Abstract
Background
The TIR domain-containing proteins BtpA/Btp1/TcpB and BtpB are translocated into host cells by the facultative intracellular bacterial pathogen Brucella. Here, they interfere with Toll like receptor signalling to temper the host inflammatory response. BtpA has also been found to modulate microtubule dynamics. In both proteins we identified a WxxxE motif, previously shown to be an essential structural component in a family of bacterial type III secretion system effectors that modulate host actin dynamics by functioning as guanine nucleotide exchange factors of host GTPases. We analysed a role for the WxxxE motif in association of BtpA and BtpB with the cytoskeleton.
Results
Unlike BtpA, ectopically expressed BtpB did not show a tubular localisation, but was found ubiquitously in the cytoplasm and the nucleus, and often appeared in discrete punctae in HeLa cells. BtpB was able to protect microtubules from drug-induced destabilisation similar to BtpA. The WxxxE motif was important for the ability of BtpA and BtpB to protect microtubules against destabilising drugs. Surprisingly, ectopic expression of BtpA, although not BtpB, in HeLa cells induced the formation of filopodia. This process was invariably dependent of the WxxxE motif. Our recent resolution of the crystal structure of the BtpA TIR domain reveals that the motif positions a glycine residue that has previously been shown to be essential for interaction of BtpA with microtubules.
Conclusions
Our results suggest a structural role for the WxxxE motif in the association of BtpA and BtpB with microtubules, as with the WxxxE GEF family proteins where the motif positions an adjacent catalytic loop important for interaction with specific Rho GTPases. In addition, the ability of ectopically expressed BtpA to induce filopodia in a WxxxE-dependent manner suggests a novel property for BtpA. A conserved WxxxE motif is found in most bacterial and several eukaryotic TIR domain proteins. Despite the similarity between ectopically expressed BtpA and WxxxE GEFs to modulate host actin dynamics, our results suggest that BtpA is not part of this WxxxE GEF family. The WxxxE motif may therefore be a more common structural motif than thus far described. BtpA may provide clues to cross-talk between the TLR and GTPase signalling pathways.
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Background
Brucella is a Gram negative facultative intracellular bacterium that can cause brucellosis or Malta Fever, the world's most widespread zoonotic disease [1]. In humans Brucella causes an undulant fever that can be accompanied by complications such as endocarditis, arthritis and osteomyelitis [2]. Brucella is a stealth pathogen, known for its silent entry into host cells and multiple mechanisms to suppress host innate immune signalling, including non-toxic lipid A, and avoidance of oxidative burst and Toll like receptor (TLR) signalling cascades [2]-[6]. Whereas the primary task of immune cells is to phagocytose and degrade microbes, many intracellular pathogens, including Brucella, use secretion systems to introduce bacterial effector proteins directly into host cells to alter host cell biology and favour their intracellular replication [7],[8]. For full virulence, Brucella requires its VirB type IV secretion system (T4SS) to modulate endosomal trafficking, and create a replication niche in ER-derived membrane vesicles, named Brucella containing vacuoles (BCV) [9]-[11]. For many years, the effector proteins translocated by the Brucella VirB system remained elusive. After the identification of a first candidate [12], recent efforts from several laboratories, using both bioinformatics screens and translocation assays have resulted in a list of possible effectors [13]-[16]. To date, the precise role of most of these proteins in Brucella virulence is still not clear and the subject of intense research.
Recently, the proteins BtpA (Btp1/TcpB) and BtpB have been shown to be translocated by Brucella into host cells [17]. Although a TEM-1 β-lactamase assay did not show a significant difference in translocation efficiency of the proteins from wild type and virB mutant bacteria, a CyA reporter assay showed VirB-dependent protein transport into host cells, suggesting BtpA and BtpB may be substrates of the VirB T4SS. BtpA and BtpB belong to a class of bacterial proteins first described in Salmonella, Escherichia coli and Brucella that share homology with the eukaryotic Toll/Interleukin-1 receptor (TIR) domain [18],[19]. A conserved TIR domain is present in eukaryotic TLR proteins as well as their downstream signalling TIR adaptor proteins, including the central cytosolic adapter protein MyD88. The TIR domain is essential for TLR and adaptor interactions and for the onset of a signalling cascade resulting in nuclear translocation of the transcription factor NFκB, followed by the production of pro-inflammatory cytokines and type I interferons [20]. TIR domain interactions play a key role in activating conserved cellular signal transduction pathways in response to pathogen signals, and it was suggested that bacterial TIR proteins interfere with host TLR defence signalling by molecular mimicry (reviewed in [21]).
B. abortus 2308 BtpA (BAB1_0279) and the almost identical B. melitensis 16M BtpA (BMEI1674) down modulate maturation of dendritic cells [22] and inhibit TLR-induced NFκB activation. It has been suggested that this is through interference with the TLR4/MyD88/TIRAP complex [23]-[27], however the exact binding partner of BtpA is still a subject of controversy. Far less is known about BtpB, however recently it has also been shown to play a role in immune modulation [17]. Recently we, and others, published the crystal structure of the BtpA TIR domain, which showed a dimeric arrangement of a canonical TIR domain [25],[28],[ For cloning Escherichia coli DH5α was cultured at 37°C in Luria-Bertani broth (LB) (Invitrogen, Merck). Kanamycin (25 μg/ml) or chloramphenicol (30 μg/ml) were added to the media when appropriate. A 828 bp version of the btpA gene was cloned from B. melitensis 16M genomic DNA (BMEI1674, Genbank accession NP_540591 is the shorter annotated version) using PCR (primers Bgl II L1674-F 5'-GAAGATCT TATGAGTTCGTACTCTTCTAATATTG-3', and Pst IL1674-R 5'-AACTGCAG TCAGATAAGGGAATGCAGTTC-3') and cloned in frame with the GFP coding sequence in eukaryotic expression plasmid pEGFP (Clontech) using standard protocols, resulting in plasmid pIN271. A 978 version of the btpB gene was cloned from B. suis genomic DNA (BR0735, Genbank accession number AAN29664 is the shorter annotated version) using PCR (primers Bgl II 735-F 5'-GAAGATCT TATGACATCTAGTCGCGACACG-3', and Pst I 735-R 5'-AACTGCAG CTAGGTGATGAGGGCGACG-3') and cloned in frame with the GFP coding sequence in pEGFP resulting in plasmid pIN292. We amplified a longer version of btpB, which was recently also shown to complement a B. abortus btpB mutant in the control of NF-κB translocation into the nucleus [17]. Site directed mutagenesis of the WxxxE motif was done using the QuickChange Site-Directed Mutagenesis kit (Stratagene) following manufacturer's instructions, using pIN271 as a template with the following mutagenic primers for btpA (changed codons for Trp (TGG) and Glu (GAA) are italicised): W213A (5'-TTTAGCAAGCAAGCG CCCGCAAGAGAATTAG-3'), W213S (5'-TTTAGCAAGCAATCG CCCGCAAGAGAATTAG-3'), W213F (5'-TTTAGCAAGCAATTC CCCGCAAGAGAATTAG-3'), E217A (5'-CAATGGCCCGCAAGAGCA TTAGATGGACTGAC-3'), E217D (5'- CAATGGCCCGCAAGAGAT TTAGATGGACTGAC-3'), I226S (5'- CTGACGGCAATGGAAAGT GGCGGACAGACGC-3'), G183A (5'- CATATACGTTGAAGGTCGCT GACAGCCTTCGGCG-3'), and btpB (pIN292 as a template): W263S (5'- CTATCAGCGAAAAGACTCG TGCGGCGTCG-3') and E267A (5'- CTGGTGCGGCGTCGCG TTCCGCGCGATTCG-3'). Constructs were verified by DNA sequencing (Eurofins MWG operon, Germany). Table 2 summarizes the plasmids used in this study. HeLa cells were grown in RPMI 1640 (Gibco) supplemented with heat-inactivated 10% fetal bovine serum (FBS, Lonza, Switzerland). Transfections were performed using Lipofectamine 2000 (Invitrogen). For immuno fluorescence studies, cells were seeded on coverslips (BD Bioscience) and cultured overnight in 12-well dishes. Cells were transfected and, after 16–20 hours, treated with nocodazole (Sigma M1404) at a concentration of 1 μg/ml for 30 min if desired. Cells were fixed with 4% PFA, and processed for immunocytochemistry. Monoclonal mouse anti-β-tubulin (Sigma Aldrich, T4026), mouse anti-FK2 (Enzo Life science, BML-PW8810), Texas Red anti-mouse (Vector Laboratories, TI-2000), Rhodamin phalloidin (Invitrogen R415), Rabbit anti-BtpA (a gift from Marty Roop, East Carolina University, Greenville), FITC anti-rabbit (Vector Laboratories, FI-2000) were used for immune labelling. Immuno fluorescence microscopy was performed using a LEICA DM/IRB microscope using filter sets L5 (band pass (BP) 480/40; Beam splitter (BS) 505; emission BP527/30) and N2.1 (515–560; BS 580; emission long pass (LP) 590), respectively. For imaging we used a Coolsnap fx (Roper Scientifique) and MetaVue software, and images were further processed using Adobe Photoshop. Confocal analysis was performed at the RIO imaging platform in Montpellier, with a Biorad MRC1024 confocal microscope. Brucella genomes were obtained and compared on the PATRIC website [33]. Sequence alignments were performed using T-Coffee analysis, and further analysed using Jalview. Structural figures were generated with Pymol (www.pymol.org).Materials and methods
Bacterial strains
Plasmids and site-directed mutagenesis
Cell culture, transfection, immunocytochemistry, and fluorescence microscopy
Bio informatic analysis
Additional files
Abbreviations
- GEF:
-
Guanidine exchange factors
- LPS:
-
Lipopolysaccharide
- MTOC:
-
Microtubule-organizing centre
- NC:
-
Nocodazole
- TIR:
-
Toll/Interleukin-1 receptor
- TLR:
-
Toll like receptor
- T4SS:
-
Type IV secretion system
- T3SS:
-
Type III secretion system
- UPS:
-
Ubiquitin Proteasome system
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Acknowledgements
The authors thank Marty Roop for BtpA antibodies, and Philippe Fort and Géraldine Pawlak for helpful discussions. We thank Montpellier RIO imaging for confocal microscopy. U1047 received funding from INSERM, Université Montpellier 1, the Region Languedoc-Roussillon, the Agence Nationale de Recherche (ANR-MIME; T4SS, ANR-MIE; BruCell). Work in the LT group was funded by the ATIP-Avenir program from the CNRS and Ligue Contre le Cancer.
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Competing interests
The authors declare that they have no competing interests.
Authors' contributions
Conceived and designed the experiments: CF, LT, DOC, ACV. Performed the experiments: CF, BKT, TK, SR. Analysed the data: CF. SR, BKT, LT, DOC, ACV. Contributed to writing the paper: CF, LT, DOC, ACV. All authors read and approved the final manuscript.
Electronic supplementary material
12964_2014_53_MOESM1_ESM.pdf
Additional file 1: Figure S1.: Evolutionary tree of Brucella species and distribution of BtpA and BtpB proteins. The alignment was made in Patric and the tree was optimized using Mega Software. btpA and btpB genes are highly conserved throughout the genus. We found only four variations in 407 sequences (showing 47 species). All B. melitensis strains carry a single amino acid change (leading to A163V), while three strains carry a variation in R167H (B. ovis), G200D (B. suis ATCC23445) or V182G (B. neotomae). BtpB is also highly conserved, with only two amino acids that show variation. The Trp residue, as part of a WxxxE motif, in BtpB was replaced by an Arg residue in B. melitensis BtpB (W263R). (PDF 492 KB)
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Additional file 2: Figure S2.: Confocal imaging of BtpA in HeLa cells shows tubular localisation at cell periphery. HeLa cells were transiently transfected with a plasmid encoding GFP-BtpA, fixed after 16 h and analysed by confocal microscopy. The coloured image (green) shows the merged stack images of 14 Z-slices, the other images show the individual Z-images (1 μm depth) of HeLa cells expressing GFP-BtpA. Bar, 25 μm. (PDF 277 KB)
12964_2014_53_MOESM3_ESM.pdf
Additional file 3: Figure S3.: Ectopic expression of BtpA in HeLa cells shows colocalisation with microtubules HeLa cells were transiently transfected with a plasmid expressing GFP-BtpA. At 16 h cells were fixed and processed for immuno labelling with mouse anti β-tubulin antibody, detected with anti-mouse Texas Red to visualize microtubules (red fluorescence). The large image shows a merged image of 6 Z-Stack confocal images (1 μm depth) of cells with microtubules (in red) and GFP-BtpA in one of the cells. Arrow heads indicate colocalisation of GFP-BtpA with the microtubule network (yellow), at the periphery of the cell. The other images represent the individual Z-stack images. Scale bar, 25 μm. Data are representative of 3 or more independent experiments. (PDF 309 KB)
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Additional file 4: Figure S4.: Expression phenotypes of GFP-BtpB in HeLa cells. HeLa cells were transiently transfected with a plasmid expressing GFP-BtpB. At 16 h cells were fixed, processed, and analysed using fluorescence microscopy. The individual images show the different observed expression patterns ranging from a diffuse signal, to cells with a diffuse fluorescent signal as well as accumulation of BtpB in punctae, heterogeneous in size and number. Scale bar, 25 μm. (PDF 192 KB)
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Additional file 5: Figure S5.: Colocalisation of BtpB and conjugated ubiquitin. HeLa cells were transiently transfected with a plasmid expressing GFP-BtpB. At 16 h cells were fixed and processed for immune labelling with mAb FK2, which detects mono- and poly conjugated ubiquitin, and labelled with anti-mouse Texas Red for fluorescence detection. A. Confocal images showing stacks of overlay and individual GFP-BtpB and FK2 images. Scale bar, 10 μm. B. Two fluorescence images (red and green filters) showing colocalisation of BtpB and FK2, also at the intercellular bridge in late cytokinesis (arrow, and boxed areas). Scale bar, 25 μm C. Image showing transfected and non-transfected cells with and without FK2 positive foci. Cells indicated with numbers in the overlay panel (on the right) are enlarged below as individual green and red fluorescent images. Scale bar, 25 μm. 1, 2. Transfected cells with a punctate pattern for BtpB, colocalising with FK2. FK2 positive, BtpB negative foci are also observed. 3, 4: Non-transfected, FK2 positive foci. 5. Transfected, no visible BtpB punctae, FK2 positive. (PDF 805 KB)
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Felix, C., Kaplan Türköz, B., Ranaldi, S. et al. The Brucella TIR domain containing proteins BtpA and BtpB have a structural WxxxE motif important for protection against microtubule depolymerisation. Cell Commun Signal 12, 53 (2014). https://doi.org/10.1186/s12964-014-0053-y
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DOI: https://doi.org/10.1186/s12964-014-0053-y