The developmental cycle of Ehrlichia chaffeensis in vertebrate cells

Abstract

SummaryEhrlichia chaffeensis, an obligatory intracellular bacterium, has two forms in mammalian cells: small dense-cored cells (DC) with dense nucleoid and larger reticulate cells (RC) with uniformly dispersed nucleoid. We have determined by electron microscopy that DC but not RC attaches to and enters into the host cells and RC but not DC multiples inside the host cells. Analysis of outer membrane protein expression by confocal microscopy showed that RC expressed the 28 kDa outer membrane protein (p28), the intermediate form, which were transforming from RC to DC, expressed both gp120 and p28, and the mature DC expressed gp120 only. The TCID 50 of DC is 6 log10 higher than RC. We conclude that E. chaffeensis has a developmental cycle, in which the DC attaches to and enters into the host cells, and transforms into RC and the RC multiplies by binary fission for 48 h and then matures into DC at 72 h.

Regulation of Type IV Secretion Apparatus Genes during Ehrlichia chaffeensis Intracellular Development by a Previously Unidentified Protein

“…Morphological observation and real-time PCR analysis demonstrated the reliability of this method for synchronization. It was previously reported that in DH82 cells each E. chaffeensis bacterium progresses through its cycle in a discrete membrane-bound inclusion and is released upon host cell lysis (34). In contrast, in human THP-1 cells, we have found that a large inclusion is formed in the host cell by 24 h p.i., which then appears to disperse into multiple small inclusions at 48 h p.i.…”

“…1A and 1B). Because the DC and RC forms described previously (34) were morphologically similar to our sonication-resistant and -sensitive forms, these forms were called DCs and RCs, respectively. Approximately 80% of the DC population was viable after more intense son- ication (as described above), based on staining with the LIVE/ DEAD BacLight bacterial viability kit (Fig.…”

“…In DH82 cells (a canine histiocytoma cell line) at 1 day p.i., an E. chaffeensis cell is transformed from a small DC (0.4 to 0.6 m) to a large RC (0.7 to 1.9 m) and starts to multiply (34). After 3 days of culture, the bacterial population converts back to DCs to initiate new infection of host cells.…”

“…After 3 days of culture, the bacterial population converts back to DCs to initiate new infection of host cells. The infectivity of the DC-rich population has been reported to be 3.5 million times greater than that of the RC-rich population (34). To establish a synchronized culture in THP-1 cells, we used controlled high-intensity sonication to rupture the RC population and enrich the infectious DC population.…”

“…E. chaffeensis manipulates host monocytes/macrophages throughout its intracellular developmental cycle (25), and this bacterium has a biphasic developmental cycle in mammalian cells that alternates between a small "dense-core cell" (DC) and a large "reticulate cell" (RC), as defined by morphological features, differential surface protein expression, and infectivity (34). The presence of biphasic developmental stages which differ in cell size, infectivity, and the expression of surface protein VirB9 of the type IV secretion (T4S) apparatus has been demonstrated for Anaplasma phagocytophilum, which is closely related to E. chaffeensis (21).…”