On the nature of obligate intracellular symbiosis of rickettsiae--Rickettsia prowazekii cells import mitochondrial porin.

Mitochondrial porin was identified in Rickettsia prowazekii by Western blot analysis of whole cells and membrane fractions with monoclonal antibody against porin VDAC 1 of animal mitochondria. Using the BLAST server, no protein sequences homologous to mitochondrial porin were found among the rickettsial genomes. Rickettsiae also do not contain their own porin. The protein imported by rickettsiae is weakly extracted by nonionic detergents and, like porin in mitochondria, is insensitive to proteinase K in whole cells. Immunocytochemical analysis showed that it localizes to the outer membrane of the bacterial cells. These data support an earlier suggestion about import by rickettsiae of indispensable proteins from cytoplasm of the host cell as a molecular basis of obligate intracellular parasitism. They are also consistent with the hypothesis invoking a transfer of genes specifying surface proteins from the last common ancestor of rickettsiae and mitochondria to the host genome, and preservation by rickettsiae of the primitive ability to import these proteins.

[A simple method for counting Rickettsia cells]. / Prostoi metod podscheta kletok rikketsii.

A simple modification of the method for counting Rickettsiae is described. The Escherichia coli cells (ECC) which served as reference particles were stained in suspension with methylene blue mixed with Rickettsia prowazekii (RP) and quickly sprayed over the glass slide. After fixation the samples were stained according to the technique of Gimenez and examined in the light microscope under oil immersion. Through a grid in the eye-piece it was not so difficult to count red-coloured RP and dark-blue ECC against a background formed by impurities. To calculate RP concentration, the reference particles' concentration was multiplied by the dilution factor of RP suspension by the ratio of RP to ECC enumerated. The statistical approach has shown that the wash of the slides during staining procedure does not change this ratio. Differential staining of Rickettsiae with fuchsin is the main clue of this new method to count them even in the crude preparations of infected yolk sacs.

Penicillin-induced unstable intracellular formation of spheroplasts by rickettsiae.

Penicillin G (greater than or equal to 20 micrograms/ml) is rapidly rickettsiacidal for intracellular Rickettsia prowazekii. Light and electron microscopic examinations revealed that penicillin G in culture medium induced a predictable transformation into typical enlarging spheroplasts deficient in the internal, putative peptidoglycan layer of the outer membrane. Under certain conditions, spheroplasts ruptured to discharge contents into host cell cytoplasm and to leave empty shells of defective outer membrane and diffuse amorphous intracytoplasmic antigen. Host cell destruction often accompanied spheroplast rupture. Penicillin G (100 micrograms/ml) caused similar spheroplast formation by Rickettsia rickettsii, but 1,000 micrograms/ml caused neither growth inhibition nor spheroplast formation in Rickettsia tsutsugamushi. The clinical and epidemiological significance of a practical rickettsiacidal drug for the treatment of louse-borne typhus fever is discussed. Practical pharmacologic considerations preclude the use of penicillin for the treatment of typhus or spotted fever.

[Current data on the polymorphism of Rickettsia prowazekii and burneti in cultured cells]. / Novye dannye o polimorfizme rikketsii Provacheka i Berneta pri kul'tivirovanii v kul'ture kletok

In cultivation of Rickettsia prowazeki (strains Breinl and E) in the cell cultures of guinea pig kidneys (GPK) and chick embryo fibroblasts (CEF) ultrastructure of rickettsia of unusual shape (filamentous, irregularpleomorphic and spheroplast-like) were revealed along with rickettsia of the usual shape and size. The polymorphism was less pronounced in the GPK and the CEF cells of Rickettsia burneti (strain M-44). It is supposed that rickettsial polymorphism was not associated with their developmental cycle and served as a morphological expression of the changes in the microorganism under the effect of unfavourable ecological conditions. The appearance of filamentous forms could be associated with disturbed cell division process; changed rigidity of the cell wall could serve as the cause of appearance of pleomorphic rickettsia. In difference from polymorphism, the cycle of rickettsial development is considered to be (in the basis of modern electron microscopic data) as a biological replacement of the vegetative (rod-like, bacillary) forms by those more stable in the external environment, resting (coccoid).

Ultrastructural studies of Rickettsia prowazeki from louse midgut cells to feces: search for "dormant" forms.

An electron microscope study of infected human louse gut cells and feces was made to determine whether a valid correlation exists between the increased resistance of Rickettsia prowazeki (in the louse feces) to adverse environmental influences and changes in the organism which might be reflected in its ultrastructure. Upon fine structural examination of this intracellular parasite as it passed from the louse midgut cell to the feces, it was apparent that no such morphological changes had occurred.

Electron microscopy of the cell wall of Rickettsia prowazeki.

Purified Rickettsia prowazeki were found to undergo morphological changes resembling plasmolysis when stained with uranyl acetate, resulting in rod-like forms. Sequential electron micrographs of disintegrating organisms provide evidence for the cell wall origin of these rod-like forms. The substructure of the cell wall was discerned by using negative-contrast electron microscopy. The wall was found to be composed of repetitive subunits with a periodicity of 13 nm and was surrounded by a thin membrane.