Localization of electron-dense tracers during entry of Rickettsia tsutsugamushi into polymorphonuclear leukocytes

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Abstract
The invasion of Rickettsia tsutsugamushi, Gilliam strain, into guinea pig polymorphonuclear leukocytes (PMNs) and the localization and distribution of tracers were followed during the process by electron microscopy. The seven tracers used were: cationized ferritin, ferritin, thorium dioxide (ThO2), carbon particles, latex spheres, paraffin oil, and Escherichia coli. These markers were added to the incubation medium containing the PMNs before or simultaneously with R. tsutsugamushi-infected BHK-21 cells. Both morphologically intact and degenerating rickettsiae were present in the phagosomes in PMNs, but only the viable-appearing rickettsiae were free in the cytoplasm. The intact rickettsiae were singly and selectively phagocytized in tightly enclosed phagosomal membranes which usually excluded the tracers, except when ThO2 or ferritin was used. When ThO2, which labels the plasma membrane of PMNs, was used. ThO2-labeled phagosomal membranes enclosing rickettsiae were observed and short membrane fragments still labeled with this tracer were found in the vicinity of rickettsiae in the cytoplasmic matrix of PMNs. When ferritin or ThO2 was used as a tracer, some of the phagosomes contained rickettsiae still enclosed in an envelope of BHK-21 cytoplasm and cell membrane. Phagolysosomes preloaded with electron-dense markers fused with subsequently formed phagosomes containing degenerated rickettsiae but not with those containing intact rickettsiae. These results support our interpretation that viable rickettsial entry into PMNs is by selective phagocytosis and escape from these phagosomes.