Invasion and Intracellular Development of the Human Granulocytic Ehrlichiosis Agent in Tick Cell Culture

Abstract

Human granulocytotropic ehrlichias are tick-borne bacterial pathogens that cause an acute, life-threatening illness, human granulocytic ehrlichiosis (HGE). Ehrlichias within neutrophil granulocytes that invade tick bite sites are likely ingested by the vector, to be transmitted to another mammalian host during the tick’s next blood meal. Thus, the cycle of replication and development in the vector is prerequisite to mammalian infection, and yet these events have not been described. We report tick cell culture isolation of two strains of the HGE agent directly from an infected horse and a dog and have also established a human isolate from HL60 culture in tick cells, proving that the blood stages of the HGE agent are infectious for tick cells, as are those replicating in the human cell line HL60. This required changes to the culture system, including a new tick cell line. In tick cell layers, the HGE agent induced foci of infection that caused necrotic plaques and eventual destruction of the culture. Using the human isolate and electron microscopy, we monitored adhesion, internalization, and replication in vector tick cells. Both electron-lucent and -dense forms adhered to and entered cells by a mechanism reminiscent of phagocytosis. Ehrlichial cell division was initiated soon after, resulting in endosomes filled with numerous ehrlichias. During early development, pale ehrlichias with a tight cell wall dominated, but by day 2, individual bacteria condensed into dark forms with a rippled membrane. These may become compacted into clumps where individual organisms are barely discernible. Whether these are part of an ehrlichia life cycle or are degenerating is unknown.

Ixodes ovatus Ehrlichia Exhibits Unique Ultrastructural Characteristics in Mammalian Endothelial and Tick‐derived Cells

“…Culture was also successful in the tickderived cell line ISE6, a line that has been used to propagate other Ehrlichia and Anaplasma species. 16,25 In those cases, however, IOE were not recovered in tick cells from the primary in- oculum of infected blood, but only after transfer from RF/6A cells. Whether that result was due to the prior mouse-to-mouse passage history of the isolate and concomitant adaptation to the mammalian host, or whether it reflected a lower degree of susceptibility of the tick cells relative to the RF/6A cells, will require additional investigation.…”

“…In this study, we demonstrate the culture of IOE in the rhesus monkey (Macaca mulatta) endothelial cell line RF/6A, 15 and in the tick cell line ISE6. 16 Furthermore, our data reveal unique ultrastructural features of IOE that may be associated with the organism's high virulence in laboratory mice.…”

“…The tick cell line ISE6 was routinely propagated in the same medium, but without HEPES and NaHCO 3 , in tightly capped flasks at 34 • C. For IOE culture, however, 25 mM HEPES and 0.25% NaHCO 3 were included in tick cell culture medium as well, as described. 16 Subsequent passage of RF/6A was performed in Complete Tumor Medium, as described previously. 17…”

Peptidoglycan in obligate intracellular bacteria

“…This family also contains the nematode-borne pathogen Neorickettsia sennetsu (Dittrich et al, 2015) and the prolific and promiscuous insect symbiont Wolbachia (Sicard et al, 2014). This family is unlike the Rickettsiaceae in primarily residing in erythrocytes, neutrophils and monocytes/macrophages and having a vacuolar cellular localisation (Carlyon and Fikrig, 2003;Rikihisa, 2003;Munderloh et al, 1999). Similar to the Chlamydiales, Anaplasmataceae exhibit a morphologically distinct biphasic life cycle, transitioning between nonreplicative and infectious dense-core particles and replicating reticulate cells (Troese and Carlyon, 2009).…”

Monoubiquitinated proteins decorate theAnaplasma phagocytophilum-occupied vacuolar membrane

“…In addition to human promyelocytic HL-60 cells, A. phagocytophilum also infects and resides in ApVs in the monkey choroidal endothelial cell line RF/6A and the I. scapularis embryonic cell line ISE6 (Munderloh et al, 1999(Munderloh et al, , 2004Herron et al, 2005). To determine if the AVM is ubiquitinated in each of these cell lines, A. phagocytophilum-infected RF/6A and ISE6 cells were screened with FK2 in conjunction with antibody against Msp2 (P44), which is the most abundant A. phagocytophilum surface protein (Rikihisa, 2010), and examined by confocal microscopy.…”