Renografin Density Gradient Centrifugation Research Articles

Protein and DNA synthesis demonstrated in cell-free Ehrlichia chaffeensis organisms in axenic medium

Ehrlichia chaffeensis, a tick-transmitted rickettsial bacterium, is the causative agent of human monocytic ehrlichiosis. Biochemical characterization of this and other related Rickettsiales remains a major challenge, as they require a host cell for their replication. We investigated the use of an axenic medium for E. chaffeensis growth, assessed by protein and DNA synthesis, in the absence of a host cell. E. chaffeensis organisms harvested from in vitro cultures grown in a vertebrate cell line were fractionated into infectious dense-core cells (DC) and the non-infectious replicating form, known as reticulate cells (RC) by renografin density gradient centrifugation and incubated in the axenic medium containing amino acids, nucleotides, and different energy sources. Bacterial protein and DNA synthesis were observed in RCs in response to glucose-6-phosphate, although adenosine triphosphate, alpha-ketoglutarate or sodium acetate supported protein synthesis. The biosynthetic activity could not be detected in DCs in the axenic medium. While the data demonstrate de novo protein and DNA synthesis under axenic conditions for E. chaffeensis RCs, additional modifications are required in order to establish conditions that support bacterial replication, and transition to DCs.

Cultivation and Laboratory Maintenance of Chlamydia trachomatis

Chlamydia species are obligate intracellular bacteria that require growth inside mammalian cells for propagation and survival. As a result, Chlamydia cannot be grown on conventional bacteriological medium. This property makes Chlamydia difficult organisms to grow and maintain in the laboratory. Up until 1965, passage in the yolk sac of the embryonated hen egg was the only way to isolate and propagate the organism. Since then, a tissue culture system has been available that allows easier laboratory culture of the Chlamydia species. However, with the exception of the LGV serovars, most C. trachomatis strains do not readily infect tissue culture cells. Chemical or mechanical assistance is used to increase their infectivity. Today, large numbers of infectious organisms can be purified through Renografin density gradient centrifugation of infected cell lysates. The ability to propagate C. trachomatis in the laboratory has greatly increased the understanding of the pathogenesis of C. trachomatis organisms.

Purification and antigenic characteristics of a rickettsia-like organism from the oyster Crassostrea ariakensis

A rickettsia-like organism (RLO) has been suggested to be the etiological agent responsible for heavy losses of the oyster Crassostrea ariakensis Gould in China. Because of the lack of molluscan cell lines for in vitro culture of intracellular prokaryotes, antigenic analysis of RLOs has been limited by the inherent difficulties of their purification. In this report, we describe the use of differential speed centrifugation and renografin density gradient centrifugation to purify the RLO directly from infected oyster tissues. The purity and integrity of purified prokaryotes were validated by transmission electron microscopy. Thirteen major constituent proteins, with molecular weights ranging between 17 and 99 kDa, were electrophoretically identified by silver staining, and 8 major proteins were identified with Coomassie blue R staining. Specific mouse polyclonal antiserum was prepared for serological characterization of the RLO and was used in an immunoblot assay, and 3 major antigen groups were identified. The present results advance our knowledge of RLO protein antigens, and several proteins have been identified that could potentially be useful for diagnostic assays or for production of experimental immunostimulants.

Purification and biological features of a rickettsia-like prokaryote from the scallop Argopecten irradians in China

Three methods were tried to purify rickettsia-like prokaryotes (RLPs) from infected Argopecten irradians tissues in China and Renografin density gradient centrifugation was found to be the optimal method. The purified organisms were of two morphological cell types: a large cell type (LC) and a small cell type (SC). In Renografin density gradient centrifugation, LCs and SCs in the band at the 20–25% interface were pure and intact. The prokaryotes exhibit two trilaminar membranes, of which the outer membrane is thicker, rippled or even. The prokaryotes possess a nucleoid structure in the center and a high electron-dense ribosome zone in the periphery. The intact RLP purified by Renografin density gradient centrifugation has a thick and obvious slime layer associated with the outer membrane in the negatively stained sections under electron microscopy. LCs averaging 960.6±275.5×823.0±222.7 nm (n=38) are polymorphic, usually coccoid, ellipsoidal or rod-shaped. SCs averaging 251.6±101.9×204.3±74.5 nm (n=24) are usually coccoid and ellipsoidal. Two propagation modes, transverse binary fission and budding, was observed.

Identification of the Antigenic Constituents of Ehrlichia chaffeensis

Ehrlichia chaffeensis, the novel etiologic agent of human ehrlichiosis in the United States, was first isolated in 1990 and reported in 1991. To analyze the antigenic components of E. chaffeensis, we cultivated these obligate intracellular bacteria in DH82 cells, purified the ehrlichiae by renografin density gradient centrifugation, and examined the antigens by Western immunoblotting. Rabbit and human antisera to E. chaffeensis revealed more than 20 bands ranging from 20 to 200 kD. The distinct 22-kD protein was heat labile. The rest of the major immunoreactive components were heat stable. The immunoblots of E. chaffeensis were highly similar when probed with antisera to E. chaffeensis, E. canis, and E. ewingii, indicating the close antigenic relationships among the three species. The 22-kD protein cross-reacted only with anti-E. canis serum. The antibody against E. sennetsu reacted strongly with the 66-, 64-, 55-, and 44-kD antigens of E. chaffeensis. The E. risticii antisera reacted strongly with the 55- and 44-kD bands but only faintly with the 66-kD band. The major immunoreactive antigens of E. chaffeensis (66, 55, and 44 kD) showed cross-reactions with all the different antisera tested. The results indicated that E. chaffeensis is antigenically most closely related to E. canis, is less closely related to E. ewingii, and is only distantly related to E. sennetsu and E. risticii.

Purification of Cytoecetes phagocytophila, the causative agent of tick-borne fever, from Infected ovine blood

Cytoecetes phagocytophila, the causative agent of tick-borne fever, was successfully separated by Percoll and Renografin density gradient centrifugation and by cellular affinity chromatography, from the peripheral blood leucocytes of experimentally infected sheep. After centrifugation on Renografin or Percoll density gradients, infectious particles of C. phagocytophila banded at buoyant densities which ranged between 1.063 to 1.140. Rickettsiae separated by wheat germ lectin cellular affinity chromatography retained their morphology but often lost their infectivity. Cell-free C. phagocytophila remained infective to susceptible sheep for 6 months when kept at -114 degrees C in sucrose phosphate buffer with 10 per cent dimethylsulphoxide as a cryopreservative.

Cyanophycin Granule Size Variation in Aphanocapsa

Techniques were developed for the rapid and simple electron microscopic visualization of isolated cyanophycin granules to allow analysis of their size from cells grown under different conditions. Granules were purified by differential and renografin density-gradient centrifugation from cells grown for various times in chloramphenicol-containing media, or in nitrogen-limited media to which nitrogen was then added back, or under light limitation. Granules were then observed by electron microscopy after staining with uranyl acetate, and random granule diameters were measured. Granule size increased significantly with time following chloramphenicol treatment and following light limitation. This suggests that much of the increase in amount of cyanophycin in cells treated with chloramphenicol or light limitation is caused by increase in granule size rather than in granule number. Insignificant changes in granule size were observed during nitrogen repletion, suggesting increase in granule number, not size.

Comparison of renografin density gradient centrifugation and ion-exchange chromatography for purification of phony peach bacterium from plant extracts

Renografin density gradient centrifugation and carboxymethyl (CM)-and diethylaminoethyl (DEAE)-cellulose ion-exchange chromatography were compared for purification of cells of phony peach, rickettsia-like bacterium (RLB) from plant extracts. Centrifugation on 30–40% linear gradients resulted in four bands. Cells of RLB sedimented into the third band located 4.9–5.4 cm below the meniscus. Phase-contrast microscopy, plant peroxidase activity, and viable bacterial assays showed the resulting cells ofRLB to be relatively free of host contaminants. A typical purification from 120 ml of extract containing 7.2×109 RLB cells resulted in the recovery of 7.0×107 RLB cells. Cells ofRLB were not successfully purified byCM andDEAE chromatography. Unitil a medium is available for cultivation of plantRLB, Renografin density gradient centrifugation should be a useful tool for obtaining cells ofRLB for biochemical and serological investigations.

Chitin Synthesis in Candida albicans : Comparison of Yeast and Hyphal Forms

Chitin synthesis was studied in both yeast and hyphae of the dimorphic fungus Candida albicans. Incorporation of N-acetyl-d-[1-(3)H]glucosamine ([(3)H]GluNAc) into an acid-alkali-insoluble fraction was 10 times greater in hyphal-phase cells. A crude preparation of chitin synthetase was obtained from sonically treated protoplasts of both forms of Candida. Enzyme activity, which was determined by using [(14)C]UDP-GLuNAc as a substrate, was exclusively associated with the 80,000 x g pellet from sonically treated protoplasts of both forms. It was determined that enzyme activity (nanomoles of [(14)C]UDP-GluNAc incorporated per milligram of protein) was approximately 2 times greater in hyphae versus yeast cells. Enzyme activity in both yeast and hyphae increased six- to sevenfold when the enzyme preparations were preincubated with trypsin. A vacuolar fraction, obtained from yeast cells but not from hyphae, stimulated enzyme activity when incubated with either yeast or hyphal enzyme preparations. Membrane fractions from protoplasts coated with [(3)H]concanavalin A before disruption were isolated by Renografin density gradient centrifugation. Chitin synthetase activity was preferentially associated with the concanavalin A-labeled fraction, suggesting that the enzyme was located on the plasma membrane. In addition, enzyme activity in protoplasts treated with cold glutaraldehyde before disruption was significantly greater than in protoplasts that were sonically disrupted and then treated with cold glutaraldehyde, indicating that the enzyme resides on the inner side of the plasma membrane.

Biological Properties of Rickettsia prowazekii Strains Isolated from Flying Squirrels

Four strains of Rickettsia prowazekii, isolated from flying squirrels (Glaucomys volans volans) from Florida and Virginia, were compared with other strains of the typhus biotype, two previously established strains each of R. prowazekii and R. typhi and one strain of R. canada, for similarities in a number of unrelated phenotypic characteristics. R. akari served as a spotted fever biotype control. All strains produced small plaques on chicken embryo cell monolayers that were clearly recognized only after 10 days of incubation at 32 degrees C. All strains were highly susceptible to erythromycin. The Renografin density gradient centrifugation procedure of separating rickettsiae from the infected yolk sacs of surviving chicken embryos was equally satisfactory in all cases and resulted in moderate to large yields of purified rickettsiae. There was relatively small variation in specific hemolytic activity or specific CO(2) formation from glutamate. None of the strains catabolized glucose. There was some strain variation in virulence for the chicken embryo, but none of the above tests separated the three species of the typhus biotype. On the other hand, R. akari was clearly distinguished by its more rapid plaque formation and by higher resistance to erythromycin. It is concluded that by the tests conducted thus far, the biological properties of the flying squirrel strains do not differ substantially from those of other strains of the typhus biotype.

Characterization of the Madrid E strain of Rickettsia prowazekii purified by renografin density gradient centrifugation.

The avirulent Madrid E strain of Rickettsia prowazekii cultivated in chicken yolk sacs could be purified successfully with a Renografin density gradient method developed previously for Rickettsia typhi. Recovery during purification, viability, and lack of contamination with host cell components were similar for the two species, although yields of R. prowazekii per yolk sac were lower. Purified typhus rickettsiae provided satisfactory antigens in the complement fixation, Ouchterlony double-diffusion, and microagglutination tests. The retention of the typhus soluble group antigen during purification was readily demonstrated by complement fixation tests. However, removal of the soluble group antigen by ether treatment was not always adequate for the demonstration of type-specific particulate antigens. Heat-killed R. prowazekii cells gave higher serum microagglutination titers than untreated or formalized cells, a difference was noted for R. typhi cells. Although the protein profiles of whole cells and extracts of R. typhi and R. prowazekii on sodium dodecyl sulfate-polyacrylamide gels were relatively similar, a small but reproducible, difference in the electrophoretic mobilities of their malate dehydrogenases was detected. Purification of typhus rickettsiae on Renografin gradients has no apparent adverse effects on their metabolic or antigenic properties.

Enzymatic activities leading to pyrimidine nucleotide biosynthesis from cell-free extracts of Rickettsia typhi.

Cell-free extracts from Rickettsia typhi were examined for the presence or absence of pyrimidine phosphotransferase enzymes and compared with the enzymes of mouse L cells and Salmonella typhimurium. The organisms were grown in mouse L cells and in the yolk sacs of chicken embryos, purified by Renografin density gradient centrifugation, and ruptured in a French pressure cell. The enzymes for the reutilization of uridine and thymidine, uridine kinase (EC 2.7.1.48) and thymidine kinase (EC 2.7.1.21), were not detected in R. typhi extracts with the phosphate donors effective for control enzymes. The following enzyme activities were demonstrated in R. typhi: uridine-5'-monophosphate kinase (UMPK, EC 2.7.4.4), deoxythymidine-5'-monophosphate kinase (dTMPK, EC 2.7.4.9), and nucleosidediphosphate kinase (NDPK, EC 2.7.4.6). Physicochemical and enzymatic analyses demonstrated that the pyrimidine nucleotide kinases of R. typhi were not of host origin and that the source (yolk sac and mouse L cells) did not influence the relative enzymatic activities. The specific activities of UMPK and dTMPK were higher when the rickettsiae were harvested before embryo death, whereas NDPK levels were slightly decreased. The specific activities of UMPK, dTMPK, and NDPK were comparable to those of S. typhimurium, and consequently the rickettsiae have potential for the anabolism of monophosphates, as do the host-independent bacteria. These results suggest that R. typhi cannot utilize host uridine or thymidine pools directly but must rely on themonophosphorylated molecules of the host cell or must synthesize the monophosphates de novo.

Enzymatic activities of cell-free extracts of Rickettsia typhi.

Cell-free extracts of Rickettsia typhi were tested for activities of enzymes of the tricarboxylic acid cycle, of glutamate catabolism, and of glycolysis. The organisms were grown in the yolk sacs of chicken embryos, harvested shortly before the time of embryo death, purified by Renografin density gradient centrifugation, and ruptured in a French pressure cell. The following enzymatic activities were demonstrated: high levels of malate dehydrogenase (MDH), moderate levels of glutamate-oxaloacetate transaminase, glutamate, succinate, and isocitrate dehydrogenases, and citrate synthase, and low levels of glutamate-pyruvate transaminase. The specific activities of some of these enzymes were higher when the rickettsiae were harvested at a time of active proliferation, 3 to 4 days prior to embryo death. Rickettsial MDH was differentiated from host MDH by its migration pattern on polyacrylamide gel electrophoresis. The activities of MDH and two other dehydrogenases, demonstrable after the cells had been disrupted, were absent from purified, intact rickettsial preparations. No activity was detected for glucose-6-phosphate, 6-phosphogluconate, glyceraldehyde-3-phosphate, lactate dehydrogenases, phosphoglucose isomerase, fructoaldolase, or pyruvate kinase. Our results suggest that extracts of R. typhi that contain demonstrable enzymes involved in the catabolism of glutamate and tricarboxylic acid cycle intermediates, unlike Coxiella burnetti, lack detectable glycolytic activity.

Separation of viable Rickettsia typhi from yolk sac and L cell host components by renografin density gradient centrifugation.

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.

Separation of Viable Rickettsia typhi from Yolk Sac and L Cell Host Components by Renografin Density Gradient Centrifugation

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.