Analysis of hydrolytic products from choline-labeled host cell phospholipids during growth of Rickettsia prowazekii.

A phospholipase activity has been associated with the interaction of Rickettsia prowazekii with the surface of erythrocytes and competent host cells as well as during the growth of the rickettsiae within their host cells. Both fatty acid and lysophosphatides have been found in the interaction of rickettsiae with the surface of eucaryotic cells; this finding provided strong evidence for the activity of a phospholipase A. However, fatty acids, but not lysophosphatides, were found during the growth of rickettsiae within cells in which the phospholipids had been radiolabeled with oleic acid; this observation left the type of phospholipase activity in doubt. In this study, the water-soluble components of phospholipid hydrolysis by phospholipase A plus lysophospholipase and phospholipase C were determined following the growth of rickettsiae in host cells in which the phospholipids had been radiolabeled with choline. In infected cells relative to mock-infected cells, there was a loss of phosphatidylcholine with a corresponding increase not in lysophosphatidylcholine but in the water-soluble components. There was a large increase in glycerylphosphorylcholine (185%) and a smaller increase in phosphorylcholine (16%). These results indicate that both phospholipase A activity (plus a lysophospholipase activity) and phospholipase C were increased during infection by R. prowazekii and that the former was the predominant activity.     

用16S rRNA探针定量测定普氏立克次体在细胞内的繁殖量

目的以普氏立克次体为研究对象，建立一种定量测定胞内微生物繁殖的方法。方法从立克次体感染细胞提取总ＲＮＡ为样品或用硫氰酸胍裂解感染细胞，以裂解物为样品，用３２Ｐ标记的１６ＳｒＲＮＡ特异探针做ＲＮＡ酶保护分析。根据探针分子结合数，计算出检测到的靶序列分子数，反映胞内微生物的繁殖量。结果从０．５μｌ直接裂解物可检测到３．９４×１０４个１６ＳｒＲＮＡ分子；从６ｐｇ的总ＲＮＡ中可检测到５．８２×１０４个１６ＳｒＲＮＡ分子。用本法试测立克次体在宿主细胞内的生长曲线，只表现出迟缓期及对数生长期，而无稳定期及衰退期。结论用特异性探针通过ＲＮＡ酶保护分析法可以定量测定胞内微生物在宿主细胞内的繁殖量。由本法测定到的普氏立克次体生长曲线表明难以沿用细菌的生长曲线来描述胞内微生物在宿主细胞内的生长过程     

Comparative ultrastructural study on the cell envelopes of Rickettsia prowazekii, Rickettsia rickettsii, and Rickettsia tsutsugamushi.

Rickettsia tsutsugamushi differs from other rickettsiae in its cell envelope organization. The differences were made evident through a comparative study of the outer envelope of R. tsutsugamushi, R. prowazekii, and R. rickettsii by electron microscopy.     

Isolation of Rickettsia prowazekii from blood by shell vial cell culture.

A blood sample from a patient who returned from Algeria with a fever inoculated on human embryonic lung fibroblasts by the shell vial cell culture technique led to the recovery of Rickettsia prowazekii. The last clinical strain was isolated 30 years ago. Shell vial cell culture is a versatile method that could replace the classic animal and/or embryonated egg inoculation.     

Methods for purification of Rickettsia prowazekii separated from the host tissue: a step-by-step comparison

Two methods for purification of Rickettsia prowazekii strains E, E Vir, and Breinl grown in chick embryo yolk sacs are described. These methods combine either differential centrifugation or sucrose mix, centrifugation through sucrose cushion, 10 mmol/l MgCl2 treatment, filtration through a glass filter AP-20 and 2 cycles of verografin discontinuous density gradient centrifugation. The purification procedure including sucrose mix allowed to recover about 38-42% biologically active rickettsiae, a yield which was by 10% higher than that obtained by the method beginning at differential centrifugation. The rickettsiae free of host cell components preserved their infectious activity. The obtained biomass was suitable for immunological and biological characterization of Rickettsia prowazekii and for isolation of its total DNA.     

Cytokine sensitivity and methylation of lysine in Rickettsia prowazekii EVir and interferon-resistant R. prowazekii strains.

Modified Rickettsia prowazekii strains have been derived from the avirulent Madrid E strain by passage in the lungs of white mice (strain EVir) or by selection for resistance to gamma interferon (IFN-gamma) (strains 427-19 and 87-17) or alpha/beta interferon (IFN-alpha/beta) (strains 83-2P, 60P, 103-2P, and 110-1P). Compared with the Madrid E strain, strain EVir has increased virulence (N. M. Balayeva and V. N. Nikolskaya, J. Hyg. Epidemiol. Microbiol. Immunol. 17:11-20, 1973) and a different lysine methylation profile in its surface protein antigen (A. V. Rodionov, M. E. Eremeeva, and N. M. Balayeva, Acta Virol. 35:557-565, 1991). The other six strains differ from the Madrid E strain in their resistance to IFN and their ability to grow well in untreated macrophagelike RAW264.7 cells. In the present study, to determine which properties are shared by these strains, we examined R. prowazekii EVir for the following: (i) the sensitivity of its growth in L929 cells to the cytokines IFN-alpha/beta, IFN-gamma, tumor necrosis factor alpha (TNF-alpha), and IFN-gamma plus TNF-alpha; (ii) the ability to grow in untreated RAW264.7 cells; and (iii) the ability to induce interferon in L929 cell cultures; we also evaluated strains 83-2P and 87-17 for lysine methylation. Multiplication of strain EVir in growing L929 cells was not markedly inhibited by either IFN-alpha/beta or IFN-gamma. In X-irradiated L929 cells, growth of strain EVir was slightly inhibited (11%) by TNF-alpha alone, somewhat inhibited (38%) by IFN-gamma alone, and markedly inhibited (87%) by IFN-gamma plus TNF-alpha. Nitrite production was induced in X-irradiated, strain EVir-infected L929 cell cultures treated with TNF-alpha alone or IFN-gamma alone; however, more nitrite was produced in infected cultures treated with IFN-gamma plus TNF-alpha. Nitrite production, the dramatic inhibitory effect of IFN-gamma plus TNF-alpha, and the modest inhibitory effect of IFN-gamma on the growth of strain EVir in X-irradiated L929 cells were all alleviated by the addition of the nitric oxide synthase inhibitor NG-methyl-L-arginine. Strain EVir grew very well in untreated macrophagelike RAW264.7 cells and appeared defective in the ability to induce IFN in L929 cell cultures. All strains grown in L929 cells in the presence of radiolabeled lysine had similar percentages of their radioactivity as methylated lysines.(ABSTRACT TRUNCATED AT 400 WORDS)     

Improved plaque assays for Rickettsia prowazekii in Vero 76 cells.

Typhus group rickettsiae, including Rickettsia prowazekii and R. typhi, produce visible plaques on primary chick embryo fibroblasts and low-passage mouse embryo fibroblasts but do not form reproducible plaques on continuous cell culture lines. We tested medium overlay modifications for plaque formation of typhus group rickettsiae on the continuous fibroblast cell line Vero76. A procedure involving primary overlay with medium at pH 6.8, which was followed 2 to 3 days later with secondary overlay at neutral pH containing 1 microgram of emetine per ml and 20 micrograms of NaF per ml, resulted in visible plaques at 7 to 10 days postinfection. A single-step procedure involving overlay with medium containing 50 ng of dextran sulfate per ml also resulted in plaque formation within 8 days postinfection. These assays represent reproducible and inexpensive methods for evaluating the infectious titers of typhus group rickettsiae, cloning single plaque isolates, and testing the susceptibilities of rickettsiae to antibiotics.     

Isolation of species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii for immunodiagnosis and immunoprophylaxis

A simple procedure for the selective isolation of the protective species-specific protein antigens (SPAs) of Rickettsia typhi and Rickettsia prowazekii was developed to permit use of the SPAs in the immunodiagnosis and immunoprophylaxis of typhus infections. Although the SPAs were readily extracted from lysozyme- or detergent-treated rickettsiae, as measured by rocket immunoelectrophoresis, other polypeptides were also present, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast, both water and seven buffers, each at a 10 mM concentration and pH 7.6, were nearly equally effective in the selective release of the SPAs from whole cells by extraction for 30 min at 45 degrees C. High-ionic-strength buffers and MgCl2 abolished this SPA release, thus suggesting that divalent cations were important in the binding of the SPAs to the cell envelope. The efficacy of the dilute buffer extraction procedure for isolation of large amounts of SPAs was tested by further characterization of the supernatants obtained by centrifugation (200,000 x g) of two successive tris-(hydroxymethyl)aminomethane-hydrochloride buffer (Tris) extracts. With this procedure, between 10 and 15 mg of SPA was obtained from 100 mg of purified rickettsiae. Although low-molecular-weight ribonucleic acid fragments were released into the Tris extracts in significant amounts, only the SPAs were detected, in significant quantities, as measured by polyacrylamide gel electrophoresis and rocket immunoelectrophoresis. The Tris extracts contained the same major and minor SPA polypeptides as those observed previously in SPA preparations obtained by extensive diethylaminoethyl-cellulose column chromatography, but the Tris SPAs were more satisfactory antigens in an enzyme-linked immunosorbent assay.     

External layers of Rickettsia prowazekii and Rickettsia rickettsii: occurrence of a slime layer.

Using a simple specific-antibody stabilization procedure on organisms gently liberated from their host cells, we have demonstrated by electron microscopy that Rickettsia prowazekii and Rickettsia rickettsii possess a coat of variable thickness, external to the outer leaflet of the cell wall and the structure designated by others as a "microcapsule," which corresponds most closely to the slime layer of certain other bacteria. Reactions in the methenamine silver and ruthenium red staining procedures and the failure to be visualized by standard procedures suggest that the slime layer is largely polysaccharide in nature. It is postulated that this slime layer accounts in large part for the large, electron-lucent, halo-like zone which is found by electron microscopy to surround organisms of the typhus and spotted fever groups in the cytoplasm of their host cells, that it may be the locus of some major group-specific antigens, and that it may function as an antiphagocytic mechanism, as an aid for attachment of rickettsiae to potential host cells, or both. Moreover, because the attenuated E strain of R. prowazekii has been shown to possess a substantial slime layer, the basis for attenuation is not likely to be a simple smooth-to-rough variation.     

Phospholipase A activity associated with the growth of Rickettsia prowazekii in L929 cells.

Cultured L929 cells infected with Rickettsia prowazekii had a greatly increased rate of hydrolysis of fatty acid from the oleic acid-radiolabeled phospholipids of the host cell membranes. The incorporation of fatty acid into phospholipid in an infected cell was only moderately inhibited relative to a mock-infected cell. Thus, even if the release of fatty acid from phospholipid represented a steady state between hydrolysis and resynthesis of phospholipids, the increase in release of fatty acid was due principally to increased phospholipase A activity. The increased rate of hydrolysis did not occur only late in the rickettsial infection; this activity began early in infection and continued throughout the course of infection. The addition of tetracycline or chloramphenicol (antibiotics which inhibit rickettsial protein synthesis) to the infected cells caused a rapid and total abatement of this increased rate of phospholipid hydrolysis. In contrast, high concentrations of penicillin affected the morphology of the intracellular rickettsiae, but did not inhibit the phospholipase activity. This phospholipase A activity clearly damages the host cell during the rickettsial infection and may represent the activity by which R. prowazekii escapes from the host cell.     

Peculiarities of Rickettsia prowazekii in the cell culture as revealed by cryoultramicrotomy

Ultrastructure of Rickettsia prowazekii has been followed in L-929 cells 4 days post-infection (p.i.) by cryoultramicrotomy. Groups of rickettsiae were present in the cytoplasm outside of vacuoles forming microcolonies. The size of rickettsiae amounted to 400 X 700 nm, the average thickness of the cell wall was 5 nm, that of periplasmic space and cytoplasmic membrane 14 and 6 nm, respectively. Within intracytoplasmic colonies the rickettsiae were tightly packed and their cell walls were closely adjacent to each other. No halo or capsule-like coating around them was detected. No ultrastructural details were observed in the light translucent spaces between cells. Marginal rickettsiae of the microcolonies were often in close contact with the host cell mitochondria.     

Rickettsia-macrophage interactions: host cell responses to Rickettsia akari and Rickettsia typhi

The existence of intracellular rickettsiae requires entry, survival, and replication in the eukaryotic host cells and exit to initiate new infection. While endothelial cells are the preferred target cells for most pathogenic rickettsiae, infection of monocytes/macrophages may also contribute to the establishment of rickettsial infection and resulting pathogenesis. We initiated studies to characterize macrophage-Rickettsia akari and -Rickettsia typhi interactions and to determine how rickettsiae survive within phagocytic cells. Flow cytometry, microscopic analysis, and LDH release demonstrated that R. akari and R. typhi caused negligible cytotoxicity in mouse peritoneal macrophages as well as in macrophage-like cell line, P388D1. Host cells responded to rickettsial infection with increased secretion of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. Furthermore, macrophage infection with R. akari and R. typhi resulted in differential synthesis and expression of IL-beta and IL-6, which may correlate with the existence of biological differences among these two closely related bacteria. In contrast, levels of gamma interferon (IFN-gamma), IL-10, and IL-12 in supernatants of infected P388D1 cells and mouse peritoneal macrophages did not change significantly during the course of infection and remained below the enzyme-linked immunosorbent assay cytokine detection limits. In addition, differential expression of cytokines was observed between R. akari- and R. typhi-infected macrophages, which may correlate with the biological differences among these closely related bacteria.     

Persistence of Rickettsia prowazekii in cotton rat macrophage cultures.

Rickettsia prowazekii is able to multiply and persist for a long time in cotton rat macrophage culture (29-days observation period). Electron microscopic studies showed that the structure of Rickettsiae remained intact at different intervals post-inoculation (p.i.). In the course of persistence Rickettsiae revealed a reduced capacity to infect chick embryos and guinea pigs, however, the infectious agent could be isolated at all stages of persistence of cultured cells such as fibroblasts of the guinea pig embryo, macrophages of intact cotton rats.     

Activated complex of L-cells and Rickettsia prowazekii with N-ethylmaleimide-insensitive phospholipase A.

The interaction of large numbers of viable Rickettsia prowazekii cells with L-cells results in the expression of a phospholipase A activity with the concomitant release of free fatty acids and lysophosphatides from the phospholipids of the L-cell. About 50% of rickettsiae present in the suspension that was centrifuged onto an L-cell monolayer at 0 degree C to effect this interaction formed a tight L-cell-rickettsiae association from which the rickettsiae could not be removed by simple washing. Both the L-cell-rickettsiae association and the rickettsiae before association with L-cells interact with N-ethylmaleimide, so that the subsequent expression of the phospholipase A activity was inhibited (treatment of the L-cells with N-ethylmaleimide before centrifugation does not inhibit phospholipase activity). However, treatment of this association with 2,4-dinitrophenol and KCN caused much less inhibition of this phospholipase A activity than did treatment of the rickettsiae with these agents before centrifugation onto the L-cells. Incubation of the L-cell-rickettsiae association for a short time at 35 degrees C resulted in a very low level of free fatty acid formation and changed this association to an activated complex in which the phospholipase A activity was no longer sensitive to the inhibitory effects of N-ethylmaleimide. The characteristics of the association and activated complex were stable: after a 2-h incubation at 0 degrees C, the association and the activated complex retained both their basal phospholipase A activities and their characteristic responses to N-ethylmaleimide treatment. In scanning electron micrographs of the activated complexes, the rickettsiae that were initially attached were no longer visible after 45 min at 35 degrees C, and the surface of the L-cell appeared to have been etched away. These activated complexes provide a system in which modulators of the phospholipase A can be investigated without the confusion caused by the first-step receptor interaction between rickettsiae and their host cells.     

Radioiodination of an outer membrane protein in intact Rickettsia prowazekii.

Intact Rickettsia prowazekii was radiolabeled with the glucose oxidase-lactoperoxidase method of iodination. Separation of the rickettsial extract into cytoplasmic, outer and inner membrane fractions demonstrated that the outer membrane was preferentially labeled. Analysis of the polypeptides of these fractions on high-resolution slab polyacrylamide gels showed that most of the 125I was in polypeptide T49, an outer membrane constituent. Additional outer membrane polypeptides were iodinated in broken envelope preparations, demonstrating that T49 is uniquely accessible to the external environment and the asymmetric polypeptide organization of the outer membrane.     

Isolation of Rickettsia prowazekii with reduced sensitivity to gamma interferon.

The growth of Rickettsia prowazekii Madrid E was monitored in mouse L929 cells subcultured for several weeks in the presence of gamma interferon (IFN-gamma) to determine whether the rickettsiae would be eliminated from or would persist in these cultures. R. prowazekii exhibited two distinct patterns in these IFN-gamma-treated cultures. In some cases, IFN-gamma-induced inhibition of rickettsial growth led to elimination of the rickettsiae from the L929 cell cultures; in other cases, the initial inhibition of rickettsial growth was followed by establishment of a persistent rickettsial infection in the IFN-gamma-treated L929 cells. During the first 3 days after infection, the growth rate of the L929 cells was significantly lower and higher percentages of the cells were killed in the IFN-gamma-treated, R. prowazekii-infected cultures than in the untreated, R. prowazekii-infected cultures or the mock-infected cultures, whether treated or untreated. This suppression of cell growth occurred in the infected, IFN-gamma-treated cultures that eventually exhibited the elimination pattern as well as the IFN-gamma-treated cultures that became persistently infected. It was not possible to predict the outcome of a particular infection from the early growth pattern of the culture. It was determined that the L929 cells in the persistently infected, IFN-gamma-treated cultures had not lost the ability to respond to IFN-gamma. These cells, after treatment with an antibiotic to eliminate the persistent rickettsiae, retained the ability to inhibit both the replication of vesicular stomatitis virus and the growth of R. prowazekii Madrid E after treatment with IFN-gamma. In contrast, rickettsiae isolated from two persistently infected, IFN-gamma-treated cultures were less sensitive than R. prowazekii Madrid E to the antirickettsial effects of IFN-gamma in standard L929 cells. The maintenance of the phenotype of these altered rickettsiae during plaque purification and passage in the absence of IFN-gamma suggests an alteration at the genetic level rather than phenotypic adaptation.     

Roles of the Fc receptor and respiratory burst in killing of Rickettsia prowazekii by macrophagelike cell lines.

It is known that the virulent strain of Rickettsia prowazekii grows in macrophagelike cell lines, but if the rickettsiae are treated with antirickettsial serum before infection, the intracellular rickettsiae fail to grow and are destroyed. The uptake of rickettsiae by macrophagelike cell lines was increased by treatment of the rickettsiae with immune serum and with purified immunoglobulin G (IgG) from this serum but not by treatment with the F(ab')2 fragment derived from this IgG. This suggested that the normal rickettsial pathway of entry could be augmented by the Fc receptor-mediated pathway. However, rickettsiae treated with these F(ab')2 fragments which contained no Fc region were destroyed as effectively as those treated with immune serum or IgG. Internalization of R. prowazekii (whether virulent, avirulent, treated, or untreated) did not lead to an increased release of CO2 from [1-14C]glucose, an increase that would have been indicative of a respiratory burst. Furthermore, a mutant macrophagelike cell line, incapable of a respiratory burst, was able to destroy rickettsiae treated with immune serum as effectively as did the parental cell line. Electron micrographs of macrophagelike cells which had been incubated with either antirickettsial IgG or with F(ab')2 fragments derived from this IgG both demonstrated marked deterioration of the rickettsiae, which were primarily within vacuoles but occasionally free in the cytoplasm. In contrast, untreated rickettsiae displayed morphologically normal rickettsiae which were mostly in the cytoplasm but occasionally in the intact and damaged vacuoles. These results indicated that (i) a respiratory burst was not a significant part of the mechanism used by macrophagelike cells to destroy R. prowazekii treated with immune serum, (ii) the destruction of the rickettsiae by the macrophage was not dependent on a diversion to the Fc receptor-mediated pathway of entry, and (iii) the locus of damage to the rickettsiae was most likely the phagolysosome of the macrophagelike cell line.