Annals of the New York Academy of SciencesVolume 590, Issue 1 p. 118-126 The Effect of Rickettsial Lipopolysaccharide and Insulin on S6 Phosphorylation in Guinea Pig Liver and HepG2 Cellsa W. D. PICKIN, W. D. PICKIN Department of Microbiology, University of Kansas, Lawrence, Kansas 66045Search for more papers by this authorT. HACKSTADT, T. HACKSTADT Department of Pathology, University of Texas Health Center, Galveston, Texas 77550Search for more papers by this authorD. PARETSKY, Corresponding Author D. PARETSKY Department of Microbiology, University of Kansas, Lawrence, Kansas 66045Corresponding author.Search for more papers by this author W. D. PICKIN, W. D. PICKIN Department of Microbiology, University of Kansas, Lawrence, Kansas 66045Search for more papers by this authorT. HACKSTADT, T. HACKSTADT Department of Pathology, University of Texas Health Center, Galveston, Texas 77550Search for more papers by this authorD. PARETSKY, Corresponding Author D. PARETSKY Department of Microbiology, University of Kansas, Lawrence, Kansas 66045Corresponding author.Search for more papers by this author First published: June 1990 https://doi.org/10.1111/j.1749-6632.1990.tb42214.x a This work was supported by PHS Grant AI16954 from the NIH and by the Fraternal Order of Eagles. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 Rickey, M. J., F. R. Gonzales & D. Paretsky. 1985. Infect. Immun. 48: 690–696. 2 Buchanan, B. J. & J. P. Filkins. 1976. Circ. Shock 3: 267–280. 3 Paretsky, D., C. M. Downs & C. W. Salmon. 1964. J. Bacteriol. 88: 137–152. 4 Hackstadt, T., M. G. Peacock, P. J. Hitchcock & R. L. Cole. 1985. Infect. Immun. 48: 359–365. 5 Madjar, J.-J. & R. R. Traut. 1980. Mol. Gen. Genet. 179: 89–101. 6 Krieg, J., A. R. Olivier & G. Thomas. 1988. Methods Enzymol. 164: 575–581. 7 Tabarini, D., A. G. de Herreros, J. Heinrich & O. M. Rosen. 1987. Biochem. Biophys. Res. Commun. 144: 891–899. 8 Picking, W. D., T. Hackstadt & D. Paretsky. 1989. Infect. Immun. 57: 3683–3688. 9 Bradford, M. M. 1976. Anal. Biochem. 72: 248–254. 10 Laemmli, U. K. 1970. Nature 227: 680–685. 11 Madjar, J.-J., S. Michel, A. J. Cozzone & J. P. Reboud. 1979. Anal. Biochem. 92: 174–182. 12 Abraham, A. K. & M. S. Khatim. 1989. Biochem. Biophys. Res. Commun. 161: 797–802. 13 Ballou, L. M., M. Siegmann & G. Thomas. 1988. Proc. Natl. Acad. Sci. USA 85: 7154–7158. 14 Swarup, G., S. Cohen & D. L. Garbers. 1982. Biochem. Biophys. Res. Commun. 107: 1104–1109. 15 Van Etten, R. L., P. P. Waymack & D. M. Rehkop. 1974. J. Am. Chem. Soc. 96: 6782–6785. 16 Jeno, P., N. Jaggi, H. Luther, M. Seigmann & G. Thomas. 1989. J. Biol. Chem. 264: 1293–1297. 17 Bialojan, C. & A. Takai. 1988. Biochem. J. 256: 283–290. 18 Novak-Hofer, I. & G. Thomas. 1985. J. Biol. Chem. 260: 10314–10319. 19 Pelech, S. L., B. B. Olwin & E. G. Krebs. 1986. Proc. Natl. Acad. Sci. USA 83: 5968–5972. 20 Cobb, M. H. 1986. J. Biol. Chem. 261: 12994–12999. 21 Thomas, G., J. Martin-Perez, M. Siegmann & A. M. Otto. 1982. Cell 30: 235–242. 22 Mizuta, K., E. Hashimoto, Y. Sakanoue, S.-I. Nakamure, H. Kondo & H. Yamamura. 1987. Biochem. Biophys. Res. Commun. 146: 239–246. 23 Duncan, R. & E. McConkey. 1982. Eur. J. Biochem. 123: 535–538. 24 Hansson, A. & M. Ingelman-Sundberg. 1985. Eur. J. Biochem. 151: 97–100. 25 Palen, E. & J. A. Traugh. 1987. J. Biol. Chem. 262: 3518–3523. 26 Johnson, S. P. & J. R. Warner. 1987. Mol. Cell. Biol. 7: 1338–1345. 27 Kruppa, J. & M. J. Clemens. 1984. EMBO J. 3: 95–100. 28 Stueckemann, J. A. & D. Paretsky. 1971. J. Bacteriol. 106: 920–924. 29 Edelman, A. M., D. K. Blumenthal & E. G. Krebs. 1987. Annu. Rev. Biochem. 56: 567–613. 30 Rosen, O. M. 1988. Harvey Lect. 82: 105–122. 31 Baca, O. G. & D. Paretsky. 1983. Microbiol. Rev. 47: 127–149. 32 Gonzales, F. R., M. Halevy & D. Paretsky. 1984. Infect. Immun. 43: 14–20. 33 Greengard, P. 1978. Science 199: 146–152. Volume590, Issue1Rickettsiology: Current Issues and PerspectivesJune 1990Pages 118-126 ReferencesRelatedInformation

C. burnetii possesses a battery of host-independent enzymes which mediate endergonic and exergonic reactions. The biochemical and biophysical lesions responsible for the organism's obligate intracellular parasitic state have not been identified. Clues to this fundamental problem may lie in the agent's acidophilic metabolism and proliferation within the host cell's phagolysomal vacuole. What are the modifiers of transcription and translation? What constituents of the lysosomal vacuole contribute to the parasite's metabolism? Does the parasite have intrinsic cell-wall synthesizing capability? The isolation of plasmids from C. burnetii opens lines of investigation which should lead to solution of the problem, thereby helping to answer the general question of what is the nature of obligate parasitism. An understanding of the pathobiochemistry of Q fever and of its endotoxicosis rests on elucidating closely interrelated regulatory events. Stimulated hepatic transcription and translation of certain RNA and protein species attend the development of the disease, as do phosphorylation and dephosphorylation of central RNA and protein species. Phosphorylation and dephosphorylation are central regulators in protein synthesis. Whole animal experiments differ from those with cultured cells in important responses. Glycogenolysis, hepatic steatogenesis, and lipase activation are obvious examples of such differences. Stimulation of the production of lymphokines and of hormones is absent in HepG2 cells, and insulin seems to be critical in regulating the phosphorylation-dephosphorylation equilibrium which leads to regulation of protein synthesis. Newly synthesized, so far unidentified proteins may be involved in convalescence, re-establishing the homeostasis of the uninfected state. The model of pathobiochemical regulation in Q fever and endotoxicosis may be applicable to other febrile infections and endotoxicoses.

Guinea pig endotoxicosis induced by lipopolysaccharide from Coxiella burnetii Nine Mile phase I stimulates phosphorylation of liver ribosomal protein S6, with a 50% increase at 12 h postinoculation. The responsible protein kinase (S6PK) has been partially purified from liver; its activity is independent of cyclic AMP and of Ca2+ plus phosphatidyl serine or diacylglycerol. The preparation has an apparent optimum concentration of 20 mM Mg2+, while Ca2+ and Mn2+ are each inhibitory at 2 mM. The apparent Km for ATP is 30 microM with intact ribosomes. Because of the central role of phosphorylation in metabolic regulation and a purported role of phosphorylated S6 in protein synthesis, the lipopolysaccharide-induced stimulation of S6PK suggests a significant regulatory role of such enzymes in the pathobiochemistry of Q fever infection and endotoxicosis.

Q fever stimulates hepatic transcription and translation. Products of stimulated transcription have been identified, but not of translation. Protein (Pr) synthesis and rPr S6 phosphorylation correlated. The authors now report stimulated synthesis of plasma Pr species in early febrile responses to Q fever and Coxiella burnetii lipopolysaccharide (LPS). Guinea pigs received 400 g LPS intraperitoneally and 7 hr later 250 Ci L-(TVS)met, then sacrificed 3 hr later. Plasma Pr sp act (cpm/mg Pr) increased 2.3X over controls (N). Exptl plasma Pr PAGE autorads showed intensified Pr bands at M/sub r/ 55K. Guinea pigs infected with C. burnetii (Inf) received 400 Ci (TVS)met 84 hr p.i. and were sacrificed 3 hr later. Inf plasma Pr 1D-PAGE showed bands at 55K similar to that found with LPS, with lower albumin concn. Coomassie stain and autorads of 2-D PAGEs showed intensified or new acidic peptide species in Inf plasma. PAGE autorads in vitro translations using liver mRNA and ribosomes showed major species in Inf systems at 49K (4+) and 62K (2+) compared to N. The data indicate acute phase protein induction by LPS or rickettsial infection.

Q fever, as well as the lipopolysaccharide prepared from the rickettsial agent Coxiella burnetii, stimulates the phosphorylation of guinea pig liver ribosomal protein S6. In vitro mRNA and ribosome-dependent rabbit reticulocyte lysate translation systems reconstituted with ribosomes and mRNAs from infected animal livers were more active than those with mRNAs and ribosomes from uninfected animals. Treatment of ribosomes with a ribosomal supernatant phosphatase reduced the in vitro translation activities; the largest decreases occurred in systems with ribosomes and mRNAs from infected liver. These experiments provide a basis for explaining the increased hepatic protein synthesis during Q fever and demonstrate, perhaps for the first time, the phosphorylation of ribosomal protein in response to lipopolysaccharide. The implications of these observations are discussed in the context of previous studies on stimulated transcription and translation during Q fever.

The proposal that gene expression may be regulated by phosphorylation of nonhistone chromatin proteins was tested by studying increased transcription resulting from Q fever. Certain liver nuclear phosphoprotein kinase and phosphatase activities were altered after guinea pigs were infected with Coxiella burnetii. Nonhistone chromatin proteins had increased phosphoprotein kinase activity and were differentially phosphorylated. The addition of spermine equally stimulated nuclear phosphoprotein kinases of uninfected and infected livers. Increased nuclear phosphatase activity accompanied infection. It was concluded that protein phosphorylations are altered by infection and are central events in regulating RNA and protein synthesis. A hypothesis is presented which attempts to correlate the findings in previous reports and those in the present paper regarding biochemical sequelae of Q fever. It is suggested that certain features of regulation described here also may be operative in some other infections or diseases.

HISTORY .................................................. 127 EPIDEMIOLOGY .................................................. 129 CULTIVATION AND GROWTH OF C. BURNETII.................................. 130 Entry into and Proliferation Within Cultured Cells; Comparison with Other Rickettsiae 130 Enumeration .................................................. 131 BIOLOGY OF C. BURNETII.................................................. 131 Ultrastructure/Morphology .................................................. 131 Genome .................................................. 132 Ribosomes .................................................. 133 Replication.................................................. 133 Phase Variation.................................................. 134 LPS.................................................. 135 BIOCHEMISTRY OF C. BURNETII ............................................... 135 Glycolytic and Related Enzymes ................................................. 135 Anabolic Enzymes of Amino Acids, Proteins, and Nucleic Acids ...................... 136 Acidophilic Biochemistry and Intravacuolar Existence............................... 137 PATHOBIOLOGY.................................................. 138 Humans .................................................. 138 Embryonated Eggs .................................................. 139 Guinea Pigs and Other Animals.................................................. 139 Cell Cultures ......................................................... 141 IMMUNOLOGY.................................................. 141 Vaccines .................................................. 143 SUMMARY AND PROSPECTS .................................................. 143 ACKNOWLEDGMENTS .................................................. 144 LITERATURE CITED ......................................................... 144

Temporal studies were made of factors associated with increased RNA synthesis in guinea pig liver during Q fever. DNA-dependent RNA polymerase activities increased immediately after infection. The major distribution of RNA polymerase classes shifted from class II to class I during infection. Ornithine decarboxylase activity was induced or stimulated soon after infection and remained elevated throughout the four-day period studied. S-Adenosylmethionine decarboxylase activity increased on the first day after infection and subsequently declined. Concomitantly elevated concentrations of the polyamines putrescine, spermidine and spermine reached a maximum on the first day after infection and then decreased. A model is presented to integrate these and other results to explain how RNA synthesis may be regulated during infection.

Journal Article Rickettsiology Conference Get access Robert N. Philip, Robert N. Philip Rocky Mountain Laboratory, Hamilton, Montana Search for other works by this author on: Oxford Academic PubMed Google Scholar David Paretsky, David Paretsky University of Kansas, Lawrence, Kansas Search for other works by this author on: Oxford Academic PubMed Google Scholar Emilio Weiss, Emilio Weiss Naval Medical Research Institute, National Naval Medical Center, Bethesda, Maryland Search for other works by this author on: Oxford Academic PubMed Google Scholar Charles L. Wisseman, Jr. Charles L. Wisseman, Jr. University of Maryland School of Medicine, Baltimore, Maryland Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 141, Issue 1, January 1980, Pages 112–118, https://doi.org/10.1093/infdis/141.1.112 Published: 01 January 1980

Experimental infections with Coxiella burnetii augment rates of ribonucleic acid (RNA) synthesis in guinea pigs. The activity of deoxyribonucleic acid-dependent RNA polymerase in L cells persistently infected with C. burnetii was threefold greater than that in unifected cells; the polymerase activity in infected cells was predominantly of class I, whereas that in uninfected cells was predominantly of class II. A search for regulatory factors of polymerase activity revealed that preincubation of uninfected L cells with lipopolysaccharide of C. burnetii or with putrescine, spermidine, or spermine enhanced polymerase activities. Because sonicated nuclei were assayed rather than purified enzymes, it cannot be stated definitely whether augmented polymerase activites were consequences of direct effects of infection on polymerases or a triggering of secondary regulatory factors.

Changes in plasma membrane proteins of guinea pig liver and L-929 cells were studied during infection with Coxiella burnetii. Polypeptide species resolved by disc polyacrylamide gel electrophoresis with sodium dodecyl sulfate showed quantitative but no qualitative differences between uninfected and infected samples. When the O'Farrell technique of isoelectric focusing, followed by sodium dodecyl sulfate-slab gel polyacrylamide gel electrophoresis, was employed, additional polypeptides were resolved. Livers and L cells were labeled with [3H]-glucosamine. Infected livers incorporated less [3H]glucosamine in the membrane proteins than uninfected material, presumably indicating lower glycoprotein levels. Infected L-cell membranes incorporated greater amounts of [3H]glucosamine, and also were labeled to a greater extent than uninfected membranes, employing the [125I]lactoperoxidase technique. Uninfected L cells showed a greater agglutinability with concanavalin A than did infected cells. Infected livers had much greater levels of cyclic adenosine 3',5'-monophosphate. The data indicate changes in plasma membranes as a result of infection. Possible physiological consequences of membrane changes are discussed.

Mouse fibroblasts (L-929) and Vero (green monkey kidney) cells were infected with the rickettsia Coxiella burnetti, and persistent infections developed and were studied over a 6- to 10-month period. Ultrastructural comparisons were made between the two infected cell types, and both were tested cytochemically for the presence of acid phosphatase, a marker enzyme of lysozymes. Rickettsiae were always observed within vacuoles, and some infected L cells showed flattened endoplasmic reticulum as compared with uninfected cells. Rickettsiae in Vero cells were most often seen in vacuoles containing whorls of membranes ("myelin configurations") which were also seen in uninfected cells. Rickettsiae in Vero cells were pleomorphic, with acid phosphatase reaction product in their periplasmic space. This suggests either rickettsial degradation by lysosomal enzymes which penetrated the cell envelope or a penetration after the rickettsiae were dead. Vacuoles of infected Vero cells showed much more reaction product than that in infected L cells, and most rickettsiae in L cells had a normal appearance and showed no reaction product in their periplasmic space.

Synthesis of ribonucleic acid (RNA) by the deoxyribonucleic acid-dependent RNA polymerase of Coxiella burnetii required adenosine, uridine, guanosine, and cytidine 5'-triphosphates. Cell-free preparations of this obligate intracellular procaryotic parasite had competence to phosphorylate ribonucleoside mono- and diphosphates in the presence of exogenous adenosine and guanosine 5'-triphosphates to the corresponding di- and triphosphates. C. burnetii contained about 2 nmol of adenosine 5'-triphosphate per mg of protein, which could serve as a approximately P donor for in vivo synthesis of nucleoside triphosphates. The latter were then used as substrates in the synthesis of RNA in a coordinated metabolic system with C. burnetii RNA polymerase. It is suggested that during infection the rickettsiae might obtain the nucleotides necessary for RNA synthesis from the vacuoles in which C. burnetii proliferates.

The lipopolysaccharide previously isolated from the rickettsial agent of Q fever, Coxiella burneti, phase I, has been further characterized. The sugar residues ribose, mannose, gluclose, D-glycero-D-mannoheptose, and L-glycerto-D-mannoheptose are present. Two sugars remain unidentified, one of which is a minor and the other a major constituent. Isomyristic, palmitic, and beta-hydroxymyristic acids are the major fatty acid residues of the 15 identified. The nature and content of other lipopolysaccharide constituents are presented.

Surface layers of Coxiella burneti studied at a high resoulution reveal a plasma membrane and an outer surface membrane 6 to 7 nm thick, and a thin, moderately electron-dense intermediate layer associated with the inner surface of the outer membrane of many cells. This layer appears to be unaffected by lysozyme treatment. Ruthenium red staining was used to delineate a layer of filamentous material external to the outer membrane; this fuzzy layer has a mean thickness of 20 nm and is not often seen on the surface of cells prepared by conventional means. Both antigenic phase I and II cells show a ruthenium red-binding surface layer. It is suggested that this fuzzy layer may be, among other possibilities, a highly branched mucopolysaccharide.

A lipopolysaccharide obtained in a dialyzed phenol extract from the rickettsia Coxiella burneti produced the following effects in guinea pigs after intraperitoneal injection: hyperthermia, loss of body weight, increased liver weight and concomitant lipid infiltration, elevated levels of hepatic and plasma cortisol, increased incorporation of [(3)H]orotic acid into hepatic 28S and 18S ribosomal ribonucleic acid, increased incorporation of (14)C-labeled amino acids into liver and plasma protein, and leukocytosis. Most of these events also occur during infection of guinea pigs with C. burneti, and a causal relationship between the rickettsial lipopolysaccharide and the biochemical changes that occur during Q fever is suggested.