bacterium that forms a large, lysosome‐like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking and signalling are unknown. Oxysterol‐binding protein‐related protein 1 long (ORP1L) is a mammalian lipid‐binding protein that plays a dual role in cholesterol‐dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 h of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signalling pathways. The ORP1L N‐terminal ankyrin repeats were necessary and sufficient for PV localization, indicating that ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co‐localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L‐depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest that ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.     

The truncated NLR protein TIR‐NBS13 is a MOS6/IMPORTIN‐α3 interaction partner required for plant immunity

Importin‐α proteins mediate the translocation of nuclear localization signal (NLS)‐containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN‐α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in the autoimmune mutant snc1 (suppressor of npr1‐1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense‐regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope‐tagged MOS6 from pathogen‐challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR‐NBS‐type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type‐III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co‐immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN‐α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N‐terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN‐α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.     

CK2‐regulated schwannomin‐interacting protein IQCJ‐SCHIP‐1 association with AnkG contributes to the maintenance of the axon initial segment

The axon initial segment (AIS) plays a central role in electrogenesis and in the maintenance of neuronal polarity. Its molecular organization is dependent on the scaffolding protein ankyrin (Ank) G and is regulated by kinases. For example, the phosphorylation of voltage‐gated sodium channels by the protein kinase CK2 regulates their interaction with AnkG and, consequently, their accumulation at the AIS. We previously showed that IQ motif containing J‐Schwannomin‐Interacting Protein 1 (IQCJ‐SCHIP‐1), an isoform of the SCHIP‐1, accumulated at the AIS in vivo. Here, we analyzed the molecular mechanisms involved in IQCJ‐SCHIP‐1‐specific axonal location. We showed that IQCJ‐SCHIP‐1 accumulation in the AIS of cultured hippocampal neurons depended on AnkG expression. Pull‐down assays and surface plasmon resonance analysis demonstrated that AnkG binds to CK2‐phosphorylated IQCJ‐SCHIP‐1 but not to the non‐phosphorylated protein. Surface plasmon resonance approaches using IQCJ‐SCHIP‐1, SCHIP‐1a, another SCHIP‐1 isoform, and their C‐terminus tail mutants revealed that a segment including multiple CK2‐phosphorylatable sites was directly involved in the interaction with AnkG. Pharmacological inhibition of CK2 diminished both IQCJ‐SCHIP‐1 and AnkG accumulation in the AIS. Silencing SCHIP‐1 expression reduced AnkG cluster at the AIS. Finally, over‐expression of IQCJ‐SCHIP‐1 decreased AnkG concentration at the AIS, whereas a mutant deleted of the CK2‐regulated AnkG interaction site did not. Our study reveals that CK2‐regulated IQJC‐SCHIP‐1 association with AnkG contributes to AIS maintenance.

     

Interdependent Nucleocytoplasmic Trafficking and Interactions of Dis3 with Rrp6, the Core Exosome and Importin‐α3

Subcellular compartmentalization of exoribonucleases (RNAses) is an important control mechanism in the temporal and spatial regulation of RNA processing and decay. Despite much progress towards understanding RNAse substrates and functions, we know little of how RNAses are transported and assembled into functional, subcellularly restricted complexes. To gain insight into this issue, we are studying the exosome‐binding protein Dis3, a processive 3′ to 5′ exoribonuclease. Here, we examine the interactions and subcellular localization of the Drosophila melanogaster Dis3 (dDis3) protein. N‐terminal domain mutants of dDis3 abolish associations with the ‘core’ exosome, yet only reduce binding to the ‘nuclear’ exosome‐associated factor dRrp6. We show that nuclear localization of dDis3 requires a C‐terminal classic nuclear localization signal (NLS). Consistent with this, dDis3 specifically co‐precipitates the NLS‐binding protein importin‐α3. Surprisingly, dDis3 constructs that lack or mutate the C‐terminal NLS retain importin‐α3 binding, suggesting that the interaction is indirect. Finally, we find that endogenous dDis3 and dRrp6 exhibit coordinated nuclear enrichment or exclusion, suggesting that dDis3, Rrp6 and importin‐α3 interact in a complex independent of the core. We propose that the movement and deposition of this complex is important for the subcellular compartmentalization and regulation of the exosome core.     

Synthesis,Biological Evaluation,and Molecular Docking Studies of Novel 4‐[4‐Arylpyridin‐1(4H)‐yl]benzoic Acid Derivatives as Anti‐HIV‐1 Agents

The structural similarities between N1 substituted 1,4‐dihydropyridines and the known gp41 inhibitors, NB ‐2 and NB ‐64 , were considered in the current research for the design of some novel anti‐HIV‐1 agents. A series of novel 4‐[4‐arylpyridin‐1(4H)‐yl]benzoic acid derivatives were synthesized and after a comprehensive structural elucidation were screened for in vitro anti‐HIV‐1 activity. Most of the tested compounds displayed moderate to good inhibitory activity against HIV‐1 growth and were evaluated for in vitro cytotoxic activity using XTT assay at the concentration of 100 μm . Among the tested compounds, 1c , 1d and 1e showed potent anti‐HIV‐1 activity against P24 expression at 100 μm with inhibition percentage of 84.00%, 76.42% and 80.50%, respectively. All the studied compounds possessed no significant cytotoxicity on MT‐2 cell line. The binding modes of these compounds to gp41 binding site were determined through molecular docking study. Docking studies proved 1a as the most potent compound and binding maps exhibited that the activities might be attributed to the electrostatic and hydrophobic interactions and additional H‐bonds with the gp41 binding site. The Lipinski's ‘rule of five’ and drug‐likeness criteria were also calculated for the studied compounds. All derivatives obeyed the Lipinski's ‘rule of five’ and had drug‐like features. The findings of this study suggest that novel 4‐[4‐arylpyridin‐1(4H)‐yl]benzoic acid might be a promising scaffold for the discovery and development of novel anti‐HIV‐1 agents.     

Bisindole‐oxadiazole hybrids,T3P®‐mediated synthesis,and appraisal of their apoptotic,antimetastatic, and computational Bcl‐2 binding potential

In the pursuit of novel anticancer leads, new bisindole‐oxadiazoles were synthesized using propyl phosphonic anhydride as a mild and efficient reagent. The molecule, 3‐[5‐(1H‐indol‐3‐ylmethyl)‐1,3,4‐oxadiazol‐2‐yl]‐1H‐indole ( 3a ) exhibited selective cytotoxicity to MCF‐7 cells with a cell cycle arrest in the G1 phase. The mechanism of cytotoxicity of 3a involved caspase‐2‐dependent apoptotic pathway with characteristic apoptotic morphological alterations as observed in acridine orange/ethidium bromide and Hoechst staining. The wound healing migratory assay exhibited an intense impairment in the motility of MCF‐7 cells on incubation with 3a . Docking simulations with anti‐apoptotic protein Bcl‐2, which is also involved in cancer metastasis displayed good affinity and high binding energy of 3a into the well characterized BH3 binding site. The positive correlation between the Bcl‐2 binding studies and the results of in vitro investigations exemplifies compound 3a as a lead molecule exhibiting MCF‐7 differential cytotoxicity via apoptotic mode of cell death in addition to its anti‐metastatic activity.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Molecular evidence for a novel <Emphasis Type="Italic">Coxiella</Emphasis> from <Emphasis Type="Italic">Argas monolakensis</Emphasis> (Acari: Argasidae) from Mono Lake,California, USA

Argasid ticks are vectors of viral and bacterial agents of humans and animals. Recent reports indicate that some ornithophilic argasids harbored rickettsial agents. A Nearctic tick, Argas monolakensis Schwan, Corwin, Brown is ornithophilic and has not previously been examined for rickettsial agents. Thirty adult A. monolakensis were tested by PCR for DNA from Rickettsia or Coxiella. Amplicons from a Coxiella sp. that were divergent from Coxiella burnetii were detected in 16/30 A. monolakensis. These molecular isolates were similar but not identical to C. burnetii, the Coxiella spp. of other ticks, and “Coxiella cheraxi” a pathogen of crayfish. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Control of late apoptotic events by the p38 stress kinase in l‐glutamine‐deprived mouse hybridoma cells

l ‐Glutamine (Gln) starvation rapidly triggers apoptosis in Sp2/0‐Ag14 (Sp2/0) murine hybridoma cells. Here, we report on the role played by the stress‐activated kinase p38 mitogen‐activated protein kinase (MAPK) in this process. p38 activation was detected 2 h after Gln withdrawal and, although treatment with the p38 inhibitor SB203580 did not prevent caspase activation in Gln‐starved cells, it reduced the occurrence of both nuclear condensation/fragmentation and apoptotic body formation. Similarly, transfection of Sp2/0 cells with a dominant negative p38 MAPK reduced the incidence of nuclear pyknosis and apoptotic body formation following 2 h of Gln starvation. Gln withdrawal‐induced apoptosis was blocked by the overexpression of the anti‐apoptotic protein Bcl‐xL or by the caspase inhibitor Z‐VAD‐fmk. Interestingly, Bcl‐xL expression inhibited p38 activation, but Z‐VAD‐fmk treatment did not, indicating that activation of this MAPK occurs downstream of mitochondrial dysfunction and is independent of caspases. Moreover, the anti‐oxidant N‐acetyl‐l ‐cysteine prevented p38 phosphorylation, showing that p38 activation is triggered by an oxidative stress. Altogether, our findings indicate that p38 MAPK does not contribute to the induction of apoptosis in Gln‐starved Sp2/0 cells. Rather, Gln withdrawal leads to mitochondrial dysfunction, causing an oxidative stress and p38 activation, the latter contributing to the formation of late morphological features of apoptotic Sp2/0 cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Testing the potency of anti‐TNF‐α and anti‐IL‐1β drugs using spheroid cultures of human osteoarthritic chondrocytes and donor‐matched chondrogenically differentiated mesenchymal stem cells

Inflammation plays a major role in progression of rheumatoid arthritis, a disease treated with antagonists of tumor necrosis factor‐alpha (TNF‐α) and interleukin 1β (IL‐1β). New in vitro testing systems are needed to evaluate efficacies of new anti‐inflammatory biological drugs, ideally in a patient‐specific manner. To address this need, we studied microspheroids containing 10,000 human osteoarthritic primary chondrocytes (OACs) or chondrogenically differentiated mesenchymal stem cells (MSCs), obtained from three donors. Hypothesizing that this system can recapitulate clinically observed effects of anti‐inflammatory drugs, spheroids were exposed to TNF‐α, IL‐1β, or to supernatant containing secretome from activated macrophages (MCM). The anti‐inflammatory efficacies of anti‐TNF‐α biologicals adalimumab, infliximab, and etanercept, and the anti‐IL‐1β agent anakinra were assessed in short‐term microspheroid and long‐term macrospheroid cultures (100,000 OACs). While gene and protein expressions were evaluated in microspheroids, diameters, amounts of DNA, glycosaminoglycans, and hydroxiproline were measured in macrospheroids. The tested drugs significantly decreased the inflammation induced by TNF‐α or IL‐1β. The differences in potency of anti‐TNF‐α biologicals at 24 h and 3 weeks after their addition to inflamed spheroids were comparable, showing high predictability of short‐term cultures. Moreover, the data obtained with microspheroids grown from OACs and chondrogenically differentiated MSCs were comparable, suggesting that MSCs could be used for this type of in vitro testing. We propose that in vitro gene expression measured after the first 24 h in cultures of chondrogenically differentiated MSCs can be used to determine the functionality of anti‐TNF‐α drugs in personalized and preclinical studies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1045–1058, 2018     

The Mood‐Stabilizing Agent Valproate Inhibits the Activity of Glycogen Synthase Kinase‐3

Abstract : Valproic acid (VPA) is a potent broad‐spectrum anti‐epileptic with demonstrated efficacy in the treatment of bipolar affective disorder. It has previously been demonstrated that both VPA and lithium increase activator protein‐1 (AP‐1) DNA binding activity, but the mechanisms underlying these effects have not been elucidated. However, it is known that phosphorylation of c‐jun by glycogen synthase kinase (GSK)‐3β inhibits AP‐1 DNA binding activity, and lithium has recently been demonstrated to inhibit GSK‐3β. These results suggest that lithium may increase AP‐1 DNA binding activity by inhibiting GSK‐3β. In the present study, we sought to determine if VPA, like lithium, regulates GSK‐3. We have found that VPA concentration‐dependently inhibits both GSK‐3α and ‐3β, with significant effects observed at concentrations of VPA similar to those attained clinically. Incubation of intact human neuroblastoma SH‐SY5Y cells with VPA results in an increase in the subsequent in vitro recombinant GSK‐3β‐mediated ³²P incorporation into two putative GSK‐3 substrates (~85 and 200 kDa), compatible with inhibition of endogenous GSK‐3β by VPA. Consistent with GSK‐3β inhibition, incubation of SH‐SY5Y cells with VPA results in a significant time‐dependent increase in both cytosolic and nuclear β‐catenin levels. GSK‐3β plays a critical role in the CNS by regulating various cytoskeletal processes as well as long‐term nuclear events and is a common target for both lithium and VPA ; inhibition of GSK‐3β in the CNS may thus underlie some of the long‐term therapeutic effects of mood‐stabilizing agents.     

Synthesis and Antitumor Evaluation in Vitro of NO‐Donating Ursolic Acid‐Benzylidene Derivatives

Antitumor activity of triterpenoid and its derivatives has attracted great attention recently. Our previous efforts led to the discovery of a series of NO‐donor betulin derivatives with potent antitumor activity. Herein, we prepared eight compounds derived from ursolic acid (UA). All the compounds were evaluated for their in vitro cytotoxicity against four human cancer cell lines (HepG‐2, MCF‐7, HT‐29 and A549). Among the compounds tested, compound 4a was found to be most active against HT‐29 (IC₅₀=4.28 μm ). Further biological assays demonstrated that compound 4a could induce cell cycle arrest at G1 phase and apoptosis in a dose‐dependent manner. In addition, compound 4a was found to upregulate pro‐apoptotic Bax, p53 and downregulate anti‐apoptotic Bcl‐2. All these results suggested that compound 4a is a potential candidate drug for the therapy of colon cancer.     

An AIL family protein promotes type three secretion system‐1‐independent invasion and pathogenesis of Salmonella enterica serovar Typhi

Adhesion and invasion of Intestinal Epithelial Cells (IECs) are critical for the pathogenesis of Salmonella Typhi, the aetiological agent of human typhoid fever. While type three secretion system‐1 (T3SS‐1) is a major invasion apparatus of Salmonella, independent invasion mechanisms were described for non‐typhoidal Salmonellae. Here, we show that T2942, an AIL‐like protein of S. Typhi Ty2 strain, is required for adhesion and invasion of cultured IECs. That invasion was T3SS‐1 independent was proved by ectopic expression of T2942 in the non‐invasive E. coli BL21 and double‐mutant Ty2 (Ty2Δt2942ΔinvG) strains. Laminin and fibronectin were identified as the host‐binding partners of T2942 with higher affinity for laminin. Standalone function of T2942 was confirmed by cell adhesion of the recombinant protein, while the protein or anti‐T2942 antiserum blocked adhesion/invasion of S. Typhi, indicating specificity. A 20‐amino acid extracellular loop was required for invasion, while several loop regions of T2942 contributed to adhesion. Further, T2942 cooperates with laminin‐binding T2544 for adhesion and T3SS‐1 for invasion. Finally, T2942 was required and synergistically worked with T3SS‐1 for pathogenesis of S. Typhi in mice. Considering wide distribution of T2942 among clinical strains, the protein or the 20‐mer peptide may be suitable for vaccine development.     

Phenethyl isothiocyanate potentiates anti‐tumour effect of doxorubicin through Akt‐dependent pathway

The present study aims to investigate the in vivo and in vitro anti‐tumour properties of phenethyl isothiocyanate (PEITC) alone and in combination with doxorubicin (Dox). The anti‐tumour activity was evaluated in vitro by MTT assay using cultured human breast cancer cell line (MCF‐7) and human hepatoma cell line (HepG‐2) cell lines. In vivo, Ehrlich solid tumour model was used. Tumour volume, weight and antioxidant parameters were determined. Immunohistochemistry analysis for active (cleaved) caspase‐3 was also performed. We tested the effect of PEITC treatment on pAkt/Akt ratio, NF‐κB p65 DNA binding activity and caspase‐9 enzyme activity in both MCF‐7 and HepG‐2 cell lines. Effect of PEITC treatment on cell migration was assessed by wound healing assay. PEITC and/or Dox treatment significantly inhibited solid tumour volume and tumour weight when compared with control mice. PEITC treatment significantly reduced oxidative stress caused by Dox treatment as indicated by significant increase in total antioxidant capacity and decrease in malondialdehyde level. Microscopic examination of tumour tissues showed a significant increase in active (cleaved) caspase‐3 expression in PEITC and/or Dox treated groups. PEITC showed a dose‐dependent inhibition of MCF‐7 and HepG‐2 cellular viability. PEITC inhibited Akt and NF‐κB activation and increased caspase‐9 activity in a dose‐dependent manner. PEITC treatment effectively inhibited both MCF‐7 and HepG‐2 cell migration. We can conclude that PEITC acts via multiple molecular targets to elicit anti‐carcinogenic activity. PEITC/Dox combination therapy might be a potential novel strategy, which may benefit patients with breast and liver cancers. Copyright © 2015 John Wiley & Sons, Ltd.     

Endocytic SNAREs are involved in optimal Coxiella burnetii vacuole development

Coxiella burnetii is a Gram‐negative intracellular bacterium. As previously described, both the endocytic and the autophagic pathways contribute to the maturation of Coxiella replicative vacuoles (CRVs). The large CRVs share the properties of both phagolysosomal and autophagolysosomal compartments. Vamp3, Vamp7 and Vamp8 are v‐SNAREs involved in the endocytic pathway which participate mainly in the fusion between endosomes and lysosomes. In the present study we observed that Vamp7 interacts with C. burnetii at different infection times (1 h–48 h p.i.). We have determined that a truncated mutant of Vamp7 (Vamp7 NT) and a siRNA against this SNARE protein affects the optimal development of CRVs, suggesting that Vamp7 mediates fusion events that are required for the biogenesis of CRVs. Indeed, we have observed that overexpression of Vamp7 NT inhibited the heterotypic fusion with lysosomes and the homotypic fusion between individual Coxiella phagosomes and CRVs. Moreover, we have detected in the vacuole membrane, at different infection times, the Vamp7 partners (Vti1a and Vti1b). Interestingly, treatment with chloramphenicol reduced the colocalization between C. burnetii and Vamp7, Vti1a or Vti1b, indicating that the recruitment of these SNAREs proteins is a bacteria‐driven process that favours the CRV biogenesis, likely by facilitating the interaction with the endolysosomal compartment.