Figure 1.
Ehrlichia are contained in the filopodia of DH82 cells.
(A) Filopodia extended from the polar ends of the E. chaffeensis- infected DH82 cell. Left: E. chaffeensis-infected DH82 cell probed with anti-Hsp60 antibody. Thick arrow indicates E. chaffeensis intracellular colonies and thin arrow indicates filopodium. Middle: E. chaffeensis-infected cell stained with DAPI. Thick arrow indicates morulae of E. chaffeensis stained with DAPI and thin arrow indicates host nucleus. Right: Merged figure. Scale bar, 25 micrometers. (B) Filopodia of E. chaffeensis- infected DH82 cells extended to neighboring cells. (C) When host cells were not in the immediate vicinity, the leading edge of an E. chaffeensis- infected DH82 cell formed a flattened fan-shaped structure filled with the pathogen. (D) Uninfected DH82 cell. (E) Uninfected DH82 cell stained with Diff-Quik stain. (F) E. muris-infected DH82 cell stained with Diff-Quik stain. Scale bar, 25 micrometers. (G) Transmission electron micrograph of a DH82 cell infected with E. muris. Arrows indicate morulae of E. muris. Scale bar, 1 micrometer.
Figure 2.
E. muris is associated with the filopodia of DH82 cells.
(A–C) Filopodium extending from the cell body of an E. muris- infected DH82 cell. Left: E. muris-infected DH82 cell probed with anti-Ehrlichia Hsp60 antibody. Thick arrow indicates E. muris, and thin arrow indicates filopodium. Middle: E. muris-infected cell stained with DAPI. Thick arrow indicates DNA of E. muris stained with DAPI, and thin arrow indicates host nucleus. Right: Merged figure. Scale bar, 25 micrometers. (D) Filopodium of an E. muris- infected DH82 cell extended to a neighboring cell. (E) Uninfected DH82 cells. (F) Absence of Ehrlichia Hsp60 primary antibody resulted in absence of staining E. muris in infected DH82 cells, but DAPI stained the E. muris DNA and DH82 nucleus.
Figure 3.
Actin was a major protein of filopodia induced during Ehrlichia chaffeensis infection.
Left: E. chaffeensis-infected DH82 cell probed with phalloidin. Thin arrows indicate filopodia. Middle: E. chaffeensis-infected cell stained with DAPI. Thick arrow indicates morulae of E. chaffeensis stained with DAPI, and thin arrow indicates host nucleus. Right: Merged figure. Scale bar, 50 micrometers. (A) Filopodia extended from an E. chaffeensis-infected DH82 cell. (B) Filopodium of E. chaffeensis-infected DH82 cell extended to a neighboring cell. (C, D) Ehrlichia are contained in a long filopodium that had a flattened fan-shaped structure with no host cells in the immediate vicinity. Thick arrow indicates the flattened fan-shaped structure at the leading edge of the filopodium. (E) Uninfected DH82 cell. (F) Lengths of filopodia of DH82 cells infected with E. chaffeensis (n = 25).
Figure 4.
Ehrlichia is transported through the filopodia of the host cells.
(A) Scanning electron micrographs of DH82 cells infected with E. chaffeensis. (a) E. chaffeensis are observed in the filopodia of DH82 cells. The thick arrow indicates the flattened fan-shaped structure at the leading edge of the filopodium (indicated by thin arrows). (b) Ehrlichia bacteria in a filopodium from which the cell membrane has been removed. The thick arrow indicates an Ehrlichia. (c) A flattened fan-shaped structure filled with Ehrlichia from which the cell membrane had been removed. The thick arrow indicates Ehrlichia, and the thin arrow indicates a filopodium. (d) A filopodium that extended from an Ehrlichia-infected DH82 cell. (e) Low magnification of an Ehrlichia-infected host cell filopodium in contact with a neighboring cell. The thick arrows indicate the flattened fan-shaped structures, and the thin arrows indicate the filopodia. (f) High magnification of a flattened fan-shaped structure from which the cell membrane has been removed at the leading edge of an Ehrlichia-infected cell (depicted in figure e) in contact with the neighboring host cell. The thick arrow indicates an Ehrlichia. (g) Intracellular Ehrlichia deforming the overlying cell membrane at the junction of a neighboring cell. (h) Localization of Ehrlichia (thick arrow) deforming the overlying cell membrane of adjacent cells. (i) Ehrlichia seen in adjacent cells of a cracked open DH82 host cell. (B) Actin was a major protein of filopodia during E. muris infection. (a) Thin arrows indicate filopodia; (b) thick arrow indicates Ehrlichia morula; (c) thick arrow in DAPI figure indicates Ehrlichia DNA, and the thin arrows indicate host nuclei and (d) merged figure. (C) Absence of filopodia in an uninfected DH82 cell.
Figure 5.
Cytochalasin D inhibited filopodium formation in Ehrlichia-infected cells.
(A) E. chaffeensis-infected DH82 cells treated with cytochalasin D and stained with phalloidin (left), DAPI (middle) (thick arrows indicate Ehrlichia morulae and thin arrows indicate host nuclei), and merged figure (right). (B) Uninfected DH82 cells treated with cytochalasin D and stained with phalloidin. (C, D) E. muris- infected DH82 cells treated with cytochalasin D and probed with Ehrlichia Hsp60 antibody (left) (thick arrow indicates Ehrlichia), DAPI (middle) (thick arrow indicates Ehrlichia DNA, and thin arrow indicates host cell nuclei), and merged figure (right). (E) Uninfected DH82 cells treated with cytochalasin D and probed with Ehrlichia Hsp60 antibody. (F) Scanning electron micrograph of E. muris-infected DH82 cells treated with cytochalasin D from which the cell membrane had been removed. (G) Transmission electron micrograph of E. muris-infected DH82 cell treated with cytochalasin D. Thick arrows indicate Ehrlichia morulae, N, nucleus. Scale bar, 1 micrometer. (H) A single IOE cell in mouse spleen. Arrows indicate actin filaments.
Figure 6.
Actin inhibition prevented filopodium formation in E. muris-infected DH82 cells.
(A) Untreated DH82 cells infected with E. muris. (B) Nocodazole treatment had no effect on filopodium formation. Treatment with (C) blebbistatin, (D) lantrunculin B, or (E) wiskostatin prevented filopodium formation.
Figure 7.
Inhibition of filopodium formation prevented Ehrlichia intercellular transport.
(A) Ehrlichia-infected DH82 cells were treated with CFSE and seeded with uninfected non-treated DH82 cells for 24 hours. Thick arrow indicates E. muris (probed with Ehrlichia HSP60), whereas the thin arrows indicate filopodia/pseudopodia of infected cells. DAPI stains the nucleus of both the uninfected and infected cells. The adjacent figure is the Nomarski image, which clearly showed the filopodia/pseudopodia of infected cells. (B, C) DH82 cells were treated with CFSE and seeded with infected non-treated DH82 cells for 24 hours. Thick arrow indicates E. muris (probed with Ehrlichia HSP60) whereas the thin arrows indicate filopodia/pseudopodia of infected cells. DAPI stains the nuclei of both uninfected and infected cells. The adjacent figure is the Nomarski image which showed clearly the filopodia/pseudopodia of infected cells. (D) Ehrlichia-infected DH82 cells were treated with CFSE and seeded with uninfected non-treated DH82 cells for 24 hours in the presence of cytochalasin D. Thick arrow indicates E. muris (the adjacent figure is the Nomarski image). (E) Uninfected DH82 cells were treated with CFSE and seeded with infected non-treated DH82 cells for 24 hours in the presence of cytochalasin D. Thick arrow indicates E. muris (the adjacent figure is the Nomarski image). (F). Quantitative real time-PCR of bacterial loads of E. muris-infected DH82 cells to evaluate cytotoxicity in the presence of cytochalasin D (n = 3 per group). (G). Quantitative real time-PCR of bacterial load of E. muris- infected DH82 cells seeded with uninfected DH82 cells in the presence and absence of cytochalasin D (n = 3 per group).
Figure 8.
Ehrlichia are observed in the filopodia of mouse macrophages.
(A) E. muris-infected mouse macrophages probed with Ehrlichia Hsp60 antibody (left), DAPI (middle) (thick arrows indicate DNA of E. muris, and thin arrows indicate mouse macrophage nuclei), and merged figure (right). (B) IOE-infected mouse macrophage probed with Ehrlichia Hsp60 antibody (left) (thin arrow indicates filopodium), DAPI (middle), and merged figure (right). (C) Scanning electron micrograph of E. muris-induced filopodium in a mouse macrophage; thin arrow indicates the filopodium. (D) The interior of a mouse macrophage from which the cell membrane has been removed contained E. muris. (E) Higher magnification of E. muris in a mouse macrophage. (F) Scanning electron micrograph of an E. muris bacterium. (G, H) Scanning electron micrograph of IOE-induced filopodia in mouse macrophages; thin arrows indicate the filopodia. (I) IOE microorganisms in a mouse macrophage. (J) Scanning electron micrograph of a single IOE bacterium. (K) Uninfected mouse macrophage. (L) High magnification of an opened uninfected mouse macrophage. (M) Transmission electron micrograph of a mouse macrophage that contained an E. muris morula (thick arrow), N, nucleus. Scale bar, 1 micrometer.
Figure 9.
Ehrlichia morulae inside a DH82 cell with an overlying ruptured host cell membrane.
(A) Different stages of E. muris in a mechanically opened DH82 cell. Thin arrow indicates dividing ehrlichiae; thick arrow indicates mature cells. (B) Mature ehrlichiae cells in a large morula (thick arrow). (C) Pore formation on a DH82 host cell containing many ehrlichiae that have deformed the overlying cell membrane (thin arrows) (intact DH82 cell). (D) Host cell membrane ruptured at the location of ehrlichial exit from the cell (intact DH82 cell). (E) Extracellular Ehrlichia attached with high affinity to the filopodium of neighboring host cells (thin arrows). (F) Ehrlichiae attached to the DH82 cell membrane adjacent to a cell membrane ruffle (thin arrow) (intact DH82 cell). (G) TEM of an IOE infected spleen (arrows indicate fused morula).