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Journal of Bacteriology, January 2009, p. 278-286, Vol. 191, No. 1
0021-9193/09/$08.00+0     doi:10.1128/JB.01031-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Four VirB6 Paralogs and VirB9 Are Expressed and Interact in Ehrlichia chaffeensis-Containing Vacuoles ^,

Weichao Bao ,^, Yumi Kumagai, Hua Niu, Mamoru Yamaguchi, Koshiro Miura, and Yasuko Rikihisa^*

Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Road, Columbus, Ohio 43210

Received 25 July 2008/ Accepted 11 October 2008

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The type IV secretion system is an important virulence factor in several host cell-associated pathogens, as it delivers various bacterial macromolecules to target eukaryotic cells. Genes homologous to several virB genes and virD4 of Agrobacterium tumefaciens are found in an intravacuolar pathogen Ehrlichia chaffeensis, the tick-borne causative agent of human monocytic ehrlichiosis. In particular, despite its small genome size, E. chaffeensis has four tandem virB6 paralogs (virB6-1, -2, -3, and -4) that are 3- to 10-fold larger than A. tumefaciens virB6. The present study for the first time illustrates the relevance of the larger quadruple VirB6 paralogs by demonstrating the protein expression and interaction in E. chaffeensis. All four virB6 paralogs were cotranscribed in THP-1 human leukemia and ISE6 tick cell cultures. The four VirB6 proteins and VirB9 were expressed by E. chaffeensis in THP-1 cells, and amounts of these five proteins were similar in isolated E. chaffeensis-containing vacuoles and vacuole-free E. chaffeensis. In addition, an 80-kDa fragment of VirB6-2 was detected, which was strikingly more prevalent in E. chaffeensis-containing vacuoles than in vacuole-free E. chaffeensis. Coimmunoprecipitation analysis revealed VirB9 interaction with VirB6-1 and VirB6-2; VirB6-4 interaction with VirB6-1, VirB6-2, and VirB6-3; and VirB6-2 80-kDa fragment interaction with VirB6-3 and VirB6-4. The interaction of VirB9 and VirB6-2 was confirmed by far-Western blotting. The results suggest that E. chaffeensis VirB9, the quadruple VirB6 proteins, and the VirB6-2 80-kDa fragment form a unique molecular subassembly to cooperate in type IV secretion.

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The gram-negative bacterial type IV secretion (T4S) system is ancestrally related to the bacterial conjugation system and is thought to function to transport substrate molecules across the membrane into target cells in an ATP-dependent manner. The T4S system acts as a critical virulence factor in host cell-associated pathogens such as Agrobacterium tumefaciens, Legionella pneumophila, Helicobacter pylori, Bartonella henselae, and Brucella abortus by delivering effector molecules into the host cell cytoplasm or nucleus to cause tumors, induce inflammatory cytokines, or create intracellular compartments for bacterial survival and proliferation (4). In the best-studied A. tumefaciens T4S apparatus, the single virB operon, along with virD4, encodes 12 membrane-associated proteins that form a complex traversing two bacterial membranes and presumably the host plasma membrane (7, 8). High-resolution static images, however, are not available for any T4S apparatus. Rather, dynamic assembly of the secretion apparatus in A. tumefaciens is inferred from biochemical and genetic analyses of the VirB-VirD complex (8).

Ehrlichia chaffeensis is a gram-negative obligatory intracellular bacterium that belongs to the order Rickettsiales. This bacterium causes human monocytic ehrlichiosis, an emerging tick-borne zoonosis (11, 30). E. chaffeensis infects monocytes and macrophages and replicates in membrane-bound compartments resembling early endosomes that do not fuse with lysosomes (1, 26). Genes homologous to A. tumefaciens virB genes and virD4 are found among members of the order Rickettsiales, including E. chaffeensis and a closely related bacterium, Anaplasma phagocytophilum (15, 29). virB9 and virB6-1 are expressed by A. phagocytophilum in human peripheral blood leukocytes and the promyelocytic leukemia cell line HL-60 (28, 29). Proteomic analyses revealed that VirB9 is exposed on the surface of isolated E. chaffeensis bacteria (13), like VirB9 of A. phagocytophilum (28). In Anaplasma marginale outer membrane-vaccinated cattle, VirB9, VirB10, and CTP (VirB9-2) induce a B-cell response and stimulate memory T-cell proliferation and gamma interferon secretion (25). Recently, host cytoplasm translocation of bacterial AnkA protein, which is important for A. phagocytophilum infection, was shown to be dependent on VirD4 (24). Consequently, the T4S system is considered to have an important role in the pathogenesis of this group of bacteria.

A. tumefaciens VirB6, an essential component of T4S, is a polytopic inner membrane protein with several transmembrane segments and associates with the T4S core channel consisting of VirB7, VirB8, VirB9, and VirB10 (4, 8, 16, 18, 19). VirB6 has a stabilizing effect on VirB5, a minor component of the T pilus, and thereby regulates T-pilus assembly (14). VirB6 also mediates formation of the disulfide-linked VirB7-VirB7 homodimer and VirB7-VirB9 heterodimer for biogenesis of the T pilus (14, 17). Anti-VirB6 serum precipitates native VirB9 of A. tumefaciens (17). The size of VirB6 is ca. 300 to 450 amino acids in A. tumefaciens, B. henselae, Bordetella pertussis, and Brucella spp., with no strictly conserved residues or motifis (34). Despite the reductive genome evolution in the order Rickettsiales, its members contain tandem multiple virB6 paralogs (15, 29) encoding significantly larger proteins (7, 15, 29). In the present study, we provide evidence for expression of the multiple VirB6 proteins by E. chaffeensis and protein interactions among the four VirB6 paralogs and VirB9. We also present data on a shorter fragment of one of the VirB6 paralogs, which is dissociated from bacteria and interacts with other VirB6 proteins.

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Bacterial strains and culture. E. chaffeensis Arkansas (10) was cultured in the human acute monocytic leukemia cell line THP-1 in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine in 5% CO₂ and 95% air at 37°C as previously described (22). Bacterial infection was monitored using a Diff-Quik (modified Giemsa [33]) staining kit (Baxter Scientific Products, Obetz, OH). Escherichia coli strains NovaBlue (Novagen, Madison, WI) and BL21(DE3) (Novagen) were used for DNA cloning and protein expression, respectively. The ISE6 cell line, derived from the Ixodes scapularis tick, was cultured at 31°C as described previously (27) and was used for E. chaffeensis Arkansas infection.

Reverse transcription-PCR (RT-PCR) analysis. Total RNA was extracted from E. chaffeensis-infected cells and cDNA was synthesized as described previously (5). The primers for PCR are shown in Table S1 in the supplemental material.

Construction of plasmids for expression of rVirB6 paralogs and rVirB9. E. chaffeensis chromosomal DNA was extracted using a QIAamp DNA Blood Mini kit (Qiagen, Valencia, CA). The DNA fragments encoding VirB9 and hydrophilic regions unique to each VirB6 paralog—the VirB6-2 N terminus (VirB6-2N), the VirB6-2 C terminus (VirB6-2C), VirB6-3, and VirB6-4 (Fig. 1) —were amplified by PCR with E. chaffeensis chromosomal DNA as a template using the primers listed in Table S2 in the supplemental material. PCR products were digested with restriction enzymes and ligated to a vector as shown in Table S2 in the supplemental material. The resulting plasmids were amplified in NovaBlue, and sequences of the inserts were confirmed by DNA sequencing. The recombinant proteins were expressed in E. coli BL21(DE3) as previously described (21). Recombinant VirB6-2C (rVirB6-2C) and rVirB9 were soluble proteins in E. coli, whereas rVirB6-2N, rVirB6-3, and rVirB6-4 were insoluble.

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FIG. 1. Schematic representation of the structure of the four VirB6 proteins and VirB9 of E. chaffeensis. The numbers of amino acid residues are indicated to the right of each protein. GenBank accession numbers are shown in parentheses. The blue brackets indicate the regions of recombinant proteins for antibody preparation. The percentages in parentheses indicate amino acid identity between the aligned regions (green) and A. tumefaciens VirB6.

Recombinant protein purification and antibody preparation. Soluble and insoluble proteins were purified from the E. coli soluble fraction and inclusion body, respectively, as previously described (21). The purified proteins were loaded onto a 12 or 15% sodium dodecyl sulfate-polyacrylamide gel, and protein bands were cut from the gel. Antisera against rVirB6-2N, rVirB6-3, and rVirB6-4 were raised in rabbits. Mouse antiserum against rVirB6-2C was developed in 15 8-week-old ICR female mice (Harlan, Indianapolis, IN) using TiterMax adjuvant (TiterMax USA, Norcross, GA) for the first immunization and Freund's incomplete adjuvant (Sigma-Aldrich, St. Louis, MO) for the second and third immunizations. For VirB6-1, a 14-residue peptide (CDYSQSDYEDKYKYI) was synthesized and conjugated with keyhole limpet hemocyanin, and antiserum was produced in rabbits.

Isolation of ECV and Western blot analysis. Ehrlichia-containing vacuoles (ECV) were isolated from E. chaffeensis-infected THP-1 cells by the methods used for phagosome isolation (32) with some modification. Infected cells (3 x 10⁷) were incubated with 250 U/ml lithium heparin (Sigma-Aldrich) in 5 ml of 10 mM Tris-HCl (pH 7.5)-0.34 M sucrose for 10 min on ice and homogenized 10 times in a loosely fitted Dounce homogenizer. DNase I (10 µg/ml) and 10 mM MgCl₂ were added and incubated for 10 min on ice, followed by 10 more homogenizations. The mixture was centrifuged at 500 x g for 5 min to sediment unbroken cells and nuclei. ECV were harvested from the supernatant by centrifugation at 10,000 x g for 5 min, and isolation of ECV was verified by Diff-Quik staining. Vacuole-free E. chaffeensis was isolated by disruption of infected cells through N₂ decompression (21) or by sonication (22). The bacterial numbers in ECV and vacuole-free samples were determined by quantifying the copy numbers of the 16S rRNA gene (a single-copy gene in the E. chaffeensis genome) through quantitative real-time PCR as described previously (5).

THP-1 cells, isolated vacuole-free E. chaffeensis, and ECV were lysed by incubation for 10 min at room temperature with radioimmunoprecipitation assay buffer (Pierce, Rockford, IL) supplemented with 1/100 volume of proteinase inhibitor cocktail consisting of six protease inhibitors with broad specificity for the inhibition of aspartic, cysteine, and serine proteases as well as aminopeptidases (Calbiochem, San Diego, CA), 10 µg/ml DNase I, and 10 mM MgCl₂. After cell debris was removed by centrifugation, protein samples were incubated with NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM dithiothreitol (DTT) for 30 min at 37°C and loaded onto a NuPAGE 3 to 8% Tris-acetate gel (Invitrogen) or a 6% Laemmli gel (23) to detect VirB6-1, VirB6-2, VirB6-3, and VirB6-4 or onto a 12% Laemmli gel to detect VirB9. Proteins in the gel were transferred to a sheet of nitrocellulose membrane using a Mini Trans-blot cell (Bio-Rad, Hercules, CA). The membrane was incubated with rabbit anti-VirB6-1 peptide, -rVirB6-2N, -rVirB6-3, or -rVirB6-4 antibodies or mouse anti-rVirB6-2C antibody (1:500 dilution) preabsorbed as described below, followed by incubation with secondary antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse immunoglobulin G (IgG) antibody (KPL, Gaithersburg, MD) (1:1,000 dilution). The membrane was then incubated with ECL Western blotting substrate (Pierce), and bands were visualized with a LAS-3000 luminescent image analyzer (Fujifilm, Stamford, CT).

The antisera were preabsorbed as follows. Lysates were prepared using radioimmunoprecipitation assay buffer (Pierce) from 5 x 10⁶ THP-1 cells and 10⁸ E. coli BL21(DE3) cells for each antiserum. Proteins in the lysates were precipitated with 10% trichloroacetic acid, incubated with NuPAGE LDS sample buffer supplemented with 50 mM DTT for 30 min at 37°C, and loaded onto a 6% Laemmli gel. Proteins in the gel were transferred to a nitrocellulose membrane. The membrane was incubated with a 1:500 dilution of each antiserum overnight at 4°C. To examine whether the anti-rVirB6-2N-reactive smaller fragment is a part of VirB6-2, anti-rVirB6-2N antibody was further preabsorbed with 10 µg of rVirB6-2N, rVirB9 as a control, or ECV proteins with molecular masses of less than 90 kDa on a nitrocellulose membrane.

Double-label immunofluorescence microscopy. In order to stain VirB6 proteins and VirB9, E. chaffeensis-infected THP-1 cells (3 day postinfection, 80% infectivity) were fixed with cold methanol at –20°C for 5 min and incubated at 37°C for 1 h with rabbit anti-VirB6-1 peptide, -rVirB6-3, -rVirB6-4, or -rVirB9 or mouse anti-rVirB6-2C antibody (1:160 dilution in phosphate-buffered saline [PBS] [137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1 mM KH₂PO₄, pH 7.4]) preabsorbed with THP-1 cell lysate. Cells were then incubated at 37°C for 1 h with dog anti-E. chaffeensis antiserum (1:200 dilution in PBS) preabsorbed with THP-1 cell lysate, followed by incubation with secondary antibodies (Alexa Fluor 555-conjugated goat anti-rabbit or goat anti-mouse IgG and fluorescein isothiocyanate-conjugated goat anti-dog IgG, 1:300 dilution in PBS) at room temperature for 1 h. As negative controls, E. chaffeensis-infected THP-1 cells were incubated with preimmune rabbit, mouse, or dog serum and the appropriate secondary fluorochrome-conjugated antibody. After incubation, the fluorescence images were analyzed with a Nikon Eclipse E400 fluorescence microscope with a xenon-mercury light source (Nikon Instruments Inc., Melville, NY).

Electron microscopy. Transmission electron microscopy specimens were prepared as described previously (31). The sample was embedded in a low-viscosity Epon mixture of 1,2,7,8-diepoxyoctane (Sigma-Aldrich) and nonenyl succinic anhydride (Electron Microscopy Sciences, Fort Washington, PA). The ultrathin section was stained with a saturated solution of uranyl acetate diluted with equal parts of acetone and then lead citrate and was observed under a Philips 300 transmission electron microscope at 60 kV.

Far-Western blotting. One microgram of rVirB9 was loaded onto a 12% Laemmli gel and transferred to a nitrocellulose membrane. rVirB9 on the membrane was denatured with 6 M guanidine-HCl in the basic buffer (20 mM HEPES [pH 7.5], 50 mM KCl, 10 mM MgCl₂, 1 mM DTT, 0.1% NP-40), followed by renaturing with the basic buffer containing serially diluted guanidine-HCl. After the membrane was washed with TBS (20 mM Tris-HCl [pH 7.5], 150 mM NaCl) and blocked with 5% skim milk in TBS, it was incubated with 2% dodecyl maltoside-solubilized E. chaffeensis lysate overnight at 4°C. The membrane was washed with TBS supplemented with 0.05% Tween 20 and incubated with rabbit anti-rVirB9, -VirB6-1 peptide, -rVirB6-2N, -rVirB6-3, or -rVirB6-4 antibody (1:500 dilution) followed by HRP-conjugated goat anti-rabbit IgG antibody (1:1,000 dilution).

Coimmunoprecipitation. Anti-rVirB9, -rVirB6-2N, -rVirB6-3, and -rVirB6-4 antibodies were purified by sodium sulfate precipitation (20) followed by DEAE column chromatography (12). The purified immunoglobulin were conjugated with protein A-agarose (Santa Cruz Biotechnology, Santa Cruz, CA). Freshly isolated ECV from E. chaffeensis-infected THP-1 cells (10⁸) and uninfected THP-1 cells (10⁷) were suspended in PBS (final protein concentration, 2.5 mg/ml) containing 2% dodecyl maltoside, 1/100 volume of proteinase inhibitor cocktail (Calbiochem), 10 µg/ml DNase I, and 10 mM MgCl₂ and incubated for 1 h at 4°C. Cell debris was removed by centrifugation at 10,000 x g for 5 min at 4°C. After the lysates were precleared with 20 µl/ml of normal rabbit IgG conjugated with protein A-agarose (Santa Cruz) for 1 h at 4°C, the lysates were incubated with the protein A-agarose antibody for 2 h at 4°C. The agarose beads were washed four times with 50 mM Tris-HCl (pH 8.0)-150 mM NaCl-0,1% NP-40 and once with PBS. The precipitated proteins were detected using anti-rVirB9, -VirB6-1 peptide, -rVirB6-2N, -rVirB6-3, and -rVirB6-4 antibodies (1:500 dilution) extensively preabsorbed with THP-1 cell lysate, followed by HRP-conjugated goat anti-rabbit IgG antibody (1:1,000 dilution). To detect proteins that interact with the VirB6-2 small fragment, antibodies were covalently cross-linked with protein A-agarose using 20 mM dimethyl pimelimidate in 0.2 M sodium borate buffer (pH 9.0). Coprecipitated VirB6-2 small fragment was probed with mouse anti-rVirB6-2C antibody (1:500 dilution) extensively preabsorbed with THP-1 lysate and HRP-conjugated goat anti-mouse IgG antibody (1:1,000 dilution) preabsorbed with normal rabbit serum.

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The four tandem VirB6 paralogs contain the TrbL/VirB6 motif. The four tandem virB6 paralogs of E. chaffeensis are predicted to encode proteins significantly larger than the prototypic A. tumefaciens VirB6 (Fig. 1). They are progressively larger, with predicted protein sizes ranging from 826 to 2,758 amino acids (aa), and their isoelectric points are progressively acidic, ranging from 8.3 to 3.9, based on the DNASTAR Protean analysis. All four VirB6 proteins contain a hydrophobic region, the TrbL/VirB6 motif, based on the DNASTAR Protean program and a motif search with Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan), and long hydrophilic regions unique to each paralog. A TrbL/VirB6 motif was found in the E. chaffeensis VirB6 paralogs at approximately 305 to 648 aa from the N terminus in each protein, with 13.9 to 15.8% amino acid identity with A. tumefaciens VirB6. By PSORTb v.2.0 (http://www.psort.org/) and LipoP 1.0 (http://www.cbs.dtu.dk/services/LipoP/) analyses, all four VirB6 paralogs were predicted to be membrane proteins: all four proteins contain a signal peptide, and VirB6-2 and VirB6-3 contain a lipobox and thus are predicted to be lipoproteins. VirB6-3 and VirB6-4 contain long repeat sequences at the C-terminal regions: 7 repeats in aa 1133 to 1341 of VirB6-3 and 14 repeats in aa 1580 to 2539 of VirB6-4 as detected by the Radar program provided by EMBL-EBI (http://www.ebi.ac.uk/Radar) (Fig. 1).

The four virB6 genes are transcribed by E. chaffeensis in THP-1 and ISE6 tick cell culture. Long RT-PCR products of two separate operons, consisting of virB8-virB9-virB10-virB11-virD4 and of sodB-virB3-virB4-virB6-1, were previously detected in E. chaffeensis cultured in the human myelocytic leukemia cell line THP-1 and in A. phagocytophilum cultured in HL-60 cells (29). Because expression of virB6-2, virB6-3, and virB6-4 has not been previously reported for either bacterium, we examined expression of the four E. chaffeensis virB6 paralogs downstream of virB4 in THP-1 cells and tick ISE6 cells (Fig. 2A). virB9 expression in ISE6 cells was also examined. RT-PCR analysis showed that the four virB6 paralogs and virB9 were transcribed by E. chaffeensis in THP-1 cells and in ISE6 tick cells (Fig. 2B). No amplicon was detected without reverse transcriptase, indicating the absence of genomic DNA contamination in the RNA preparation. As the predicted intergenic spaces between the four tandem virB6 paralogs are relatively short, from 3 to 198 bp, we examined whether the virB6 paralogs were cotranscribed. RT-PCR of the intergenic regions showed that the virB6 paralogs were cotranscribed with adjacent genes by E. chaffeensis in THP-1 cells and ISE6 tick cells (bottom rows for each cell type Fig. 2). Thus, the sodB, virB3, virB4, virB6-1, virB6-2, virB6-3, and virB6-4 genes are likely cotranscribed in both mammalian and tick cells.

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FIG. 2. Transcription of the four E. chaffeensis virB6 paralogs and virB9 in human leukocyte and tick cell cultures. (A) Schematic representation of the four virB6 paralogs and virB9. Open reading frame are represented as open arrows, and the direction of the arrowheads indicates their orientations. The lengths of the intergenic spaces are indicated in base pairs. The PCR target regions are shown as double arrows. (B) Total RNA was prepared from E. chaffeensis-infected THP-1 cells (80% infected cells) and ISE6 tick cells (70% infected cells). + and – indicate the presence or absence of reverse transcriptase, respectively. D, use of chromosomal DNA as a template as a positive control for the PCR. The genes or intergenic spaces and the base pair sizes of the amplified products are indicated at the top and bottom, respectively, of the panels.

Expression of the four VirB6 paralogs and VirB9 by E. chaffeensis in THP-1 cells. According to a BLAST search for short, nearly exactly matched sequences in the NCBI nonredundant database, each of the amino acid sequences of the VirB6 paralogs and VirB9 had little or no homology to any other known proteins (E values were greater than 0.1) except for orthologs present in other members of the family Anaplasmataceae and thus were unique to each proteins in E. chaffeensis. Antibodies were prepared against VirB9 and unique regions of each VirB6 paralog (the regions cloned for antibody preparation are shown in Fig. 1). Western blot analysis indicated that each antibody specifically recognized the respective recombinant protein, and absence of any cross-reaction was verified (Fig. 3A). Double-labeling immunofluorescence of E. chaffeensis in THP-1 cells with the antibodies and dog anti-E. chaffeensis showed that all five VirB proteins were colocalized with E. chaffeensis in each ECV (Fig. 3B). As negative controls, E. chaffeensis-infected THP-1 cells were incubated with the preimmune rabbit, mouse, or dog serum and the appropriate secondary fluorochrome-conjugated antibodies. There was no detectable labeling, indicating that the labeling with both dog anti-E. chaffeensis and rabbit or mouse anti-recombinant protein antisera was specific (Fig. 3B).

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FIG. 3. Antibody preparation and double-label immunofluorescence assay. (A) Antibodies reacted specifically with the respective recombinant proteins. One microgram of rVirB6-2N (2N), rVirB6-2C (2C), rVirB6-3 (3), rVirB6-4 (4), or rVirB9 (9) was loaded onto a 15% Laemmli gel. Proteins were detected with anti-VirB6-1 peptide (VirB6-1), -rVirB6-2N (VirB6-2N), -rVirB6-2C (VirB6-2C), -rVirB6-3 (VirB6-3), -rVirB6-4 (VirB6-4), and -rVirB9 (VirB9) antibodies. (B) Double-label immunofluorescence of infected THP-1 cells. Infected THP-1 cells (3 days postinfection) were fixed with methanol and incubated with the following antisera: rabbit anti-VirB6-1 peptide, -rVirB6-3, -rVirB6-4, or -rVirB9 antiserum followed by Alexa Fluor 555-labeled goat anti-rabbit IgG antibody (red, left panels); mouse anti-rVirB6-2C antiserum followed by Alexa Fluor 555-labeled goat anti-mouse IgG antibody (red, left panels); and dog anti-E. chaffeensis antiserum followed by fluorescein isothiocyanate-labeled goat anti-dog IgG antibody (green, center panels). The panels on the right are merged images. As controls for immunofluorescence labeling, E. chaffeensis-infected THP-1 cells were incubated with the preimmune rabbit, mouse, or dog serum and the appropriate secondary fluorochrome-conjugated antibody. Bar, 5 µm.

The four VirB6 paralogs and VirB9 in isolated ECV. As E. chaffeensis is an obligatory intracellular bacterium, in order to study T4S apparatus protein interaction, it is critical to enrich E. chaffeensis organisms yet to preserve the apparatus structure as intact as possible. Therefore, we isolated ECV using a method developed to isolate phagosomes from neutrophils (32). Morphological features such as morulae (dense basophilic clusters of bacteria characteristic of this group of bacteria; these are larger than individual bacteria) evident under light microscopy (Fig. 4A) and the membrane-bound vacuoles containing various numbers of small "dense-cored cells" or large "reticulate cells" (bacteria) evident under electron microscopy (Fig. 4B) indicate that the E. chaffeensis-containing vacuoles were enriched by this method. Mitochondria, which were major contaminants in the vacuole fraction (Fig. 4B), were not removed, as further purification reduced the integrity of isolated ECV.

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FIG. 4. Isolation of ECV. (A) Light micrographs of E. chaffeensis-infected THP-1 cells (left panel), isolated E. chaffeensis (middle panel), and isolated ECV (right panel) stained with Diff-Quik stain. Bar, 2.5 µm. (B) Transmission electron micrograph of the isolated ECV. Note several ECV with dense-cored cells (D) or lighter and larger reticular cells (R). Arrowheads. inclusion membrane; M, mitochondrion. Bar, 0.6 µm.

The presence and amounts of the four VirB6 paralogs and VirB9 in ECV and vacuole-free bacteria were examined by Western immunoblot analysis. VirB6-1, VirB6-3, VirB6-4, and VirB9 were detected in both ECV and vacuole-free bacteria in similar amounts, suggesting that these proteins firmly anchor to the bacterial membrane. The detected sizes of VirB6-1 and VirB6-3 were slightly larger than predicted, while VirB6-4 was smaller than predicted in a Tris-acetate gel (Fig. 5). Of note is that both anti-rVirB6-2N and -rVirB6-2C antibodies reacted with two bands, with apparent molecular masses of around 80 kDa and 120 kDa, in both ECV and vacuole-free bacteria (Fig. 5). The intensity of the 80-kDa band was stronger than that of the 120-kDa band in ECV. To verify that the 80-kDa band was a segment of VirB6-2, anti-rVirB6-2N antibody was preabsorbed with proteins of the ECV lysate with molecular masses of less than 90 kDa on a nitrocellulose membrane. After the antibody was preabsorbed, the 120-kDa band disappeared and the 80-kDa band density became lower (Fig. 5, VirB6-2N, ECV lysate). When the anti-rVirB6-2N antibody was preabsorbed with rVirB6-2N, the 120-kDa band disappeared and the 80-kDa band became much weaker than when the antibody was preincubated with rVirB9 (Fig. 5, VirB6-2N absorbed). It is unlikely that the smaller fragment was generated during sample preparation, since the proteinase inhibitor cocktail was included in all of the sample preparation procedures and the smaller fragment was not detected from other VirB6 proteins. Therefore, these results suggest that VirB6-2 was cleaved to produce an 80-kDa protein and that most of the 80-kDa fragment was disassociated from bacteria and accumulated in the ECV.

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FIG. 5. Expression and intracellular distribution of VirB6 paralogs and VirB9. Uninfected THP-1 cell lysate control (T) as well as lysates of ECV (V) and isolated E. chaffeensis (Ec) normalized to bacterial genome copy number were loaded onto a NuPAGE 3 to 8% Tris-acetate gel (TA gel), a 6% Laemmli gel (6% gel), or a 12% Laemmli gel (12% gel), and protein expression was assessed by Western blotting using anti-VirB6-1 peptide, -rVirB6-2N, -rVirB6-2C, -rVirB6-3, -rVirB6-4, and -rVirB9 antibodies extensively preabsorbed with uninfected THP-1 and E. coli BL21(DE3) lysates. Anti-rVirB6-2N antibody was further preabsorbed with ECV proteins with molecular masses of less than 90 kDa (VirB6-2N, ECV lysate underlined), rVirB6-2N (VirB6-2N absorbed, rVirB6-2N), or rVirB9 (VirB6-2N absorbed, rVirB9). Numbers on the left are molecular masses in kilodaltons.

Interaction of VirB6 paralogs and VirB9. As all of the VirB6 paralogs and VirB9 were demonstrated to be expressed by E. chaffeensis and present in ECV, we examined whether these proteins interact in isolated ECV. Far-Western blotting showed the interaction of rVirB9 and native VirB6-2, as anti-rVirB6-2N antibody revealed the rVirB9 band after the rVirB9-immobilized nitrocellulose membrane was incubated with the ECV lysate (Fig. 6A, upper panel). This was not due to cross-reaction, as the anti-rVirB6-2N antibody did not cross-react to rVirB9 (Fig. 6A, lower panel).

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FIG. 6. Interaction among VirB6 paralogs and VirB9. (A) Far-Western blotting. rVirB9 (1 µg) was electrotransferred to a nitrocellulose membrane, denatured, renatured, and incubated with or without the E. chaffeensis (Ec) lysate. Each membrane strip was incubated with anti-rVirB9, -VirB6-1 peptide, -rVirB6-2N, -rVirB6-3, or -rVirB6-4 antibody, followed by HRP-conjugated goat anti-rabbit IgG antibody. Bands were visualized by incubating the membrane with ECL Western blotting substrate for 20 s (VirB9 with Ec lysate) or 1 min (VirB6-1, VirB6-2, VirB6-3, and VirB6-4). (B) Coimmunoprecipitation. Lysates derived from uninfected THP-1 cells (T) and freshly isolated ECV (V) were incubated with protein A-agarose-conjugated anti-rVirB9, -rVirB6-2N, -rVirB6-3, -rVirB6-4, or normal rabbit IgG (Control IgG). Precipitated proteins were separated by electrophoresis on a 12% Laemmli gel (for detection of VirB9), a 6% Laemmli gel (for detection of VirB6-1, 120-kDaVirB6-2, VirB6-2 80-kDa fragment, and VirB6-3), and a 3 to 8% Tris-acetate gel (for detection of VirB6-4). Precipitated proteins were detected using anti-rVirB9, -VirB6-1 peptide, -rVirB6-2N (120 kDa), -rVirB6-2C (80-kDa fragment), -rVirB6-3, or -rVirB6-4 antibody followed by HRP-conjugated goat anti-rabbit IgG antibody or HRP-conjugated goat anti-mouse IgG antibody. Lysate, each protein in the lysates of uninfected THP-1 cells (T) and ECV (V) was detected by Western blotting.

By coimmunoprecipitation of isolated ECV with anti-rVirB9, -rVirB6-2N, -rVirB6-3, and -rVirB6-4 antibodies, the interaction of VirB9 and 120-kDa VirB6-2 was reciprocally detected (Fig. 6B). The interaction of VirB6-3 and VirB6-4 was also reciprocally detected. The 80-kDa fragment of VirB6-2 interacted with VirB6-3 and VirB6-4. As in the case of A. tumefaciens (17), VirB9 and VirB6-1 were found to interact in ECV, although the interaction was not detected by far-Western blotting. Perhaps the protein level of VirB6-1 in ECV lysate was insufficient to be detected by far-Western blotting. Furthermore, though not reciprocal, the interaction of VirB6-1 and VirB6-4 as well as VirB6-2 and VirB6-4 was detected by coimmunoprecipitation. Normal rabbit IgG did not precipitate any VirB6 or VirB9 proteins (Fig. 6B). The apparent molecular sizes of VirB6-1 and VirB6-3 in a 6% Laemmli gel were 100 kDa and 150 kDa, respectively, which were slightly smaller than those on a 3 to 8% Tris-acetate gel (Fig. 5).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
As these tandem virB6 paralog loci are one of the rare regions of synteny conserved in the genomes of all sequenced members of the order Rickettsiales (15), there must be evolutionary pressure to conserve this structure and these gene products in this group of pathogens. The present study was initiated to investigate whether these predicted virB6 paralogs encode functional proteins that interact with each other and another T4S protein, VirB9. The four virB6 genes were demonstrated to be cotranscribed by E. chaffeensis in mammalian THP-1 cells as well as in ISE6 tick cells, suggesting that the four VirB6 paralogs cooperate in an individual bacterium in both mammalian and tick cells.

In A. tumefaciens the five VirB proteins VirB6, -7, -8, -9, and -10 are postulated to be the "core components" of the T4S apparatus (4, 8, 18, 19). VirB9 is one of the key molecules for the core complex assembly (9, 17). As in the case of A. tumefaciens (3), VirB9 is exposed on the bacterial surface in both A. phagocytophilum (28) and E. chaffeensis (13). E. chaffeensis VirB9 interacts with VirB6-1 and VirB6-2, and VirB6-3 interacts with VirB6-4. In addition, E. chaffeensis VirB6-4 appears to interact with VirB6-1 and VirB6-2. E. chaffeensis VirB9 interaction with VirB6-1 and VirB6-2 is similar to Agrobacterium VirB9 and VirB6 interaction (17). The interactions of the quadruple VirB6 paralogs and VirB9 suggest that these proteins form a functional complex of T4S in an individual bacterium. The involvement of four distinct E. chaffeensis VirB6 proteins in the T4S apparatus subassembly demonstrated here represents a novel form of the T4S system, and this subassembly of T4S is likely to be conserved in the bacteria belonging to the order Rickettsiales, as they have multiple virB6 paralogs. However, a precise assembly mechanism remains to be elucidated; for example, an additional factors(s) may be involved to form the subassembly consisting of the VirB6s and VirB9.

It has been suggested that in A. tumefaciens VirB6 directly interacts with a minor component of the T-pilus protein, VirB5, to stabilize this protein (14). A. tumefaciens VirB6 mediates formation of the VirB7-VirB7 homodimer (14) and the VirB7-VirB9 heterodimer (17). In contrast, virB1, virB5, and virB7 genes have not been identified in the E. chaffeensis genome (6, 15), whereas large quadruple virB6 genes have been acquired, which is contrary to the dogma of the ongoing reductive genome evolution in members of the order Rickettsiales (15). Perhaps the ViB6s evolved to confer unique regulation, membrane localization, architecture, and functions to the T4S apparatus in these obligatory intracellular pathogens. For example, the large VirB6 paralogs might compensate for the missing VirB proteins by forming VirB6s-VirB9 complexes. In addition, a larger amount of the 80-kDa fragment of VirB6-2 was detected in ECV than in vacuole-free bacteria, suggesting disassociation of the 80-kDa fragment from bacteria and its release to ECV. The extrabacterial localization of the VirB6-2 80-kDa fragment enables us to propose a hypothesis that the fragment has a function analogous to that of A. tumefaciens VirB5. Since multiple genes of virB2 encoding a major component of the pilus were identified in the E. chaffeensis genome, the 80-kDa fragment may interact with VirB2 and form a T4S pilus structure, compensating for the lack of VirB5. Moreover, unlike the case for A. tumefaciens VirB6, interaction of the 80-kDa fragment with VirB6-3 and VirB6-4 suggests that the latter two proteins are bacterial surface exposed.

The 80-kDa fragment enriched in ECV was shown to be a part of the 120-kDa VirB6-2 protein, suggesting the specific cleavage of VirB6-2 to generate the 80-kDa fragment, although we cannot exclude the possibility that the 80-kDa fragment is independently translated from an alternative translation start site. The A. tumefaciens full-length VirB1 protein associates predominantly with the inner membrane, while the cleaved C-terminal fragment (VirB1*) becomes soluble. VirB1* is in part secreted outside of bacteria, whereas bacterium-associated VirB1* interacts with the VirB7-VirB9 heterodimer (2). Despite the lack of homology between E. chaffeensis VirB6-2 and A. tumefaciens VirB1, E. chaffeensis VirB6-2 may also be proteolytically cleaved. Since the 80-kDa fragment of VirB6-2 was detected by both anti-rVriB6-2N and -rVirB6-2C antibodies, it contains part or all of both the hydrophilic N-terminal and C-terminal regions of the120-kDa VirB6-2. Further study is required to clarify how the 80-kDa fragment is generated and how it interacts with VirB6-3 and VirB6-4. Since the 80-kDa fragment of VirB6-2 is likely an extrabacterial component of the T4S system and T4S is suggested to be a prerequisite for intracellular bacteria to survive in host cells, these proteins may serve as vaccine candidates to prevent human monocytic ehrlichiosis.

Finally, one important achievement of the present study is the isolation of ECV from infected cells. This technique enabled us to assess protein interaction by coimmunoprecipitation by preserving the protein complex as intact as possible and by removing most of host cell-derived proteins which may interfere with the assay. This technique also would further facilitate studying inclusion proteins as well as bacterial and host protein interactions for this group of intracellular bacteria.

 
This work was supported by National Institutes of Health grant R01 AI054476.

We thank Urlike Munderloh for the uninfected ISE6 tick cells and advice on tick cell culture. We also thank Qingming Xiong and Takane Kikuchi for their assistance in mouse antibody preparation.

 
* Corresponding author. Mailing address: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210-1093. Phone: (614) 292-5661. Fax: (614) 292-6473. E-mail: rikihisa.1{at}osu.edu

Published ahead of print on 24 October 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

These authors contributed equally to this study.

Present address: Arnold School of Public Health, University of South Carolina, 800 Sumter Street, Columbia, SC 29208.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Barnewall, R. E., Y. Rikihisa, and E. H. Lee. 1997. Ehrlichia chaffeensis inclusions are early endosomes which selectively accumulate transferrin receptor. Infect. Immun. 65:1455-1461.[Abstract]
2
2. Baron, C., M. Llosa, S. Zhou, and P. C. Zambryski. 1997. VirB1, a component of the T-complex transfer machinery of Agrobacterium tumefaciens, is processed to a C-terminal secreted product, VirB1. J. Bacteriol. 179:1203-1210.[Abstract/Free Full Text]
3
3. Bayliss, R., R. Harris, L. Coutte, A. Monier, R. Fronzes, P. J. Christie, P. C. Driscoll, and G. Waksman. 2007. NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system. Proc. Natl. Acad. Sci. USA 104:1673-1678.[Abstract/Free Full Text]
4
4. Cascales, E., and P. J. Christie. 2003. The versatile bacterial type IV secretion systems. Nat. Rev. Microbiol. 1:137-149.[CrossRef][Medline]
5
5. Cheng, Z., Y. Kumagai, M. Lin, C. Zhang, and Y. Rikihisa. 2006. Intra-leukocyte expression of two-component systems in Ehrlichia chaffeensis and Anaplasma phagocytophilum and effects of the histidine kinase inhibitor closantel. Cell. Microbiol. 8:1241-1252.[CrossRef][Medline]
6
6. Cheng, Z., X. Wang, and Y. Rikihisa. 2008. Regulation of type IV secretion apparatus genes during Ehrlichia chaffeensis intracellular development by a previously unidentified protein. J. Bacteriol. 190:2096-2105.[Abstract/Free Full Text]
7
7. Christie, P. J. 1997. Agrobacterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional transporters in eubacteria. J. Bacteriol. 179:3085-3094.[Free Full Text]
8
8. Christie, P. J., K. Atmakuri, V. Krishnamoorthy, S. Jakubowski, and E. Cascales. 2005. Biogenesis, architecture, and function of bacterial type IV secretion systems. Annu. Rev. Microbiol. 59:451-485.[CrossRef][Medline]
9
9. Das, A., and Y. H. Xie. 2000. The Agrobacterium T-DNA transport pore proteins VirB8, VirB9, and VirB10 interact with one another. J. Bacteriol. 182:758-763.[Abstract/Free Full Text]
10
10. Dawson, J. E., B. E. Anderson, D. B. Fishbein, J. L. Sanchez, C. S. Goldsmith, K. H. Wilson, and C. W. Duntley. 1991. Isolation and characterization of an Ehrlichia sp. from a patient diagnosed with human ehrlichiosis. J. Clin. Microbiol. 29:2741-2745.[Abstract/Free Full Text]
11
11. Demma, L. J., R. C. Holman, J. H. McQuiston, J. W. Krebs, and D. L. Swerdlow. 2005. Epidemiology of human ehrlichiosis and anaplasmosis in the United States, 2001-2002. Am. J. Trop. Med. Hyg. 73:400-409.[Abstract/Free Full Text]
12
12. Fahey, J. L. 1967. Chromatographic separation of immunoglobulins, p. 321-332. In A. Williams and W. Chase (ed.), Methods in immunology and immunochemistry, vol. 1. Academic Press, New York, NY.
13
13. Ge, Y., and Y. Rikihisa. 2007. Surface-exposed proteins of Ehrlichia chaffeensis. Infect. Immun. 75:3833-3841.[Abstract/Free Full Text]
14
14. Hapfelmeier, S., N. Domke, P. C. Zambryski, and C. Baron. 2000. VirB6 is required for stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens. J. Bacteriol. 182:4505-4511.[Abstract/Free Full Text]
15
15. Hotopp, J. C., M. Lin, R. Madupu, J. Crabtree, S. V. Angiuoli, J. Eisen, R. Seshadri, Q. Ren, M. Wu, T. R. Utterback, S. Smith, M. Lewis, H. Khouri, C. Zhang, H. Niu, Q. Lin, N. Ohashi, N. Zhi, W. Nelson, L. M. Brinkac, R. J. Dodson, M. J. Rosovitz, J. Sundaram, S. C. Daugherty, T. Davidsen, A. S. Durkin, M. Gwinn, D. H. Haft, J. D. Selengut, S. A. Sullivan, N. Zafar, L. Zhou, F. Benahmed, H. Forberger, R. Halpin, S. Mulligan, J. Robinson, O. White, Y. Rikihisa, and H. Tettelin. 2006. Comparative genomics of emerging human ehrlichiosis agents. PLoS Genet. 2:e21.[CrossRef][Medline]
16
16. Jakubowski, S. J., V. Krishnamoorthy, E. Cascales, and P. J. Christie. 2004. Agrobacterium tumefaciens VirB6 domains direct the ordered export of a DNA substrate through a type IV secretion system. J. Mol. Biol. 341:961-977.[CrossRef][Medline]
17
17. Jakubowski, S. J., V. Krishnamoorthy, and P. J. Christie. 2003. Agrobacterium tumefaciens VirB6 protein participates in formation of VirB7 and VirB9 complexes required for type IV secretion. J. Bacteriol. 185:2867-2878.[Abstract/Free Full Text]
18
18. Judd, P. K., R. B. Kumar, and A. Das. 2005. The type IV secretion apparatus protein VirB6 of Agrobacterium tumefaciens localizes to a cell pole. Mol. Microbiol. 55:115-124.[CrossRef][Medline]
19
19. Judd, P. K., D. Mahli, and A. Das. 2005. Molecular characterization of the Agrobacterium tumefaciens DNA transfer protein VirB6. Microbiology 151:3483-3492.[Abstract/Free Full Text]
20
20. Kekwick, R. A. 1940. The serum proteins in multiple myelomatosis. Biochem. J. 34:1248-1257.[Medline]
21
21. Kumagai, Y., Z. Cheng, M. Lin, and Y. Rikihisa. 2006. Biochemical activities of three pairs of Ehrlichia chaffeensis two-component regulatory system proteins involved in inhibition of lysosomal fusion. Infect. Immun. 74:5014-5022.[Abstract/Free Full Text]
22
22. Kumagai, Y., H. Huang, and Y. Rikihisa. 2008. Expression and porin activity of P28 and OMP-1F during intracellular Ehrlichia chaffeensis development. J. Bacteriol. 190:3597-3605.[Abstract/Free Full Text]
23
23. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.[CrossRef][Medline]
24
24. Lin, M., A. den Dulk-Ras, P. J. J. Hooykaas, and Y. Rikihisa. 2007. Anaplasma phagocytophilum AnkA secreted by type IV secretion system is tyrosine phosphorylated by Abl-1 to facilitate infection. Cell. Microbiol. 9:1368-1378.
25
25. Lopez, J. E., G. H. Palmer, K. A. Brayton, M. J. Dark, S. E. Leach, and W. C. Brown. 2007. Immunogenicity of Anaplasma marginale type IV secretion system proteins in a protective outer membrane vaccine. Infect. Immun. 75:2333-2342.[Abstract/Free Full Text]
26
26. Mott, J., R. E. Barnewall, and Y. Rikihisa. 1999. Human granulocytic ehrlichiosis agent and Ehrlichia chaffeensis reside in different cytoplasmic compartments in HL-60 cells. Infect. Immun. 67:1368-1378.[Abstract/Free Full Text]
27
27. Munderloh, U. G., S. D. Jauron, V. Fingerle, L. Leitritz, S. F. Hayes, J. M. Hautman, C. M. Nelson, B. W. Huberty, T. J. Kurtti, G. G. Ahlstrand, B. Greig, M. A. Mellencamp, and J. L. Goodman. 1999. Invasion and intracellular development of the human granulocytic ehrlichiosis agent in tick cell culture. J. Clin. Microbiol. 37:2518-2524.[Abstract/Free Full Text]
28
28. Niu, H., Y. Rikihisa, M. Yamaguchi, and N. Ohashi. 2006. Differential expression of VirB9 and VirB6 during the life cycle of Anaplasma phagocytophilum in human leucocytes is associated with differential binding and avoidance of lysosome pathway. Cell. Microbiol. 8:523-534.[CrossRef][Medline]
29
29. Ohashi, N., N. Zhi, Q. Lin, and Y. Rikihisa. 2002. Characterization and transcriptional analysis of gene clusters for a type IV secretion machinery in human granulocytic and monocytic ehrlichiosis agents. Infect. Immun. 70:2128-2138.[Abstract/Free Full Text]
30
30. Paddock, C. D., and J. E. Childs. 2003. Ehrlichia chaffeensis: a prototypical emerging pathogen. Clin. Microbiol. Rev. 16:37-64.[Abstract/Free Full Text]
31
31. Rikihisa, Y., and S. Ito. 1979. Intracellular localization of Rickettsia tsutsugamushi in polymorphonuclear leukocytes. J. Exp. Med. 150:703-708.[Abstract/Free Full Text]
32
32. Rikihisa, Y., and D. Mizuno. 1977. Binding of markers of either side of plasma membranes to the cytoplasmic side of paraffin oil-loaded phagolysosomes. Exp. Cell Res. 110:93-101.[CrossRef][Medline]
33
33. Skipper, R., and D. B. DeStephano. 1989. A rapid stain for Campylobacter pylori in gastrointestinal sections using Diff-Quik. J. Histotechnol. 12:303-304.
34
34. Yeo, H. J., and G. Waksman. 2004. Unveiling molecular scaffolds of the type IV secretion system. J. Bacteriol. 186:1919-1926.[Free Full Text]

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Journal of Bacteriology, January 2009, p. 278-286, Vol. 191, No. 1
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