Vol. 20, No. 3

INFECTION AND IMMUNITY, June 1978, p. 632-639 0019-9567/78/0020-0632$02.00/0 Copyright © 1978 American Society for Microbiology

Printed in U.S.A.

Contrast of Glycogenesis and Protein Synthesis in Monkey Kidney Cells and HeLa Cells Infected with Chlamydia trachomatis Lymphogranuloma Venereum DOUGLAS A. WEIGENT* AND HOWARD M. JENKIN The Hormel Institute, University of Minnesota, Austin, Minnesota 55912 Received for publication 4 January 1978

Glycogen metabolism of monkey kidney (LLC-MK-2) cells and HeLa 229 cells infected with a Chlamydia trachomatis lymphogranuloma venereum 440 L (LGV) was studied. The growth cycle of LGV in both host cells was similar; however, a greater number of infectious organisms developed intracellularly and were released into the medium during LGV infection of HeLa 229 cells than MK-2 cells. A rapid infection accompanied by a high rate of glycogen synthesis and a short period of accumulation was found in HeLa 229 cells infected with LGV. LGVinfected MK-2 cells started to accumulate glycogen about the same time as HeLa 229 cells; however, the rate of glycogen synthesis was lower and the period of accumulation was longer. The LGV agent grew in cycloheximide-treated cells in the absence of host cell protein synthesis. Protein synthesis associated with LGV throughout the developmental cycle was similar in both cell types and could be abolished by chloramphenicol. The continued synthesis of glycogen in the presence of cycloheximide suggested that the synthesis of glycogen was directed by the organism in both MK-2 cells and HeLa 229 cells. sis and accumulation and the role that glycogen serves in the infectious process remains limited. Two reports concerning the regulatory mechanisms of glycogenesis of chlamydial organisms have appeared which suggest that chlamydiae produce an enzyme that directs polysaccharide synthesis; however, the host cell controls the amount of glycogen formed in the inclusion (6, 18). Jenkin and Fan (18) observed an increase in the activity of an adenosine 5'-diphosphate (ADP)-glucose-dependent glycogen synthetase in C. trachomatis infections of HeLa 229 cells. Although these data were obtained for unfractionated lysates of infected cells, they imply the presence of a procaryotic glycogen synthetase and/or that the cell had a limited amount of glycogen synthesis could be delineated as a re- substrate which was available for use by the sult of infections by the growth of three different chlamydiae. In another study (6), the relationchlamydial strains in HeLa 229 cells. C. tra- ship between the multiplicity of infection and chomatis lymphogranuloma venereum (LGV) the yield of organisms with the amount of polyshowed a rapid infection accompanied by a high saccharide produced was examined. The results rate of glycogen synthesis and a short period of showed that, regardless of the multiplicity of accumulation. A slower rate of infection, along infection, the same amount of polysaccharide is with a slower rate and lower amount of glycogen produced by each infected cell. This observation synthesis and high glycogen retention, was ob- suggests that the polysaccharide is a product of served with C. trachomatis TW-3. C. psittaci the host cell. meningopneumonitis (MN) grew rapidly, but a Several questions arose in the course of our low amount of glycogen with slow turnover oc- previous studies. For example, is there an assocurred without any glycogen accumulation. Our ciation between host susceptibility and glycogen understanding of the pattern of glycogen synthe- accumulation in specific C. trachomatis infec-

Chlamydiae are obligate intracellular microorganisms that are classified into two main species, Chlamydia trachomatis and C. psittaci (22). Within the genus, a distinguishing species characteristic is the presence (C. trachomati) or absence (C. pittaci) of iodine- (27) or Schiffperiodate-staining (11) inclusions associated with unsaturated compounds or aldehydrogenic compounds including glycogen. The polysaccharide elaborated in trachoma inclusion conjunctivitis (TRIC) agent infections isolated from baby hamster kidney cells by Garrett has been characterized as glycogen with an average chain length of 14 to 16 glucose units (10). In an earlier publication from our laboratory (18), it was suggested that three patterns of

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tions which could be associated wth pathogenesis in the natural disease? Can definite evidence be found as to whether the host or the organism synthesizes the accumulated glycogen? This communication is a continuation of our earlier glycogen metabolic studies showing differences in ['4C]glucose uptake into glycogen, using a single chlamydial strain (LGV) to infect monkey kidney and HeLa 229 cells. In addition, we report the results of our experiments to distinguish the source of the glycogen from the organism and/or the host cell. MATERIALS AND METHODS Cultivation of monkey kidney (MK-2) and HeLa 229 cells. The MK-2 and HeLa 229 cells used in previous investigations in this laboratory (8, 18) were employed in all experiments. The method of cultivation of these cells has been described by Fan and Jenkin (8, 18). Counts of viable cells were made by the trypan blue exclusion test (20). Control cultures of uninfected cells were acquired from the same stock cultures as infected cells and treated identically. Growth of LGV in host cells. The 440L isolate (28) of strain LGV, received from J. Schachter, was serially passed five times at 45-h intervals in MK-2 cells from yolk sac suspensions as previously described (8). The percentage of infected cells was determined by microscopic examination of host cells stained with May-Grunwald-Giemsa (9) 45 h after infection. Multiplicity of infection used in the experiments was approximately 10 inclusion-forming units per cell and produced from 90 to 100% infection of cells (15). Infection and labeling of host cells. Monolayers of host cells were formed after 18 h of incubation at 370C by using an inoculum of 2 x 106 cells per 25-cm2 Falcon flask as previously described (18). Chloramphenicol (12 /g/ml) and cycloheximide (2 ,g/ml) were routinely added 2 h before infection. A 0.5-ml amount of diluted inoculum was added to each flask, and the infection was performed as previously described (8). Cells were pulsed with radioisotopes as described in the figure legends. A second set of host cells, equivalent in number to the samples in the isotopic tests, was run at the same time to have enough spent medium after pulsing with glucose for chase experiments. Incorporation of U-14C-labeled protein hydrolysate into total protein of normal and LGV-infected host cells. "4C-labeled protein hydrolysate (7 x 105 cpm/flask) was added to uninfected and infected host cell cultures as described in the legend to Fig. 2. After designated periods of incubation at 370C, cells from duplicate 25-cm2 Falcon flasks were harvested by pouring off the medium and rinsing the cells twice (5 ml each) with Hanks balanced salt solution (14) without calcium and magnesium (GKNP). The cells were removed in 1 ml of 0.05% trypsin containing 0.05% ethylenediaminetetraacetic acid at pH 7.4 to 7.6. A 0.1-ml sample was removed for cell counts, which were performed with the aid of a hemacytometer (17). Total protein was determined by the method of Lowry et al. (19). The samples were quickly frozen in a dry iceethanol bath until all samples were collected. The samples were rapidly thawed, and 2 ml of 6% trichlo-

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roacetic acid was added to precipitate the protein. The tubes were placed in a boiling-water bath for 10 min. An additional 2 ml of 6% trichloroacetic acid was then added to each sample, and the precipitates were collected under vacuum on 0.45-/Am membrane filters (type HA, Millipore Corp., Bedford, Mass.). The tubes were washed three times with 2 ml of 6% trichloroacetic acid. The filters were washed twice with 5 ml each of 6% trichloroacetic acid and twice with 80% ethyl alcohol. Dried filters were counted in 10 ml of a dioxane-based liquid scintillation fluid prepared from Permablend I (Packard Instrument Co., Inc., Downers Grove, Ill.). Liquid scintillation counting was done in a Packard liquid scintillation spectrophotometer (model 2405). Incorporation of D-[U-14C]glucose into glycogen of normal and LGV-infected MK-2 and HeLa 229 cells. [14C]glucose (2 x 106 cpm/flask) was added to uninfected and infected host cell cultures as described in the legend to Fig. 4. At the designated times, cells from duplicate 25-cm2 Falcon flasks were harvested as described for samples labeled with 14C-protein hydrolysate, and the glycogen was isolated from infected and uninfected cells by the method of Fan and Jenkin (7). Measurement of infectivity in eggs. Monolayers of MK-2 and HeLa 229 were obtained after 24 h of incubation, using an inoculum of 107 cells per 32-oz (ca. 1-liter) glass prescription bottles. The cells were infected with LGV and, at various times after infection, the medium was aspirated and LGV-infected host cells were removed from the bottles with glass beads and disrupted by sonic oscillation as previously described (8). The amount of extracellular and intracellular infectious LGV was determined by titrating the medium and disrupted cells, respectively, by the procedure of Wang and Grayston (29). The method of Reed and Muench (26) was used to obtain the 50% egg lethal dose per milliliter of the original suspensions. Antibiotics and isotopes. Cycloheximide was a gift from the Upjohn Co., Kalamazoo, Mich.; chloramphenicol was purchased from Sigma Chemical Co., St. Louis, Mo.; [U-_4C]glucose (specific activity, 182 mCi/mmol) was from New Engand Nuclear Corp., Boston, Mass.; '4C-protein hydrolysate (specific activity, 57 mCi/matom) was from Amersham/Searle, St. Louis, Mo.

RESULTS Growth cycle of LGV in host cells. The intracellular and extracellular infectivity changes during the growth cycle of LGV in HeLa 229 and MK-2 cells are shown in Fig. 1. The pattern of LGV development was similar in both cell types. In HeLa 229 cells, there was an initial slight reduction of intracellular infectivity followed by at least a 4-logi0 increase in LGV between 12 and 48 h after infection. The appearance of infectious LGV in the medium commenced approximately 36 h after infection and reached a maximum increase by 48 h. The intracellular infectivity decreased rapidly after 72 h, whereas the extracellular infectivity re-

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FIG. 1. Growth of LGV in HeLa 229 and MK-2 cells as determined by the 50%o egg lethal dose end point. The medium and LGV-infected host cells were removed separately from 32-oz (ca. 1-liter) flasks at various intervals of time after infection, as described in the text. The medium and cell homogenates from duplicate flasks were assayed for infectivity at three dilution levels. Symbols: 0, intracellular LGV-infected HeLa; 0, extracellular LGV-infected HeLa; A, intracellular LG V-infected MK-2; A, extracellular LG V-infected MK-2.

mained nearly constant during the 96 h of observation. In MK-2 cells, there was also an initial loss of intracellular infectivity followed by only a 2.5logio increase in LGV between 24 and 48 h after infection. The appearance of extracellular LGV was observed 36 h after infection and reached a maximum 48 h after infection. Although the pattern of LGV development was similar in MK2 and HeLa 229 cells, it is clear that greater production and release of infectious units occurred during LGV infection of HeLa 229 cells than with MK-2 cells. Cycloheximide inhibition of protein synthesis in LGV-infected and uninfected host cells. Cycloheximide inhibits the incorporation of amino acids into proteins in eucaryotic cells (5). This property is demonstrated in Fig. 2A and B for HeLa 229 and MK-2, respectively. Cycloheximide (2 ,ug/ml) inhibited protein synthesis in both cell types immediately and was almost 92% inhibited after 50 h of incubation. The data also show the effect of infection with the LGV organism on the rate of host protein synthesis. Examination of the results from both cell types suggests that infection with the LGV agent did not inhibit host protein synthesis. The

sum of the incorporation of "4C-protein hydrolysate into the protein of cycloheximide-treated, infected host cells and the incorporation into uninfected host cells equaled the incorporation into infected, untreated host cells. During the 45- to 48-h infection period, there appears to have been a linear rate of accumulation of label in both cell systems with and without cycloheximide (Fig. 2). MK-2 cells had a lower rate of protein synthesis than HeLa cells (Fig. 2), which correlates with its slower rate of growth. Cycloheximide at 2 tug/ml killed infected and uninfected host cells more rapidly than cells maintained in the absence of the drug (Fig. 2). The greatest lethal effect of cycloheximide was observed when the drug was added to infected

cells. To determine the time of synthesis of LGVspecific proteins, LGV-infected and uninfected MK-2 and HeLa 229 cells were treated with cycloheximide before infection and pulse-labeled with radioactive protein hydrolysate. The incorporation of radioactive amino acids into the LGV proteins was detected throughout 15 to 60 h of infection (Fig. 3). The patterns of protein syntesis in both cell types were similar. During the initial 12 h after infection, LGV-specific protein

VOL. 20, 1978

GLYCOGEN METABOLISM OF LGV C

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FIG. 2. Incorporation of 4C-labeledprotein hydrolysate into infected and uninfected cycloheximide-treated and untreated HeLa 229 (A) and MK-2 (B) cells. Cycloheximide (2 pg/ml) was added 2 h before infection and 4 h before "C-labeled protein hydrolysate (7.5 X 105 cpm/25-cm' bottle). At various times after infection, cells from duplicate 25-cm2 flasks were harvested and analyzed as described in the text. The data were extrapolated to zero hour after infection from the first '4C samplings at 5 h (A) and 12 h (B). The lower parts of (A) and (B) plot the results of '4C-protein hydrolysate incorporation. The upper parts show cumulative host cell viability. Symbols: 0, LGV-infected cells; 0, uninfected cells; A, LGV-infected, cycloheximide-treated cells; A, cycloheximide-treated, uninfected cells. These data are representative of results of three independent experiments performed in duplicate.

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synthesis was not detectable. This was probably due to the low rate of protein synthesis by minimum numbers of LGV early in the infection, which was masked by residual cell protein synthesis. After 12 h of incubation, the rate of protein synthesis markedly increased, especially in LGV-infected HeLa 229 cells, until 30 h after infection. After that time, protein synthesis plateaued in MK-2 cells until 36 h after infection and then decreased, whereas there was a gradual

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(B) cells in the presence of cycloheximide and/or chloramphenicol. Cycloheximide (2 pg/ml) and/or chloramphenicol (12 jig/ml) was added to host cells 2 h before infection and was present throughout the experiment. At various times after infection, cells were exposed to "C-protein hydrolysate (7.5 x 105 cpm/25-cm2 bottle) and labeled for 5-h pulses. Cells were harvested and analyzed as described in the text. Each point on the curves represents the incorporation that occurred in the preceding 5-h interval. The data are representative of two independent experiments. Symbols: 0, LGV-infected, cycloheximide-treated cells; x, LGV-infected, cycloheximide- and chloramphenicol-treated cells; A, cycloheximide-treated, uninfected cells.

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decrease in the activity in HeLa cells 30 h after infection. The pattern of ''C uptake we observed resembled that observed by Alexander (1) with MN in mouse L cells. The effect of chloramphenicol on LGV-specific protein synthesis is also shown in Fig. 3. Synthesis of LGV proteins appeared to be completely inhibited when the infected, cycloheximide-treated cells were exposed to chloramphenicol. These results suggest that cycloheximide-treated MK-2 and HeLa 229 cells could be utilized to evaluate host variation of glycogen synthesis by LGV. Incorporation of [U-1'Clglucose into glycogen in MK-2 cells and HeLa 229. The results of ["C]glucose incorporation into glycogen of normal and LGV-infected HeLa 229 cells are shown in Fig. 4A. LGV-infected HeLa 229 16 A Hela

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36 48 60 72 Hours FIG. 4. Incorporation of [U- '4C~glucose into glycogen of uninfected and LGV-infected HeLa 229 (A) and MK-2 (B) cells. Cells were exposed to [U-''CJglucose (2 x 106 cpm/25-cm2 flask) and pulsed for 12 h. After the pulse, the samples were washed with Hanks balanced salt solution without calcium and magnesium, and spent medium from the same incubation time was added to the cells for an additional 12-h chase before harvesting. Each point represents the radioactivity at the end of the pulse or chase period from the average of duplicate samples. These data are representative of results from three independent experiments performed in duplicate. Symbols: x, pulse uninfected; 0, pulse LGV-infected; 0, chase LGV-infected. O

INFECT. IMMUN.

cells started to accumulate glycogen at approximately 30 h after infection, with a rapid drop in glycogen accumulation by 50 h. A similar result was reported by Jenkin and Fan (18). In another set of experiments contrasting HeLa 229 and LGV-infected MK-2 cells, the data show a different pattern of glycogen metabolism (Fig. 4B). LGV-infected MK-2 cells started to accumulate glycogen about the same time as HeLa 229 cells after infection; however, the cells maintained the glycogen throughout the 72-h observation period. The lower rate of glycogen synthesis and the greater retention paralleled the pattern of glycogen metabolism observed with strain TW3 infection of HeLa 229 (18). In LGV-infected HeLa 229 cells, glycogen synthesis is 2.3-fold greater 30 to 42 h after infection than that observed in LGV-infected MK-2 cells. The activity of [14C]glycogen in uninfected cells throughout the observation period was considerably lower than in infected cells (Fig. 4). Normal HeLa 229 cells showed more glycogen accumulation than normal MK-2 cells. Labeled glycogen did not appear to turn over in infected MK-2 cells throughout the 72-h observation period, but turnover was observed after 48 h in infected HeLa cells (Fig. 4). Synthesis of glycogen in LGV-infected host cells. The incorporation of ["4C]glucose into glycogen in HeLa 229 cells and MK-2 cells was studied in experiments comparable to those previously described for 14C-labeled protein hydrolysate. Figure 5 shows the results for uninfected and LGV-infected HeLa 229 cells. Cycloheximide, which inhibited host cell protein synthesis, eliminated the uptake of [14C]glucose into glycogen of uninfected HeLa cells. Cycloheximide only slightly inhibited the incorporation of [I4C]glucose by infected cells. The mechanism of this inhibition is not known. A combination of cycloheximide and chloramphenicol blocked the synthesis of glycogen in infected cells. A similar experiment was performed with MK-2 cells, as described above, to determine the antibiotic effect on glycogen synthesis and to examine the pattern of glycogen synthesis late in the infection (Fig. 6). The data show that chlamydial protein synthesis was required for glycogen to be synthesized. Cycloheximide had no inhibitory effect on glycogen synthesis by infected cells and, after 72 h, the amount of glycogen synthesis dropped considerably. Accumulation of the glycogen synthesized from [14C]glucose was observed in a pulse-chase experiment (Fig. 6). LGV-infected cells were incubated for an additional 12 h in the absence of labeled glucose. The data showed a small decrease in accumulation of glycogen after 70 h. Incorporation of ['4C]glucose in the presence of cyclohex-

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Hours FIG. 5. Incorporation of [U-'4CJglucose into glycogen in infected and uninfected cycloheximidetreated and untreated HeLa 229 cells. Cells were FIG. 6. Incorporation of [U-'4C]glucose into glyexposed to [U-'4Cjglucose (1.8 x 10' cpm/25-cm2 cogen in infected and uninfected cycloheximideflask) and labeled for 12-h pulses. Cycloheximide (2 treated and untreated MK-2 cells. The experiment pg/ml) and/or chloramphenicol (12 pg/ml) was was performed and the data wereplotted as described added 2 h before infection and 4 h before ['4Cjglu- in the legend to Fig. 5, except that 1.5 x 106 cpm of cose. At the designated times, cells from duplicate 25['4CJglucose was added per 25-cm2 flask. Chase samcm2 flasks were harvested and analyzed for their ples were treated as described in the legend to Fig. 5. glycogen content as described in the text. Symbols: Symbols: x, uninfected, untreated HeLa; A, cyclohexx, uninfected, untreated HeLa; A, cycloheximideimide-treated, uninfected HeLa; *-*, LGV-intreated, uninfected HeLa; 0, LGV-infected HeLa; 0, fected HeLa; 0, LG V-infected, cycloheximide-treated LGV-infected, cycloheximide-treated HeLa; and A, HeLa; 0----0, chase LGV-infected HeLa; and A, LGV-infected cycloheximide- and chloramphenicol- LGV-infected, cycloheximide- and chloramphenicoltreated HeLa. The data represent typical results of treated HeLa. The data represent typical results of two independent experiments. two independent experiments. imide was inhibited at all labeling periods when chloramphenicol and cycloheximide were added simultaneously to LGV-infected MK-2 cells. DISCUSSION This study and a previous indirect study (18) suggest that the enzyme responsible for glycogen synthesis during LGV infection of tissue culture cells is coded by the organism. Our results directly demonstrate that LGV can synthesize its proteins in cycloheximide-treated host cells. Similar results with other chlamydial agents have been reported by Alexander (1) and Becker (2, 3). In the absence of 90% of host protein synthesis, LGV can synthesize glycogen. Results from experiments described in Fig. 2 showed

that host protein synthesis is not inhibited by infection with the LGV agent. Cycloheximideinsensitive incorporation of labeled protein hydrolysate and labeled glucose in infected host cells which can be abolished by chloramphenicol is assumed to be the result of LGV-specific protein synthesis. The growth cycle of the LGV agent in MK-2 and HeLa 229 cells consisted of three major stages and was in agreement with earlier reports (8). In this respect, LGV resembled MN grown in L cells (16) and the T'ang strain (TE-55) of trachoma agent (TRIC/1RC-PK/OT) cultivated in FL cells (4). The intracellular development of infectious progeny was detectable about the same time as specific protein synthesis by

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the agent. In MK-2 cells, LGV agent could pro- identified two enzyme reactions to be necessary duce 10% of the infectivity observed in HeLa 229 for the biosynthesis of a-1,4-glucosidic linkages cells. The explanation for the 2-logio difference of glycogen in bacteria (13, 24). One of the enin intracellular development of infectious prog- zymes, ADP-glucose pyrophosphorylase, cataeny is not obvious. The cause of this variation lyzes the synthesis of ADP-glucose while the may be nutritional because, in human kidney other enzyme, glycogen synthetase, transfers the cells, C. trachomatis TE-55 could produce 80% glucosyl moiety from ADP-glucose to a preexistof its infectivity in cells incubated with medium ing a-glucan primer. The in vitro regulation of without seine, but in FL cells no strain TE-55 bacterial glycogen synthesis is at the level of development took place in the same medium ADP-glucose pyrophosphorylase (23, 24), and (21). It should be pointed out that our data glycolytic intermediates have been shown to be measure infectious organisms only and that the activators of the reaction, whereas adenosine 5'production of noninfectious LGV is unknown. monophosphate, (AMP), ADP, and inorganic Since ["4C]glucose incorporation into glycogen phosphate inhibit ADP-glucose synthesis. Gowas essentially abolished in infected, cyclohexi- vons et al. (12) have isolated Escherichia coli mide-treated cells by chloramphenicol, the cy- mutants with different abilities to synthesize cloheximide-resistant incorporation was proba- glycogen which contain ADP-glucose pyrophosbly catalyzed by a chlamydial enzyme. This re- phorylases altered in their allosteric properties sult is in agreement with earlier published data (25). The mutants accumulate two- to threefold demonstrating a 200-fold increase in ADP-glu- more glycogen than the parent strain and concose acceptance by glycogen synthetase in in- tain ADP-glucose pyrophosphorylases having fected cell lysates (18). The inhibition of glyco- higher affinity for the activator fructose diphosgen synthesis by chloramphenicol further sug- phate and lower affinity for the allosteric inhibgests that the organism does not induce the host itor AMP. In our system, the amount of glycogen to synthesize the new enzyme. Another alterna- synthesis 30 to 40 h after infection was two- to tive is that the organism synthesizes a factor threefold higher ihi HeLa cells than in MK-2 which alters the host enzyme specificity. Addi- cells. It is tempting to speculate, therefore, that tional investigation is required to evaluate this the difference we observed may have been caused by allosteric regulation of the ADP-gluhypothesis. The results of LGV infection in HeLa and cose pyrophosphorylase within the inclusion MK-2 cells on glycogen accumulation were strik- body. To determine the intracellular concentraingly different. The rate of glycogen synthesis tion of several allosteric compounds and their between 30 and 42 h after infection was higher diffusion across the inclusion membrane, addiand the breakdown of glycogen was earlier and tional investigation is needed. more dramatic in HeLa 229 cells than in MK-2 ACKNOWLEDGMENTS cells. The metabolism of glycogen in MK-2 cells did not correspond to the peak time of protein We thank Lloyd Anderson and Christy Bjornson for their synthesis or release of infectious organisms from expert technical assistance. This investigation was supported by Public Health Srvice the host cell, as observed in HeLa 229 cells. The research grant HL 08214 from the Program Projects Branch, amount of ['4C]glucose incorporated into glyco- Extramural National Heart, Lung and Blood Instigen in LGV-infected HeLa cells with and with- tute, and by Programs, The Hormel Foundation. out cycloheximide was approximately twofold lower between 24 and 48 h after infection in the LITERATURE CITED presence of cycloheximide. Although this differ- 1. Alexander, J. J. 1968. Separation of protein synthesis in ence was not observed in MK-2, it suggests that meningopneumonitis agent from that in L cells by differential susceptibility to cycloheximide. J. Bacteriol. the host cell does contribute to the organism in 95:327-332. the amount of glycogen synthesized, probably Y. 1974. The agent of trachoma, p. 99. In J. L. by the availability of nutritional factors. It is 2. Becker, Melnick (ed.), Monographs in virology, vol. 7, 1st ed. S. unclear whether the amount and/or the rate of Karger, Basel, Switzerland. glycogen synthesized is related to the number of 3. Becker, Y., and Y. Asher. 1972. Synthesis of trachoma agent proteins in emetine-treated cells. J. Bacteriol. infectious particles produced or vice versa. The 109:966-970. data demonstrate significant differences by host 4. Bernkopf, H., P. Mashiah, and Y. Becker. 1962. Corvariation on chlamydial synthetic activities with relation between morphological and biochemical a single chlamydial strain. changes and the appearance of infectivity in FL cultures infected with trachoma agent. Ann. N.Y. Acad. Sci. The biochemical basis for the different pat98:62-81. terns of glycogen accumulation and degradation 5. Ennis, H. L., and M. Lubin. 1964. Cycloheximide: aspects in LGV infection of HeLa 229 cells and MK-2 of inhibition of protein synthesis in mammalian cells. cells is not known. Previous investigators have Science 146:1474-1476.

VOL. 20, 1978 6. Evans, A. 1972. The development of TRIC organisms in cell cultures during multiple infection. J. Hyg. 70:39-48. 7. Fan, V. S. C., and H. M. Jenkin. 1970. Glycogen metabolism in chlamydia-infected HeLa cells. J. Bacteriol. 104:608-609. 8. Fan, V. S. C., and H. M. Jenkin. 1974. Lipid metabolism of monkey kidney cells (LLC-MK-2) infected with Chlamydia trachomatis strain lymphogranuloma venereum. Infect. Immun. 10:464-470. 9. Furness, G., D. M. Graham, and P. Reeve. 1960. The titration of trachoma and inclusion blennorrhoea viruses in cell cultures. J. Gen. Microbiol. 23:613-619. 10. Garrett, A. J. 1975. Some properties of the polysaccharide from cell cultures infected with TRIC agent (Chlamydia trachomatis). J. Gen. Microbiol. 90:133-139. 11. Gordon, F. B., and A. L. Quan. 1965. Occurrence of glycogen in inclusions of the psittacosis-lymphogranuloma venereum-trachoma agents. J. Infect. Dis. 115:186-196. 12. Govons, S., R. Vinopal, J. Ingraham, and J. Preiss. 1969. Isolation of mutants of Escherichia coli B altered in their ability to synthesize glycogen. J. Bacteriol. 97:970-972. 13. Greenberg, E., and J. Preiss. 1964. The occurrence of adenosine diphosphate glucose: glycogen transglucosylase in bacteria. J. Biol. Chem. 239:4314-4315. 14. Hanks, J. H., and R. E. Wallace. 1949. Relation of oxygen and temperature in the preservation of tissues by refrigeration. Proc. Soc. Exp. Biol. Med. 71:196-200. 15. Hatch, T. P. 1975. Competition between Chlamydia psittaci and L cells for host isoleucine pools: a limiting factor in chlamydial multiplication. Infect. Imun. 12:211-220. 16. Higashi, N., N. Notake, and T. Fukada. 1959. Growth characteristics of the meningopneumonitis virus in strain L cells. Annu. Rep. Inst. Virus Res. Kyoto Univ. 2:23-56. 17. Jenkin, H. M., and L. E. Anderson. 1970. The effect of oleic acid on the growth of monkey kidney cells. Exp. Cell Res. 59:6-10. 18. Jenkin, H. M., and V. S. C. Fan. 1971. Contrast of

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Contrast of Glycogenesis and protein synthesis in monkey kidney cells and HeLa cells infected with Chlamydia trachomatis lymphogranuloma venereum.

Vol. 20, No. 3 INFECTION AND IMMUNITY, June 1978, p. 632-639 0019-9567/78/0020-0632$02.00/0 Copyright © 1978 American Society for Microbiology Print...
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