lipid composition of in monkey kidney cells in defined · unpublished data). lipid components of...

JOURNAL OF BACrERIOLOGY, July 1970, p. 62-70Copyright 0 1970 American Society for Microbiology

Vol. 103, No. 1Printed in U.S.A.

Lipid Composition of Chlamydia psittaci Grown inMonkey Kidney Cells in Defined Medium

S. MAKINO, H. M. JENKIN, H. M. YU, AND D. TOWNSEND

The Hormel Institute, University ofMinnesota, Austin, Minnesota 55912Received for publication 29 December 1969

The lipid compositions of (i) monkey kidney (MK-2) cells cultivated in Eagle'sminimal essential medium (MEM) with 5% calf serum, (ii) MK-2 cells cultivated inWaymouth medium supplemented with 20 gug of sodium oleate and 2 mg of bovinealbumin per ml, (iii) Chlamydia psittaci strain 6BC grown in the latter host system,and (iv) calf serum were compared. Strain 6BC contains 31 % phosphatidyl ethanol-amine (PE) and 15% phosphatidyl glycerol (PG), whereas the host cell containsalmost the same amount of PE (27%) and no PG. A high concentration of totallipid was observed in strain 6BC (29 to 34%), whereas MK-2 cells contain only 9 to15% and calf serum contains 4.5% total lipid. The fatty acids of the total lipid fromstrain 6BC contain branched-chain acids. These fatty acids were found mostly in PE(33.0%) and PG (37.0%). No branched-chain fatty acid was found in the MK-2cells. There was an increase in triglyceride content when MK-2 cells cultivated inMEM (19.2%) were compared with cells cultivated in Waymouth medium (28.0%).A high concentration (62.0%) of octadecenoic acid (C18 :1) was found in the triglyc-eride of MK-2 cells cultivated in Waymouth medium. The level of polyunsaturatedfatty acids observed in MK-2 cells cultivated in Waymouth medium (10.8%) and inthe chlamydiae grown in these cells (13.3%) was low compared with the level inMK-2 cells (28.8%) cultivated in MEM with 5% calf serum and the level in calf se-

rum itself (50.8 %). A higher ratio of sterol ester to free sterol was found in calf serumthan in MK-2 cells or in chlamydiae. Host contribution to lipid composition ofstrain 6BC is discussed.

Chlamydiae, which are obligate intracellularbacteria, have been reported to contain a highlevel (30 to 50%) of lipids (11, 22; Jenkin et al.,unpublished data). Lipid components of theseorganisms have been indirectly associated withcomplement-fixing antigens of the cell wall andintact particles (8, 15). Results from qualitativeand quantitative analysis of lipid classes ofChlamydia psittaci, strain meningopneumonitis,grown in chorioallantoic membrane (CAM) cellsof chick embryos, suggested that some lipids areassociated specifically with the organism orinfected host cells, or with both (7, 13; Jenkinet al., unpublished data).

Jenkin and Anderson (14) recently were ableto cultivate monolayers of MK-2 cells in thedefined medium of Waymouth (23) supplementedwith sodium oleate, after the cells had beeninitially cultured in a serum-containing medium.MK-2 cells in this system supported growth ofchlamydiae (Jenkin et al., unpublished data).With this new host system, more definitive studiesof lipid composition and metabolism were pos-sible.

The analyses of the lipid composition of strain6BC and the appropriate host controls are thesubject of this report.

MATERIALS AND METHODSOrganism. C. psittaci strain 6BC (16) was used to

infect MK-2 cells. The egg inocula were preparedaccording to the procedure of Moulder and Weiss(17). The stock inoculum was in its 11th passage inwhite Leghorn chick embryos and was passed twomore times in MK-2 cells (10) cultivated in Eagle'sminimal essential medium (MEM; 5) with 2% new-born calf serum.The cell culture stock inoculum of strain 6BC was

prepared in the MK-2 cells as described by Jenkin forthe preparation of trachoma agents in HeLa cells (12).

Cultivation of cells. A population of 2.5 X 106 to3.5 X 106 MK-2 cells suspended in 30 ml of MEMcontaining 5% calf serum was added to 32-oz (0.95-liter) prescription bottles and incubated at 37 C. Thesecells were used as an inoculum for roller drum tubes.The tubes were seeded with 2.5 X 107 to 3.0 X 107cells suspended in 80 ml of Waymouth medium con-taining 0.5% calf serum, and were placed in a rollerdrum (685 mm X 64 mm outer diameter; Flow Lab-oratories Inc., Rockville, Md.).

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LIPID COMPOSITION OF CHLAMYDIA PSITTACI

The Waymouth medium with 0.5% calf serum wasremoved from the cell monolayer after 24 hr of incu-bation at 37 C and was replaced with Waymouthmedium containing 20 ,ug of sodium oleate (9-18:1)per ml and 2 mg of bovine albumin (fatty acid poor,Pentex Inc., Kankakee, Ill.) per ml (14). The cellswere further cultivated in the Waymouth mediumand formed a monolayer after 3 to 4 days of incuba-tion. These cells were used for infection and normalcontrol studies. Cells further cultivated in fresh MEMwith 5% calf serum were also used as a control. Thesecells were handled in the same way as cells cultivatedin Waymouth medium.

Cells were routinely checked for mycoplasma con-tamination before use in any experiment. All fractionsfor analyses were examined for freedom from bacterialcontamination in thioglycolate broth and TrypticaseSoy Broth, and on blood-agar slants, before the lipidanalysis was performed. Direct smears of the fractionswere made with the use of crystal violet and Machie-vello stains as a further check for the presence offastidious organisms (diptheroids) which might notgrow in the above media.

Infection of cells. The medium was decanted fromthe MK-2 cell cultures, the cells were washed twotimes with 20 ml of Hanks balanced salt solution(BSS), and 5 ml of strain 6BC (108.7 to 109 0 egg LD50)in MEM with 2% calf serum was inoculated intoeach of 14 roller tubes. The cells exposed to the in-oculum were rotated in the roller drum at 37 C. After2 hr, 80 ml of Waymouth medium with 20 Ag ofsodium oleate and 2 mg of bovine albumin per ml

was added, and the cells were further incubated at37 C for 72 hr.

Preparation of strain 6BC. The supernatant fluidfrom infected cell cultures (14 tubes) was collected.Tissue debris in the fluid was removed by sedimenta-tion at 800 X g for 10 min. The supernatant fluid wasfurther centrifuged at 8,000 X g for 30 min. Strain6BC remaining in the latter pellet was resuspendedin 2% ammonium acetate (NH4Ac) and was desig-nated strain 6BC (see Fig. 1).

Removal of strain 6BC from infected cells. The in-fected cell monolayer was removed from the glasssurface with glass beads (4-mm diameter) in BSS(10 ml/tube). The cells harvested from 14 tubes werepooled. The beads were washed with BSS and thewashings were added to the first pool. The pool wassedimented at 800 X g for 10 min. The resulting cellpellet was treated with a Sonifier (Branson Instru-ments, New Bedford, Mass.) at 4 C for 3 min withthe use of intermittent 30-sec pulses. The sonicallytreated cells were again centrifuged at 800 X g for 10min. The pellet was suspended in 2% NH4Ac andcentrifuged at 800 X g for 10 min. This procedure wasrepeated. The final pellet was suspended in 2%NH4Ac and designated infected MK-2 debris.

Lipid analysis. The method of Bligh and Dyer (3)was employed for total lipid extraction. Total lipidswere extracted with chloroform-methanol-water(10:20:8) three times for 15 min at room tempera-ture. Chloroform and water were added to the com-bined extracts to give a final chloroform-methanol-water ratio of 20:20:18. After low-speed centrifuga-

Infected cells

Supernatant fluid Infected cells

Centrifuge 800 X g, 1O min Roll off with glass beads in BSS

Supernatant fluid Centrifuge 800 X g, 10 min

Centrifuge 8,000 X g, 30 min Pellet

Pellet Resuspend in BSS sonicated 20 kc, 3 minat 30-sec intervals, 4 C

Resuspend in 2% NH4Ac

Strlain 6B3CCentrifuge 800 X g, 10 min

Pellet

Wash 2X with 2% NH4Ac

Centrifuge 800 X g, 10 min

Pellet

Resuspend in 2% NH4Ac

Infected MK-2 debrisFIG. 1. Flow sheet ofpreparation of strain 6BC and infected MK-2 debris. NH4AC, ammonium acetate; BSS,

Hanks balanced salt solution.

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MAKINO ET AL.

tion, the lower phase was removed; the upper aque-ous phase was washed three times with chloroformand combined with the lower chloroform layer. Thechloroform layer was dried under N2, by use of arotary evaporator. The samples were then made upto a specific volume with chloroform.

Total lipids (40 to 50 mg) were separated on twothin-layer chromatographic (TLC) plates coatedwith a layer 500 /Am thick of Silica Gel H (E. MerckAG, Darmstadt, Germany). By use of 2% aceticacid in diethyl ether, the total lipids were separatedinto total neutral lipid near the solvent front andtotal polar lipid at the origin; both bands were locatedby spraying the plate with water. The bands wereremoved separately by scraping.The total neutral lipid fraction was extracted from

the gel with diethyl ether and was further fractionatedby means of TLC with a solvent system containingpetroleum ether-diethyl ether-acetic acid (90:15:2).TLC plates were sprayed with 2',7'-dichlorofluores-cein (Eastman Organic Chemicals, Rochester, N.Y.)to locate the lipid bands which were scraped from theplate and extracted with diethyl ether. Sterol ester,triglyceride, diglyceride, free sterol, and monoglyc-eride were collected as individual neutral lipid classesand identified by comparison with authentic stand-ards.The polar lipid fraction was extracted with chloro-

form-methanol-water (3:5:2). Chloroform andwater were added to the polar lipid to make thechloroform-methanol-water ratio 20:20:18. Thelower phase was evaporated to near dryness underN2 and dissolved in chloroform. Individual phospho-lipids and glycolipids were fractionated by TLC withchloroform-methanol-7 M NH40H (65:30:4) orchloroform-methanol-acetic acid-water (25:15:4:2).Purity of individual fractions was confirmed by atwo-dimensional solvent system of chloroform-metha-nol-7 M NH40H, 65:30:4, and chloroform-methanol-acetic acid, 170:75:25 (21). Individual polar lipidclasses were identified by comparison of RF withauthentic standards and by their reactions afterspraying with specific reagents.

Phospholipids were stained by the procedure ofDittmer and Lester (4), with the use of molybdenumblue. The Dragendorif reagent was used to identifyqualitatively phosphatidyl choline, sphingomyelin,and lysophosphatidyl choline by means of the pro-cedure of Beiss (2). The ninhydrin reagent (18) wasused to stain phosphatidyl ethanolamine and phos-phatidyl serine and their lyso forms. An a naphtholspray (20) was used to locate and tentatively identifyglycolipids. Infrared spectrophotometry (PerkinElmer, model no. 21) was used to verify further thepurity and structure of the individual polar lipids.Individual neutral lipid and polar lipid classes werequantitated on the basis of dry weight with an elec-tronic balance (model G, Cohn Division, VentronInstruments Corp., Paramount, Calif.). Phosphoruscontent of calf serum phospholipids was determinedby the method of Bartlett (1). All analyses had astandard error of less than 5% from triplicate samples.

Fatty acid methylation and gas-liquid chromatog-raphy of the esters. Lipid samples were hydrolyzed

by use of 0.2 ml of 33%h0 KOH-5 ml of ethyl alcoholat 50 C for 90 min. The nonsaponifiable fraction wasremoved with petroleum ether, the solution wasacidified with 6 N HCl, and free fatty acids were ex-tracted with petroleum ether. Fatty acids thus ob-tained were then methylated by the diazomethanemethod of Schlenk and Gellerman (19) and wereanalyzed by gas-liquid chromatography. Identificationof individual fatty acid methyl esters was based onthe equivalent chain length (9) and the relative reten-tion time (6), and on the results of mass spectro-photometry (Jenkin et al., unpublished data). Chainlength and presence of unsaturated fatty acid esterswere confirmed by hydrogenation. Victoreen model4000 and Packard gas chromatographs with flameionization detectors were used. Aluminum or glasscolumns (1.8 to 3 meters long, 0.32-cm outer diam-eter) packed with 15% ethylene glycol succinateplus 2% phosphoric acid on Gas Chrom P (80 to 100mesh) were used isothermally at 180 C. Reed anddigital logarithmic electrometers (Victoreen Corp.,Cleveland, Ohio) were also employed.

Authentic compounds. TLC and gas-liquid chroma-tography standards were obtained from the LipidsPreparation Laboratory of The Hormel Institute,University of Minnesota, Austin. Phospholipidstandards were prepared by silicic acid column chro-matography, by TLC, and by enzymatic hydrolysisfrom beef heart. These compounds were identified bytwo-dimensional TLC and infrared spectrophotom-etry.

RESULTS

Comparison of lipid composition. Table 1illustrates the results of quantitative lipid dataobtained from analysis of calf serum, MK-2cells cultivated in MEM plus 5% calf serum,MK-2 cells cultivated in Waymouth mediumsupplemented with 20 ,ug of sodium oleate per ml,strain 6BC grown in Waymouth MK-2 cells,and strain 6BC-infected MK-2 debris. Per-centages of extractable total lipid were found tobe 29.1 to 35.4% in strain 6BC and 44.7 to 50.1%in strain 6BC-infected MK-2 debris, whereasuninfected MK-2 cells contained 14.8% (inMEM plus 5% calf serum) and 9.1% (in Way-mouth medium). Calf serum contained 4.5%total lipid.The proportion of neutral lipid (18.6 to 25%),

phospholipid (75 to 85%), and glycolipid (7 to8%) was relatively constant for strain 6BC andfor uninfected MK-2 cells cultivated in eithermedium. Calf serum had almost equal amounts ofneutral lipid and phospholipid (Table 2).The ratio of sterol esters to free sterols was

greater than one only in calf serum (Table 3).A higher proportion of triglycerides was

observed in MK-2 cells cultivated in Waymouthmedium supplemented with sodium oleate thanin cells cultivated in MEM.

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TABLE 1. Lipid content of calf serum, MK-2 cells, and Chlamydia psittaci strain 6BC

Prepn Total vol, no. of cells, Total extractable Total lipidor infectivity Total dry wt (mg) lipid (mg) (% of dry weight)

Calf serum ...................... 50 ml 3,787.5 170.3 4.5MK-2 cells in MEM-5% calf

serum ....................... 109.1 cells 516.3 76.3 14.8MK-2 cells in Waymouth medium-

sodium oleate (20 pg/ml). 108.6 cells 1,908 .8 174.4 9.1Expt 1

Strain 6BC .................... 1010 7 ELD50a 35.5 12.6 35.4InfectedMK-2 debris 111.9 56.1 50.1

Expt 2Strain6BC .................. 10'0*5 ELD50 17.2 5.0 29.1Infected MK-2 debris 58.4 26.1 44.7

a Egg LD50.

Quantitation of individual phospholipids bymeans of a Cahn balance was employed for thesucceeding experiments because the phosphorusassay and dry weight measurements in calf serumwere equivalent (Table 4). The major componentsof phosphatides of strain 6BC were phosphatidylethanolamine (31.1 %), phosphatidyl choline(23.8 %), and phosphatidyl glycerol (1 5.0%).Other components found were sphingomyelin(9.1 %), cardiolipin (6.9%), phosphatidyl serine(6.9%), and phosphatidyl inositol (6.7%). Incontrast, calf serum contained a high concentra-tion of phosphatidyl choline (57.9%) and a lowconcentration of phosphatidyl ethanolamine(4.7%). MK-2 cells cultivated in MEM andWaymouth media contained 36 and 53% ofphosphatidyl choline and 24.9 and 27.4% ofphosphatidyl ethanolamine, respectively. Thephosphatidyl choline was present in the highestconcentration (36 to 53 %). Phosphatidyl glycerolwas not detectable in cells cultivated in eithersystem, but was a major component (15%,) instrain 6BC.Fatty acid composition. The fatty acid patterns

between the host controls and the organismshowed marked differences. Strain 6BC con-tained about 20% branched-chain fatty acids inthe total lipid fraction, whereas host cells had

TABLE 2. Comparison ofneutral lipid, phospholipid,and glycolipid

Percentage of total lipid

PrepnNeutral Phos- Gly-

lipid pholipid colipid

Calf serum............. 52.5 47.5MK-2 cells in MEM-

calf serum........... 24.1 68.2 7.7MK-2 cells in Way-mouth medium-so-dium oleate (20 pg/ml) 26.7 66.2 7.1

Strain 6BC............. 18.6 74.3 7.1

only a trace, if any, present (Table 5). Analysisof individual lipid classes of strain 6BC showedthe presence of carbon number 14 to 21 saturatedbranched-chain fatty acids, some of which werenot readily observed in the total lipid (Tables6-9). Infected MK-2 debris also containedbranched-chain fatty acids (4.7% in total lipid).Odd-numbered fatty acids contained anteiso-branching, whereas even-numbered fatty acidscontained iso-branching fatty acids (Tables 5-9).The total phospholipids of strain 6BC containeda high percentage of carbon number 15 (C15 :0)

TABLE 3. Neutral lipid patterns of calf serum, MK-2 cells, and strain 6BC

Percentage of total neutral lipidSource

Sterol ester Free sterol Free fatty Triglyceride Diglyceride Mono-acid e glyceride

Calf serum.6...........................0. 17.8 9.1 4.3 7.4 1.5MK-2 cells inMEM-5% calfserum 10.1 55.5 4.2 19.2 5.4 4.9MK-2 cells in Waymouth medium-sodium oleate (20 pg/ml)........... 9.8 41.8 2.6 28.0 9.1 8.6

Strain 6BC. 13.7 36.1 21.4 25.5 3.2

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TABLE 4. Phospholipid patterns of calf serum, MK-2 cells, and strain 6BC

Percentage of total phospholipidaSource

Lyso PC Sphingomyelin PC PS PI PE PG CL

Calf serum .............. 8.2 (9.5) 26.5 (28.8) 57.9 (55.8) - Trb (Tr) 4.7 (3.8) 2.6 (1.8) -

MK-2 cells in MEM-5%calf serum............. - 21.4 36.1 2.5 7.2 24.9 7.9

MK-2 cells in Waymouthmedium-sodium oleate(20 pg/ml).....-....... . 11.0 53.9 0.3 0.8 27.4 - 6.8

Strain6BC.- 9.1 23.8 6.9 6.7 31.1 15.0 6.9

a Phosphorus assay data are given in parentheses. PC = phosphatidyl choline; PS = phosphatidylserine; PI = phosphatidyl inositol; PE = phosphatidyl ethanolamine; PG = phosphatidyl glycerol;CL = cardiolipin.

b Trace.

TABLE 5. Fatty acid anialysis of total lipids ofstrain6BC and MK-2 cell culture controls

TABLE 6. Fatty acid analysis of neutral lipids ofstrain 6BC and MK-2 cell culture controls

Fatty acid'

14:015:0 ba15:016:0 bi16:016:117:0 ba17:018:0 bi18:018:119:0 ba18:220:0 bi20:018:320:120:220:4

All saturated fatty acidsabove 21

All unsaturated fattyacids above 21

Percentage of total acidsb

Calfserum

0.5Tr0.4Tr11.7

1 .9

Tr

13.112.9

40.2

0.8

2.2

9.04.3

2.3

MK-2 cellsc

MEM Way-mouth

1.2

Tr1.0

14.15.8

0.5

15.928.2

13.5

Tr1.6

10.20.9

4.4

1.4Tr0.7

9.53.2

4.00.411.157.0

5.6

Tr2.7

Tr3.1

2.9

Strain6BC

0.69.9

2.813.7

3.9Tr0.514.819.8

.3

5.61.11.5

6.9Tr2.23.4

4.5

In-fectedMK-2debris

1.32.4

1.419.12.90.92.2

11.436.8

4.0

0.44.4

2.02.12.0

1.0

a Number to the left of the colon refers to number of carbonatoms; number to the right refers to number of double bonds;ba = anteiso branched-chain acid; bi = iso branched-chainacid.

bTr = less than 0.3%.c MK-2 cells in MEM with 5% calf serum and in Waymouth

medium-sodium oleate (20 pg/ml).

anteiso branched-chain fatty acid (13 ,Xj), whichwas found mainly in phosphatidyl ethanolamine(23%) and phosphatidyl glycerol (24%o; Tables7 and 9). Neutral lipid contained more carbonnumber 17 (C17 :0) anteiso branched-chain

Fatty acida

14:015:0 ba15:016:0 bi16:016:117:0 ba17:018:0 bi17:118:018:119:0 ba18:220:0 bi20:018:320:120:220:4




Calfserum

1.00.40.7Tr10.72.3

1.6

6.212.4

51.5

1.2

3.0

6.01.4

Tr

MK-2 cells'

MEM Way-mouth

1.8 1.7Tr 0.40.7 0.40.5 0.716.2 13.65.4 3.9

Tr 0.4Tr

1.4 -

13.6 8.724.1 54.4

7.7 2.4

0.7 0.50.6 1.4

0.48.9 1.11.3 2.8

7.8 4.7

Strain6BC

1.21.50.71.8

17.2Tr6.21.50.7

18.515.41.36.12.6Tr

6.5

0.53.6

3.5

In-fectedMK-2debris

2.90.91.01.4

21.14.51.30.80.50.612.335.8

3.5

0.61.4

5.11.02.3

3.1


b Tr = less than 0.3%.c MK-2 cells in MEM with 5% calf serum and in Way-

mouth medium-sodium oleate (20 jig/ml).

fatty acids than phospholipid and practically no

C15:0 anteiso fatty acids (Tables 6-9).The proportion of polyunsaturated fatty acids

was higher in calf serum (.49.2%) and in MK-2

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cells cultivated in MEM-5% calf serum ( ' 25.3 %)than in MK-2 cells cultivated in Waymouthmedium with sodium oleate (_11.4%; Table 5).Strain 6BC grown in MK-2 cells in Waymouthmedium and the cell debris infected with thisorganism contained >7.8% and .10.5% ofpolyunsaturated fatty acids each (Table 5).MK-2 cells in Waymouth medium supple-

mented with sodium oleate were found to havea high C18:1 percentage (57.0% in total lipid).The highest concentration of C18 :1 (62.0%)was found in the triglyceride fraction of Way-mouth-grown cells (Table 8). Strain 6BC grownin these cells, however, contained only 19.8%C18:1 in total lipid (Table 5).

DISCUSSION

For the purpose of chemical characterizationof intracellular microorganisms, it is essentialto try to establish as chemically defined anenvironment as possible in which to grow theorganisms. The present study deals with lipidanalysis of strain 6BC grown in MK-2 cellscultivated in the defined medium of Waymouthand of the related host materials which aredirectly or indirectly essential for growth ofstrain 6BC. These materials include MK-2 cellscultivated in MEM with 5% calf serum, fromwhich MK-2 cells cultivated in Waymouthmedium with sodium oleate were derived, as wellas calf serum itself.There are two points of comparison to be made:

(i) the difference between the organism and hostmaterials and (ii) the difference in lipid composi-tion of MK-2 cells cultivated in two media, eachassociated in a phase of cell growth beforeinfection.The high percentage of total lipid in strain

6BC is probably a reflection of the high lipidcontent of the MK-2 cells after infection withthis organism. This speculation may be sup-ported by the finding of a lower concentration oflipid in the same organism grown in the CAM ofchick embryos (Jenkin et al., unpublished data).However, how the lipid changes occur in suchinfected cells is unknown.

High concentrations of C15 :0 anteisobranched-chain fatty acids are found mainly inphosphatidyl glycerol and phosphatidyl ethanol-amine. The C17:0 anteiso branched-chain fattyacid is found almost entirely in neutral lipidclasses. These unusual fatty acids are char-acteristic of the organism and are not found inthe normal host. For the first time, an identifyingchemical marker is available, suggesting thatlipid metabolism of this obligate intracellularbacterium is different from that of the host.

TABLE 7. Fatty acid analysis of phospholipids ofstrain 6BC and MK-2 cell culture controls

Fatty acida

14:015:0 ba16:0 bi16:016:117:0 ba17:018:0 bi18:018:119:0 ba18:220:018:320:120:220:4




Calfserum

16.51.1

0.9

26.315.4

14.0

11.9

8.64.9

Tr

MK-2 cellsc

MEM Way-mouth

1.1

2.214.13.9

0.6

15.024.3

12.70.40.9

8.31.6

5.0

1.3Tr0.512.73.1

0.5Tr11.945.2

5.1Tr1.8

3.40.7

5.4

Strain6BC

Tr12.32.4

20.9

6.10.40.716.925.50.72.21.4

1.2

1.40.7

1.8

In-fectedMK-2debris

1.42.71.9

15.94.60.90.40.714.434.1

3.70.81.6

2.32.24.3

Tr


bTr = less than 0.3%.MK-2 cells in MEM with 5% calf serum and in Waymouth

medium-sodium oleate (20 ,ug/ml).

The necessity to examine individual lipidclasses or species becomes apparent when thetotal lipid, total neutral lipid, and total polarlipid fractions show minor differences betweenexperimental and control material. Only whenthe fatty acids of isolated lipids are analyzed dohost and parasite differences become magnified;i.e., the presence of branched-chain fatty acids isreadily detectable.The supernatant fluid derived from infected

cell culture is probably the cleanest fraction touse, because this material contains the leastcontamination from cell debris. Sonic treatmentand repeated washing of infected cells withNH4Ac may not remove all of the cell-associatedorganisms. The fraction designated infectedMK-2 debris is not entirely free from strain 6BC.Small amounts of branched-chain fatty acidsfound in the infected cell debris are most likelyassociated with the contaminants from theorganism remaining in the infected host cells.Another difference found between strain 6BC

and the host is the higher ratio of phosphatidylethanolamine to phosphatidyl choline in the

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68 MAKINO ET AL. J. BACTERIOL.

TABLE 8. Fatty acid analysis of individual neutral lipid classes

Sterol ester Free fatty acid Triglyceride

Fatty acid'

Calf MK-2 MK-2 Strain Calf MK-2 Strain Calf MK-2 MK-2 Strainserum MEMb moWuthC 6BC serum Wauyth 6BC serum MEM Way- 6BC

14:0 0.6d 1.8 2.3 4.4 1.7 1.4 1.8 1.6 0.5 1.2 2.915:0 ba 0.6 - 1.1 2.7 0.4 Tr 0.6 Tr - Tr 1.115:0 0.5 0.4 0.5 2.4 0.6 Tr 0.9 1.2 - Tr 1.616:0 bi Tr 0.4 0.5 2.1 Tr 1.2 1.6 0.4 1.1 Tr 1.216:0 5.6 14.0 15.2 20.9 18.6 12.8 35.9 27.8 9.5 7.5 23.316:1 2.2 6.1 5.4 Tr 3.7 4.2 Tr 3.1 4.5 3.3 -17:0 ba 11.8 _ 7.3 - 7.817:0 Tr 1.0 0.4 0.8 1.2 0.6 2.3 0.8 1.0 Tr 0.618:0 bi - 1.6 - 1.5 1.1 - - - 1.117:1 - 0.8 0.7 - - 0.6 1.9 Tr18:0 0.6 9.6 7.0 9.4 23.1 11.9 20.5 19.8 14.0 6.2 13.218:1 6.2 27.1 41.3 13.2 28.4 32.9 17.3 26.1 21.3 62.0 37.819:0 - 1.5 Tr 1.3 - 0.8 - Tr Tr 0.418:2 66.4 11.3 2.2 5.3 11.9 6.6 0.9 8.6 2.1 2.1 1.720:0 2.0 3.1 0.7 1.1 - 0.5 0.9 0.5 1.7 Tr 0.718:3 - - 2.7 - - - 4.7 3.620:1 3.9 - - 2.0 1.3 1.9 0.8 1.4 1.920:2 - - - - - 0.4 - 2.2 -

20:4 7.3 2.5 2.0 - 6.1 4.6 0.4 3.1 2.4 1.4 TrAll saturated fatty acids 1.4 2.5 3.9 4.6 2.1 3.9 1.8 1.1 4.3 2.2 2.1above 21

All unsaturated fatty acids Tr 14.9 10.4 7.2 Tr 8.5 2.8 1.6 17.4 5.8 1.4above 21

Number to the left of the colon refers to number of carbon atoms; number to the right refers tonumber of double bonds; ba = anteiso branched-chain acid; bi = iso branched-chain acid.

b MK-2 cells in MEM with 5% calf serum.c MK-2 cells in Waymouth medium-sodium oleate (20 ,ug/ml).d Numbers show the percentage of total acids; Tr = less than 0.3%.

organism (Table 4). The high ratio is not peculiarto strain 6BC grown in MK-2 cells, becausesimilar data were obtained for this organismgrown in CAM (Jenkin et al., unpublished data).Phosphatidyl glycerol is one of the lipids uniquelyabsent in the host, but is a major component ofstrain 6BC (Table 4). This observation is anotherexample of the capability of independent lipidmetabolism of chlamydiae.

Increase of triglyceride from MK-2 cellscultivated in MEM to MK-2 cells cultivated inWaymouth medium (19.2% to 28.0%) is ofinterest, because the percentage of C18:1 is thehighest in triglyceride of Waymouth MK-2 cells(62.0%). Oleic acid may have a stimulatory effecton triglyceride synthesis in this system.

Polyunsaturated fatty acids found in MK-2cells grown in MEM with 5% calf serum areprobably derived from calf serum because theproportion of these fatty acids in calf serum ismarkedly high.

Qualitative and quantitative data comparing

differences in the lipid components between hostand parasite are available. The quantitative datamay now be used for taxonomic purposes indistinguishing various strains of chlamydiae.Further studies of lipid metabolism can bepursued by using isotopic tracer methods tofollow the synthesis and catabolism of char-acteristic lipids.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Serviceresearch grant HE 08214 from the Program Projects Branch Ex-tramural Programs, NIH, by grant A107747 from the NationalInstitute of Allergy and Infectious Diseases, and by The HormelFoundation.We thank L. Anderson for excellent technical assistance and T.

Madson and J. Chipault for performing and interpreting theinfrared spectrophotographs.

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LIPID COMPOSITION OF CHLAMYDIA PSITTACI 69

TABLE 9. Fatty acid analysis of individual phospholipid classes

Phosphatidyl ethanolamine Phosphatidyl choline Sphingomyelin Phos-phati-

Fatty acida -dyligly-Calf MK-2 MK-2 Strain Calf MK 2 MK-2 Strain Calf MK-2 MK-2 Strain cerol,serum MEMb mWay> 6B C serum MEM Way- 6BC serum MEM Way- B strainmouthc ~~~mouth mouth BC 6BC

14:0 bi - Tr 1.1 Tr Tr Tr 1.414:0 0.7d 0.4 1.2 2.2 Tr 1.6 1.4 1.8 Tr 2.0 3.6 3.4 2.315:0 ba - Tr - 23.7 _ Tr - 0.6 - Tr Tr 0.4 24.415:0 - Tr 0.6 Tr Tr 0.4 Tr 0.4 0.8 -16:0 bi 1.4 2.6 1.6 4.8 - Tr Tr 2.2 - Tr Tr 0.7 2.416:0 4.7 7.3 6.7 13.0 14.8 18.2 13.7 19.7 15.4 20.2 23.1 28.8 16.416:1 1.8 1.4 3.9 - 1.2 6.4 4.3 - 1.1 2.5 3.6 - 0.817:0 ba - 2.3 _ 6.0 - 2.7 8.617:0 Tr Tr - 0.7 Tr - 1.2 0.6 1.418:0 bi 1.7 0.9 - 0.8 1.418:0 2.9 16.3 10.6 12.2 25.0 12.2 10.4 13.3 25.7 16.7 9.7 22.1 12.018:1 3.8 27.6 37.0 24.1 17.0 33.4 51.6 37.4 17.7 19.3 24.8 11.8 11.919:0 0.9 Tr 0.4 1.0 0.4 Tr18:2 50.3 13.6 8.5 3.4 14.7 14.4 5.3 4.5 15.2 6.0 1.7 1.4 8.020:0 bi - 1.2 - _ -20:0 - Tr Tr 2.4 - Tr Tr 0.9 - 0.4 0.4 0.9 2.018:3 Tr 4.1 0.7 0.7 2.2 - 0.9 - - 0.4 -20:1 0.5 1.0 0.6 1.0 1.420:2 _ Tr 0.3 _ 0.6 0.4 0.4 0.2 - 1.0 Tr Tr20:4 20.9 17.2 11.7 1.7 10.6 2.6 2.3 11.2 5.5 1.5 0.4 2.2

All saturated 1.9 1.2 1.7 1.7 7.7 2.3 1.4 1.9 6.8 13.2 3.7 10.0 1.1fatty acidsabove 21

All unsatu- 9.3 3.2 1.9 1.6 5.3 3.1 2.9 1.4 4.6 9.2 12.9 10.3 1.2rated fattyacids above21

a Number to the left of the colon refers to number of carbon atoms; number to the right refers tonumber of double bonds; ba = anteiso branched-chain acid; bi = iso branched-chain acid.

b MK-2 cells in MEM with 5% calf serum.G MK-2 cells in Waymouth medium-sodium oleate (20 /Ag/ml).d Numbers show the percentage of total acids; Tr = less than 0.3%.

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lipid composition of in monkey kidney cells in defined · unpublished data). lipid components of...

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