Purification of Rickettsia tsutsugamushi by Percoll density gradient centrifugation

Purification of Rickettsia tsutsugamushi has been achieved by Percoll density gradient centrifugation. The microorganisms purified showed good retention of infectivity and intracellular morphology. Budding rickettsiae in the egressing stage and intracellular rickettsiae in the multiplying process were harvested separately and purified by this technique. In electron microscopic observations, the intracellular rickettsiae obtained were surrounded with double membrane-layers of cell wall and cell membrane, and the budding rickettsiae were enveloped with an additional outermost membrane which may have originated from host cell membrane obtained in the budding process.     

Penetration of Rickettsia tsutsugamushi into cultured mouse fibroblasts (L cells): an electron microscopic observation

The mechanism of penetration of purified Rickettsia tsutsugamushi (Gilliam strain) into cultured mouse fibroblasts (L cells) was examined by electron microscopy. 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, while heavy plasmolyzed ones were never phagocytized. Additionally, rickettsiae were taken up individually into a phagosome, and phagocytosis of several rickettsiae together was rarely observed, except in the case of heat-inactivated microorganisms. In the cells, phagosomes whose membranes enclosed rickettsiae either 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. On the other hand, rickettsiae inactivated by trypsin did not attach to host cells. Inactivation by heat or UV irradiation resulted in reduction of phagocytosis, and rickettsiae treated with rifamycin could penetrate into the host cell cytoplasm to the same extent as in the case of infection with intact rickettsiae.     

Electron Microscopic Studies on Intracellular Multiplication of Rickettsia tsutsugamushi in L Cells

The mechanism and kinetics of intracellular growth of Rickettsia tsutsugamushi were investigated by electron microscopic observations, parallel with quantitative analysis by counting the rickettsiae seen in electron micrographs and by plaque assay for infectivity of the culture. The observations demonstrated the existence of electron-less dense and -dense types of rickettsiae in the early stage of infection, binary fission and the process of release of the microorganisms in the host cell cytoplasm and from the cell surface, formation of abnormally long rickettsiae, and the process of lysis of the host cell in the later stage of infection with vacuole formation between the inner and outer leaflets of the host cell nuclear membrane. Separate titrations of infectivity of the cells and the culture fluid showed a very slow increase in infectivity in the culture fluid compared with the intracellular titer, suggesting that the progeny rickettsiae stay in the cell or at the cell surface for a relatively long period. Doubling time of the rickettsia was found to be about 9 hr.     

Assembly of Rickettsia tsutsugamushi progeny in irradiated L cells.

In the assembly of Rickettsia tsutsugamushi progeny in irradiated L cells, nascent forms first appear as undemarcated foci in the host cell granular cytoplasm, in which electron-lucent filamentous (f) and electron-dense granular (g) areas differentiate. Morphological observations indicated that the assembly involves formation of a filamentous network in the f area, manufacture of rickettsial ribosomes in the g area, and formation of mildly electron-dense fuzzy zones, along which a double membrane assembles.     

Growth pattern of Rickettsia tsutsugamushi in irradiated L cells.

Irradiated L cells infected with Rickettsia tsutsugamushi were studied under the electron microscope to define the morphological growth pattern of the organism. For 2 days after inoculation, no rickettsiae were found either extra- or intracellularly; after 2 days multiple rickettsiae appeared within the host cells without morphological evidence of their entry. These observations showed that the rickettsiae within the cell were assembled in situ by segregation of portions of the granular cytoplasm and subsequent internal differentiation and surface membrane assembly of the segregated bodies. The protoplasmic (P) bodies, which seemed to be formed by shedding infected-cell granular cytoplasm, consistently appeared on the surface and within the phagosomes of the host cells. Rickettsiae were occasionally seen entering host cells in the later phase of infection; these were apparently the ones assembled within the P bodies. This suggested that the P bodies, and not the rickettsiae, were the major infectious particles that transmitted the rickettsial genetic substance among the host cells. On the basis of the present morphological observations, viral-type multiplication for R. tsutsugamushi is proposed.     

The effect of age on protein synthesis in mouse liver

1. A system of microsomes and 105000g supernatant from livers of old mice is less able to promote the incorporation of [(14)C]phenylalanine into protein than a similar system from livers of young animals. 2. The decrease in [(14)C]phenylalanine incorporation is attributable to changes in microsomes from old animals rather than in the cell-sap fraction. 3. Decreased synthetic ability is found in various classes of microsomes from older animals, namely unfractionated, light and heavy microsomes, but not in detergent-washed ribonucleoprotein particles. 4. Deletions of certain detergent-soluble microsomal proteins accompany the decreased synthetic ability of microsomes from older animals. 5. Microsomes from old mice are less responsive to a synthetic messenger RNA, polyuridylic acid, and this is partly due to a higher rate of hydrolysis in the presence of cell sap from animals of extreme age. 6. Other more direct evidence, from the priming of a cell-free protein-synthesizing system from bacteria and the examination of ribonucleoprotein particles on sucrose density gradients, suggests that senescence is accompanied by a decrease in messenger RNA content.     

Effect of rickettsial antigen-specific T cell line on the interaction of Rickettsia tsutsugamushi with macrophages

The effect of rickettsial antigen-specific T cell line on macrophages infected with Rickettsia tsutsugamushi was studied. It was suggested that the T cells could produce a factor which acts cytotoxically and selectively on infected macrophages.