Penetration of Rickettsia tsutsugamushi into Cultured Mouse Fibroblasts (L Cells): An Electron Microscopic Observation

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Abstract
The mechanism of penetration of purified R. tsutsugamushi (Gilliam strain) into cultured mouse fibroblasts (L cells) was examined by EM. After 10-40 min of infection, rickettsiae in the process of being phagocytized were often seen on the cell surface. These were restricted to the rickettsiae which seemed to be intact in morphology, heavily plasmolyzed ones were never phagocytized. Rickettsiae were taken up individually into a phagosome; phagocytosis of several rickettsiae together was rarely observed, except in the case of heat-inactivated microorganisms. In the cells, phagosomes whose membranes enclosed rickettsiae tightly or loosely were seen. Rickettsiae in the loose phagosomes often showed signs of plasmolysis and were rarely released into the cell cytoplasm. Partial disintegration of phagosomal membranes and the escape of rickettsiae from the phagosomes were seen only in tight phagosomes. Large phagosomes containing a clump of several rickettsiae were observed occasionally, in which case the microorganisms were deformed and seemed to be denatured. From the above observations and the frequency of appearance of these different penetration stages in the specimens 10, 20 and 40 min after infection, it was concluded that the rickettsiae enter initially into a tight phagosome by phagocytosis and are then released into the cell cytoplasm by disruption of the phagosomal membrane. No other mechanisms of penetration were found. Rickettsiae inactivated by trypsin did not attach to host cells. Inactivation by heat or UV irradiation resulted in reduction of phagocytosis; rickettsiae treated with rifamycin penetrated into the host cell cytoplasm to the same extent as in infection with intact rickettsiae.