Figure 1.
Two morphologically distinct cell forms of E. chaffeensis in infected macrophages or tick cells.
This Figure included TEM images to represent uninfected macrophages (A) and tick cells (B) and E. chaffeensis-infected macrophages (C) and tick cell (D). Majority of the morulae in the infected macrophages harbored only reticulate cells or dense core cells. In infected tick cells, considerably more infected cells contained both cell forms of the bacteria within the same morula. (Scale bar 1 µm).
Figure 2.
Variations of morulae in infected macrophages and tick cells.
E. chaffeensis containing phagosomes within the infected macrophages (A) were more compact with organisms occupying most of the intra-morulae space. The organisms in infected tick cells (B) were mostly aggregated at one end of the morula or attached to the morula membrane, intra-morulae space is also considerable more in the tick cell phagosomes and the morula size is also larger. (Scale bar 1 µm).
Figure 3.
Extensive pleomorphic structures of E. chaffeensis in infected tick cells.
E. chaffeensis in infected tick cells have extensive pleomorphic structures. (Scale bar 1 µm).
Figure 4.
Corrugated outer membrane present in E. chaffeensis.
Both E. chaffeensis reticulate and dense core forms have ruffled outer membrane structures (Higher magnification of reticulate forms from infected macrophages (A) and infected tick cells (B) are presented. (Scale bar 1 µm).
Figure 5.
Mitochondria aggregation around morulae.
Aggregation of mitochondria was observed more frequently in infected macrophages where they were also attached to the phogosomal membrane (A). Fewer mitochondria were visible in the infected tick cells harboring E. chaffeensis (B). (Scale bar 1 µm).
Figure 6.
Attachment and internalization of dense core forms of E. chaffeensis.
In macrophage cells (A), E. chaffeensis dense core cells interaction with the host cell membrane was seen as direct attachment to the host cell and with pseudopodia formed. E. chaffeensis dense core forms in tick cells (B) also attach to the host cell membrane and get internalized with the formation of pseudopodia. (Scale bar 1 µm).
Figure 7.
E. chaffeensis reticulate cells in macrophages and tick cells exhibiting replication by binary fission (A, macrophage and B, tick cells). Tick cell grown organisms also included filamentous type cell divisions (about 20% of the cells) (C). This observation can also be seen in the image presented in Figure 1D. (Scale bar 1 µm).
Figure 8.
Release of E. chaffeensis from infected macrophages and tick cells.
Most of the infected host cells exhibited release by complete lysis. A subset of the infected cells also released organisms by exocytosis by creating an opening to the morula membrane. (A and C, infected macrophages and B and D, infected tick cells) (Scale bar 1 µm).
Figure 9.
An infected macrophage containing phagosomes with cell debris.
(Scale bar 1 µm).
Figure 10.
Inclusion in the nuclei of infected host cell.
Vacuoles with inclusions within the infected macrophage (A) and tick cell (B) nuclei. (Scale bar 1 µm).
Figure 11.
Confocal microscopy Z-stack imaging to localize E. chaffeensis within a host cell nucleus.
Immunofluorescent detection of E. chaffeensis was accomplished with mAb 56.5. Detection was made using Alexa Fluor 488 (green fluorescence) anti-mouse secondary antibody. Propidium iodide was used to stain nuclei (red fluorescence). Yellow fluorescence indicates E. chaffeensis localized within the nucleus. (Z-stack images collected at 0.8 µm sections were presented in the top left panel of the figure. The cell sections in the figure were identified with the section depth at the top left on each image. The magnification in each cell section was presented at the bottom right of each image by placing a 10 µm scale bar.) In top right panel; A is Z-projection in the X–Z direction, B is Z-projection in the Y–Z direction and the blue lines in A and B indicate the Z-depth of the 3.20 µm optical slice in C. The green and red lines in C indicate the orthogonal planes of the X–Z and Y–Z projection, respectively. Uninfected cells which were subjected to similar immunofluorescence analysis were used to serve as a negative control for this experiment (bottom right panel).
Figure 12.
Western blot analysis to identify E. chaffeensis proteins.
Total cell lysates from uninfected cells, cytoplasm (C) and nucleic (N) fractions from E. chaffeensis-infected macrophages and tick cells were assessed by immunoblot analysis using E. chaffeensis mAb 56.5 that recognizes p28 Omp 19 [22]. E. chaffeensis infected macrophage and tick cell protein fractions were also probed with β actin Ab. (U–T, uninfected cell-derived total soluble proteins; I–C, E. chaffeensis-infected cell derived cytoplasmic proteins; I–N, E. chaffeensis-infected cell derived nucleic proteins).