Four VirB6 paralogs and VirB9 are expressed and interact in Ehrlichia chaffeensis-containing vacuoles

Abstract

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.

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.

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.

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 (αVirB6-1), -rVirB6-2N (αVirB6-2N), -rVirB6-2C (αVirB6-2C), -rVirB6-3 (αVirB6-3), -rVirB6-4 (αVirB6-4), and -rVirB9 (αVirB9) 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.

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.

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 (αVirB6-2N, ECV lysate underlined), rVirB6-2N (αVirB6-2N absorbed, rVirB6-2N), or rVirB9 (αVirB6-2N absorbed, rVirB9). Numbers on the left are molecular masses in kilodaltons.

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 (αVirB9 with Ec lysate) or 1 min (αVirB6-1, αVirB6-2, αVirB6-3, and αVirB6-4). (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.