Vol. 160, No. 3

Biochemical and Immunological Properties of Coxiella burnetii CellWall and Peptidoglycan-Protein Complex Fractions

KEN-ICHI AMANO,'t JIM C. WILLIAMS,lt* THOMAS F. MCCAUL,1§ AND MARIUS G. PEACOCK2Laboratory of Microbial Structure and Function' and Epidemiology Branch,2 Rocky Mountain Laboratories, National

Institute of Allergy and Infectious Diseases, Hamilton, Montana 59480

Received 29 June 1984/Accepted 18 September 1984

Coxiella burnetii morphological cell types were fractionated into large-cell variant cell walls, two fractions ofsmall-cell variant cell walls, and one fraction of small-cell variant whole cells. Based on the contents ofpeptidoglycan (PG)-constituents and the yields of the sodium dodecyl sulfate-insoluble PG-protein complex(PG-PC) from cell walls, the fraction of large-cell variant cell walls contained significantly less PG than did thefraction of small-cell variant cell walls. The yields of PG-PC from the fractions of large-cell variant cell wallsand small-cell variant cell walls were 2 and 32%, respectively. These results indicated that the PG of the large-cell variant cell walls may be partially digested by PG-lytic enzymes or incompletely synthesized, whereas thesmall-cell variant cell walls appeared to have intact PG. Proteins associated with PG-PC were resistant toproteolysis by trypsin, protease VI, and proteinase K. Saturated and unsaturated fatty acids were detected inwhole cells and cell walls but not in PG-PC, which contained a 3-deoxy-D-mannooctulosonic acid-likecomponent that is also present in phase I lipopolysaccharide. Immunogenicity of the fractions was tested bymeasuring the temporal sequence of phase II and phase I antibody responses in vaccinated rabbits. Both phaseII and phase I antibody responses were demonstrated with all fractions except the sodium dodecyl sulfatesupernatant of the small-cell variant cell walls, whereas PG-PC elicited a pure phase II antibody response up to29 days postvaccination. The immunogenicity of these fractions may reflect a quantitative difference in antigenconcentration or may be due to a qualitative difference in phase II and I determinants.

Coxiella burnetii, the etiological agent ofQ fever, has beenreported to exist in a number of morphologically variantforms. The pleomorphic nature of C. burnetii was firstdescribed by Davis and Cox (9) and Cox (8), who observedby light microscopy minute coccoid and granular forms,bacillary forms, and filter-passing particles. In 1972, Wiebeet al. (24) speculated that two cell variants, which could beseparated with CsC12, sucrose, or isopycnic Renografingradients, represented two stages in a complex developmen-tal cell cycle similar to that of Chlamydia psittaci. Theseauthors further speculated that partial degradation of thesmall-cell variants (SCVs) by lysozomal enzymes of hostcells rendered them large-cell variants (LCVs). Recently,McCaul and Williams (15) studied the morphological hetero-geneity of the cell variants and discovered a dense body inthe periplasmic space of LCVs. Thus, a putative develop-ment cycle was proposed to include cellular differentiation ofC. burnetii into endospore, SCV, and LCV cell types.

In the present study, whole cells of C. burnetii wereseparated into LCV cell walls (LCW-CW) two fractions ofSCV cell walls (SCV-CW), and one fraction of SCV wholecells (SCV-WC). Some chemical and immunological proper-ties of the cell variants were studied while focusing on theamount of peptidoglycan (PG) associated with SCV andLCV cell walls. PG-protein complex (PG-PC) of C. burnetiiwas successfully purified from SCV-CW but not from LCV-

* Corresponding author.t Present address: Department of Bacteriology, Hirosaki Univer-

sity School of Medicine, Hirosaki, Aomori 036, Japan.t Present address: Rickettsial Diseases Laboratory, Aerobiology

Division, U.S. Army Medical Research Institute of InfectiousDiseases, Fort Detrick, Frederick, MD 21701.

§ Present address: Department of Medical Microbiology, LondonSchool of Hygiene and Tropical Medicine, London WC1 7HT,United Kingdom.

CW. Most of the PG-associated proteins were tightly boundto the PG, and these proteins were resistant to proteolysis.The temporal sequence of phase II and phase I antibodyresponses in rabbits vaccinated with various fractions indi-cated that only the PG-PC elicited a pure phase II antibodyresponse up to 29 days postvaccination. Our studies suggestthat special features of the PG-PC may be important bio-chemical determiners of immunogenicity, resistance to envi-ronmental conditions, and intracellular digestion of C. bur-netii by eucaryotic cells.

MATERIALS AND METHODS

Organism and growth conditions. The cultivation andseparation of phase I C. burnetii (Ohio strain [CBOI]) fromhost components were previously described (26).

Subfractions of C. burnetii. C. burnetii cell variants wereseparated according to susceptibility and resistance to os-motic shock, ultrasonication, and pressure treatment fol-lowed by low- and high-speed centrifugations. Purified C.burnetii variants were stored at -20°C in phosphate-bufferedsaline-sucrose (26). The cells were thawed at room tempera-ture, and subfractions were prepared as follows. Cells weresedimented at 16,300 x g for 30 min. The supernatant wasdiscarded, and the pellet was suspended in 800 ml of water at45°C with shaking at 250 rpm for 2 h. Cells were sedimentedat 16,300 x g for 30 min, and the supernatant was saved. Thecell pellet was washed three times with water, and thesupernatants were pooled. Pooled supernatants were used toprepare LCV-CW (Fig. 1A and 2A). The cell pellet was usedto prepare SCV-CW (Fig. 1B and C and Fig. 2C and E).Electron microscopy was used by methods described previ-ously (14, 15) to check on the fractionation procedure.

Preparation of PG-PC. PG-PC preparation was performed

982

JOURNAL OF BACTERIOLOGY, Dec. 1984, p. 982-9880021-9193/84/120982-07$02.00/0Copyright C) 1984, American Society for Microbiology

C. BURNETII FRACTIONS 983

B

I Pellet. WC

suspended in 50% sucrose for24 h at 4 C.10,400 x g, 2 h

I

A

c

R p9 wc1Ribi cell fractionator, 10,400 x g, 20 min.

r ~~~~~~~~~~~I5 (Discard) P6

(Osmotic shock)10,400 x g 20 min.

s10 p9A(wash 1 X)

(wash 1 X) 1 p9B| S8 I7 DNase, RNase

pP2 (Sonic disruption)

10.400 x g, 20 min.DNase, RNase(wash 1X) S 10,1 12 9C

S7P3p8wash 2 x) 82.500 Is 9, 1 h (Discard)

53 P3 (wash 2SX) p.(wash 2X)

l I -'19S4 S8 | Pg WC 13

20-45% RGG (see Fig. tC) D,L DNase, RNase70,000 x g, 2 h (wash 1X)

collect bands1-4 (wash 2X) S6-8

D,L 82,500 x g, 1 h 82,500 X g, I h

I i 1 ~~ ~~~~~~~~~~~~~~~~~1498Ep5 S p10 (Discard)

D.L DNase, RNase(wash 1 x)82,500 x 9. I h

LCV-S,mg LCV-CW, 168 mg I-SCV- S 154 | mg | s,8S mg scv-cw,97 mg

FIG. 1. Flow diagram for the subfractionation of C. burnetii phase I (Ohio strain). (A) Pooled supernatants (S,,) obtained from lysed LCV;(B) pellet (Pj) obtained from whole-cell fractions; (C) P9, whole cells used to prepare SCV-CW and resistant cells (see the text). S,Supernatant; CW, cell wall; wash 1 x, particulates were washed at 20°C in water at 82,500 x g for 1 h; DNase and RNase treatments were in 10mM Tris buffer (pH 7.4)-20 mM MgCl2 at 37°C for 1.5 h, using 0.2 mg of the enzymes of ml; RGG, 20 to 45% Renografin gradient (26); D, dial-ysis against water, using dialysis tubing with a molecular weight restriction of 5,000; L, lyophilization. Osmotic shock was carried out in 1.6 li-ter of water with stirring at room temperature for 2 h. Sonic disruption was carried out in a sealed atmospheric treatment chamber with acooling jacket at 4°C, using a pulse of the 25% duty cycle for 45 min at no. 9 output. The Ribi cell fractionator (model RF-1) was used to disruptcells at 50,000 lb/in2 (14).

essentially as described for E. coli by Braun and Rehn (5).Cell walls prepared from LCV and either or both SCVfractions were suspended in 4% sodium dodecyl sulfate(SDS) at 1 mg (dry weight)/ml, boiled for 1 h, and cooled toroom temperature. This suspension was cooled to 20°C andcentrifuged at 100,000 x g for 1 h. The PG-PC pellet waswashed six times at 20°C with sterile, demineralized distilledwater. All of the supernatants were pooled, dialyzed against1-liter volumes of distilled water for 7 days, and lyophilized.The pellets (20 mg [dry weight]) were incubated with 20 ml of100 mM Tris buffer (pH 7.2) containing 10 mM MgCl2, 1 mgof DNase I (Sigma Chemical Co., St. Louis, Mo.) and 2 mgof RNase A (Sigma) at 370C overnight. Four milligrams oftrypsin (Sigma) was added, and incubation was performed at37°C for 8 h. Four milligrams of protease VI (Sigma) wasadded, and the mixture was further incubated for 16 h at370C. The mixtures were centrifuged at 100,000 x g for 1 h(20°C) and washed twice with 20-ml volumes of distilledwater. Precipitates were dissolved in 50 ml of 4% SDS andboiled for 1 h. After cooling at room temperature, themixtures were centrifuged at 100,000 x g for 1 h and washedfive times with 50-ml volumes of distilled water. The PG-PCfrom SCV-CW was used in the following studies.

Proteolytic enzyme treatments of PG-PC. Samples (0.4 mg)were incubated at 370C with 50 Fg of trypsin, protease VI,and proteinase K (E. Merck AG, Darmstadt, West Germa-ny) as previously described (1, 2).

Analytical methods. Amino acids and amino sugars wereanalyzed in a Beckman 118-CL amino acid analyzer by using

1 mg of each sample hydrolyzed in 6 N HCl at 100°C for 15 hin sealed glass ampoules. Protein was determined by themethod of Lowry et al. (13) and amino acid analysis. Totalphosphorus was determined by the method of Lowry et al.(12). Neutral sugars were measured by the phenol-H2SO4method (9), with glucose as a reference standard. Reducinggroups were determined by the method of Park and Johnson(18) with glucose as a reference standard. 3-Deoxy-D-man-nooctulosonic acid (KDO)-like components were analyzedby the method of Osborn (3a, 17).

Fatty acids were analyzed as methyl esters in a Perkin-Elmer 900 gas chromatograph on a glass-GasChrom P col-umn (100 to 200 mesh) containing 10% ethylene glycolsuccinate (Applied Science Laboratories, Park Ridge, Ill.).One milligram of sample was hydrolyzed in 4 N HCl at 100°Cfor 4 h in sealed ampoules and then esterified by treatmentwith boron trichloride-methanol (Applied Science). Compo-nents were eluted with petroleum ether and identified bycomparison with the retention times of commercial fatty acidmethyl esters (Applied Science).

Test of immunogenicity. C. burnetii whole cells and sub-fractions were used to immunize New Zealand White rab-bits. Groups of two rabbits weighing 2 to 2.5 kg (June LaneRabbitry, Missoula, Mont.) were prebled and inoculatedsubcutaneously in the axillary nodal region with 100 ,ug ofantigenic preparations suspended in 1 ml of incompleteFreund adjuvant (Difco Laboratories, Detroit, Mich.). Rab-bits were injected intramuscularly on day 7 with 100 ,ug inthe right rear leg and again with 100 ig on day 27 in the left

VOL. 160, 1984

984 AMANO ET AL. J. BACTERIOL.

FIG. 2. Electron micrographs of C. burnetii fractions showing cell walls and whole cells. (A) LCV-CW, bar = 0.1 ,um; (B) whole cells(WC), bar = 0.5 ,um; (C) I-SCV-CW, bar = 0.1 ,um; (D) WC, bar = 0.5 ,um; (E) SCV-CW, bar = 0.1 pm; (F) SCV-WC, bar = 0.1 p.m.

rear leg. Sera collected on 0, 7, 15, 22, 29, 43, and 54 dayspostvaccination were evaluated for seroconversion by usingwhole-cell antigen (26) in a microagglutination test (11).

RESULTS

SUbfracti6ns of C. burnetii. We previously demonstratedthe presence of three morphological cell types, LCVs,SCVs, and endospores of C. burnetii and proposed a devel-opmental cycle consisting of vegetative and sporogenicdifferentiation (14, 15, 26). In the present study, C. burnetiic,lls were fractionated into four subfractions (Fig. 1): LCV-

CW, intermediate SCV-CW (I-SCV-CW), SCV-CW, andSCV-WC. The fractions were then compared for their chem-ical compositions and ultrastructures (Fig. 2A through F).LCVs were disrupted by osmotic shock in distilled water,and the mixture of cells and cell walls was separated bycentrifugation into two fractions, LCV-CW and unbrokencells (Fig. 2A and B). Based on the ultrastructure describedpreviously (14), the cell wall fraction coincided with the cellwall derived from LCVs. The unbroken cells were exposedto ultrasonication and separated into a cell wall fraction andunbroken cells (Fig. 2C and D). The I-SCV-CW revealedmultilayer membranes as well as the outer membrane struc-ture of the SCVs previously described (14). The undisrupted

f.

im ffA.. .;Ar ;, z. 1..15. ... 1,

Mvl.?-';

v

W.

m

NW

TABLE 1. Chemical analyses of C. burnetii whole cells and cell wall fractions

Dry wtTotal proteins (%)b by: Neutral Total KDO-like Total fatty

Fraction (mg)a Amino acid Lowry sugar phosphorus compoundc acids (%)analysis method (nmol/mg) (nmol/mg) (nmol/mg)

Whole cells 3,555 (100) 53 57 527 738 26 14LCV-CW 168 (5) 49 59 553 242 14 16I-SCV-CW 301 (9) 51 55 279 276 8 10SCV-CW 97 (3) 35 NDd 465 243 3 13SCV-WC 290 (8) 39 ND 456 669 15 16

a Percentages are shown within parentheses.b Milligrams percent (dry weight).Thiobarbituric acid positive (17).

d ND, Not determined.

whole cells were subjected to more drastic methods by using were distributed in all fractions, with the greatest amounts inthe Ribi cell fractionator, and subfractions were collected by whole cells, LCV-CW, and SCV-WC, whereas lessercentrifugation. In the supernatant (Fig. 2E), the cell wall amounts of the KDO-like components were found in I-SCV-fraction corresponded to the SCV-CW previously described CW and SCV-CW. Fatty acids were found in all fractions,(14), whereas SCV-WC was found in the pellets (Fig. 2F) ranging from 10 to 16%. Unbranched fatty acids ranged from(14, 15). 25 to 29%, whereas branched fatty acids were 66.4 to 72.9%Chemical compositions of subfractions. Fractionation of of the total fatty acids (Table 3).

whole cells into LCV-CW, I-SCV-CW, SCV-CW, and SCV- Characterization of PG-PC. An attempt was made to purifyWC resulted in the recovery of 5, 9, 3, and 8%, respectively, PG from LCV-CW and SCV-CW. The yield of the PG-PCof the initial dry weight (Table 1). Amino acid analysis of fraction purified from LCV-CW was only 2%, whereas theeach fraction indicated that whole cells, LCV-CW, and I- yield from SCV-CW was 32%. The PG-PC fraction fromSCV-CW contained roughly equivalent amounts of protein, SCV-CW contained 75% protein, 211 nmol of neutral sugarswhereas SCV-CW and SCV-WC contained less protein than per mg, 43 nmol of phosphorus per mg, and 3 nmol of KDO-did the first three fractions (Table 1). However, the amounts like compounds per mg, but no fatty acids were detected.and molar ratios of amino acids were different in all fractions Proteolytic enzyme treatment of the PG-PC during the(Table 2). A comparison of the meso-diaminopimelic acid purification procedure did not significantly reduce the con-and muramic acid contents of the fractions suggested that I- centration of amino acids (Table 4). Thus, PG-associatedSCV-CW and SCV-CW contained roughly 2.7-fold more PG proteins were resistant to solubilization with SDS and tothan did LCV-CW (Table 2). hydrolysis by proteolytic enzymes.

Neutral sugars, phosphate, and fatty acids were present in Antibody response to fractions in rabbits. Table 5 showsthe fractions (Table 1). Neutral sugar concentrations were the results of comparative serological tests performed onroughly equivalent in whole cells and LCV-CW, SCV-CW serial bleedings from rabbits inoculated with killed wholeand SCV-WC, but were lower in I-SCV-CW. Phosphorus cells, LCV-CW, SCV-CW, LCV-CW and SCV-CW SDSconcentrations were highest in whole cells but were roughly supernatants, PG-PC, and SCV-WC. Antibodies againstequivalent in the cell wall fractions. KDO-like components phase II and I whole cell antigens were detected on day 7,

TABLE 2. Amino acid and amino sugar compositions of C. burnetii whole cells and cell wall fractions

Amino acid or amino Amino acid molar ratioa of fraction:sugar Whole cells LCV-CW I-SCV-CW SCV-CW SCV-WC

Aspartic Acid 11.0 12.2 4.0 3.5 5.5Threonine 6.3 8.6 2.8 2.5 3.0Serine 7.3 8.6 2.9 2.6 3.4Muramic acid 0.8 0.9 0.8 1.1 0.9Glutamic acid 13.5 8.3 4.0 3.3 6.0Proline 6.5 6.6 1.8 2.1 3.2Glycine 10.3 14.0 4.2 3.3 4.5Alanine 11.9 12.7 5.2 4.9 5.9Valine 5.7 7.8 2.7 2.0 2.7Methionine 2.3 3.2 2.2 0.7 0.9Diaminopimelic acid 1.0b 1.0 1.0 1.0 1.0Isoleucine 3.7 4.6 1.5 1.2 1.6Leucine 8.3 9.7 2.9 2.5 3.3Tyrosine 1.5 7.1 1.9 1.6 1.7Phenylalanine 3.5 5.2 1.5 1.9 2.2Glucosamine 1.6 2.6 0.8 1.0 1.0Histidine 2.1 2.4 1.1 0.9 1.1Lysine 6.4 5.0 2.3 2.2 3.1Arginine 6.7 5.1 2.4 2.1 3.5

a Molar ratio = amino compound/diaminopimelic acid.b Amounts of meso-diaminopimelic acid were 35, 29, 78, 72, and 58 nmol/mg (dry weight) in whole cells, LCV-CW, I-SCV-CW, SCV-CW, and SCV-WC,

respectively.

C. BURNETII FRACTIONS 985VOL. 160, 1984

986 AMANO ET AL.

TABLE 3. Fatty acid compositions of C. burnetii whole cells and cell walls

Fatty acid content of fraction':Fatty acida

Whole cells LCV-CW I-SCV-CW SCV-CW SCV-WC

UnbranchedC14 1.9 1.1 1.4 1.9 2.0C16 9.8 10.1 9.9 7.7 7.6C17 2.7 Tr Tr 2.6 2.7C18 8.2 10.8 9.0 7.0 6.7C19 1.4 Tr Tr 1.4 1.5C20 4.3 7.0 5.2 4.7 4.5

BranchedIso-C,4 3.7 1.3 2.2 3.2 3.7Anteiso-C15 34.7 30.0 35.0 34.5 34.6Iso-C16 6.5 7.7 6.9 5.7 5.5Anteiso-C17 25.1 27.4 24.3 29.5 28.3

a C15, C16:1, and C18:1 were detected in only trace amounts. The amount of hydroxy fatty acids was not measured.bPercentage of total fatty acid.

and on all remaining test dates when whole cells, LCV-CW,SCV-CW, and SCV-WC were the immunizing antigens.Antibodies against phase II and I antigens were not detectedwhen SCV-CW SDS supernatants was the immunizing anti-gen. Interestingly, the LCV-CW SDS supernatant and PG-PC immunizing antigens elicited similar antibody responses,

with the former showing delayed anti-phase II (day 15) and I(day 22) responses, whereas the latter elicited an early (day7) anti-phase II response and late (day 43) anti-phase Iresponse. These sera also were positive for membranefluorescence, as determined with viable phase I and phase IIC. burnetii (data not shown), thus suggesting that the frac-tions contained immunogenic membrane determinants.

DISCUSSIONIn this paper, we reported some chemical compositions

and immunogenic activities of C. burnetii whole cells andsubfractions produced by osmotic shock, sonication, andpressure disruption. This study presented evidence that C.burnetii whole cells and cell walls derived from LCVs andSCVs contained similar chemical compositions and immuno-genic activities. The morphological integrity of the LCVswas apparently compromised by incomplete PG cross-link-ing since only 2% of the PG was purified by extraction ofLCV-CW with hot SDS and ultracentrifugation. In contrast,32% of the SCV-CW PG was recovered by using the same

procedures. The PG of LCVs apparently exists primarily as

soluble components and may account, in part, for theosmotic sensitivity and pleomorphic morphology of theLCVs (7, 14, 15, 24). In another study (15) addressing theultrastructure and morphological heterogeneity of C. burne-tii cell variants, a developmental cycle composed of LCVs,SCVs, and endospore formation was studied. Wiebe et al.(24) proposed that the large round cell types of C. burnetiiwere derived from degenerated rod-shaped cells by host-mediated digestion of the rigid PG layer. A later putativedevelopmental cycle suggested that the SCVs are producedfrom endospores which arise from autolysis of LCVs duringsporogenic differentiation (15). In the present study, we didnot resolve whether PG-lytic enzymes were autolytic or

lysozomal (i.e., lysozymes of host cells). The possibilityexists that either or both of these developmental cyclesaccount for the pleomorphic nature of C. burnetii cell types.The whole cells and cell wall fractions contained protein,

neutral sugar, phosphate, fatty acids, and a KDO-like com-

ponent (Table 1). Analysis of amino acid compositions of thevarious fractions indicated that whole cells and LCV-CWhad similar molar ratios, which were clearly different fromthose of I-SCV-CW, SCV-CW, and SCV-WC (Table 2).Amino acid molar ratios of the PG-PC (Table 4) weremarkedly different from those of the cell wall fractions;however, the amino acid and amino compound ratios of PGcomponents, namely, glutamic acid, alanine, diaminopimelicacid, muramic acid, and glucosamine, were very similar. Thefatty acid contents of the whole cells and cell wall fractionswere similar (Tabfes 1 and 2); however, no fatty acids weredetected in the PG-PC fraction. Our results confirm andextend the studies of Tzianabos et al. (23) of the fatty acidcomposition of C. burnetti. Saturated (C15) and unsaturated(C16:1) and C18:1) fatty acids were detected in only traceamounts. Saturated unbranched fatty acids ranged from 25

TABLE 4. Amino acid and amino sugar compositions of PG-PCof C. burnetii

PG-PC treatmenta:

Amino acid or amino With proteases Without proteasessugar Molar Molar

nmol/mg ratio nmol/mg ratio

Aspartic acid 519 1.63 590 2.41Threonine 349 1.09 369 1.51Serine 402 1.26 398 1.65Muramic acid 287 0.90 233 0.95Glutamic acid 492 1.54 369 1.50Proline 198 0.62 219 0.90Glycine 609 1.90 606 2.48Alanine 811 2.53 741 3.03Methionine NDb ND ND NDValine 418 1.31 389 1.59Diaminopimelic acid 320 1.00 245 1.00Isoleucine 112 0.35 210 0.86Leucine 344 1.08 369 1.51Tyrosine 258 0.81 261 1.07Phenylalanine 182 0.57 134 0.55Glucosamine 300 0.94 216 0.88Histidine 87 0.27 89 0.36Lysine 126 0.39 168 0.69Arginine 81 0.25 164 0.67

a During the purification procedure of PG-PC, trypsin and protease VI wereused to degrade protein in only one sample.

b ND, Not detected.

J. BACTERIOL.

C. BURNETII FRACTIONS 987

TABLE 5. Reciprocal microagglutination titers of antisera from rabbits immunized with C. burnetii fractionsMicroagglutination titer on day of bleeding:

Fraction 7 15 22 29 43 54

IIa ia II IIII I II I II I II I

Whole cells 128 32 512 128 512 128 512 128 1,024 512 256 512LCV-CW 128 32 512 128 1,024 1,024 1,024 512 2,048 1,024 512 1,024SCV-CW 128 32 128 64 256 256 128 256 128 256 128 256LCV-CW SDSb <4 <4 16 <4 64 4 64 8 1,024 32 1,024 64SCV-CW SDSb <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4PG-PC 8 <4 8 <4 128 <4 128 <4 64 16 512 16SCV-WC 128 64 512 256 1,024 1,024 512 512 512 512 512 512

a Phase I and II antibody titers expressed as reciprocal dilutions.b Supernatant.

to 29% of the total fatty acids, and the dominant fatty acidswere C16, C18, and C20. Branched-chain saturated acidsranged from 66.4 to 72.9% of the total fatty acids, and thedominant fatty acids were anteiso-C15 and anteiso-C17.These fatty acid profiles are similar to those of some gram-positive bacteria (21) and some Legionella spp. (6).

Purification of PG-PC from SCVs was easily accom-plished. Proteins associated with the PG were resistant toproteolysis by trypsin, protease VI, and proteinease K.These results were similar to the resistance properties ofproteins associated with the PG of Legionella pneumophila(1, 2). Chemical analysis of the PG-PC indicated that a KDO-like component present in phase I lipopolysaccharide (3a,20) copurified with this fraction, thus suggesting that LPSand protein were associated with the PG.The immunizing potential of the various subfractions was

tested in rabbits to determine the distribution of phase-specific antigens (20, 22, 26). Although whole cells and cellwalls elicited similar antibody responses, the PG-PC andSDS-derived soluble fractions were more effective in elicit-ing primary antibody responses against phase II than againstphase I whole-cell antigens. The elicitation of an antibodyresponse against phase II antigens by the PG-PC suggeststhat protease-resistant protein determinants may be repre-sented in this fraction. Recent studies with hybridoma-derived monoclonal antibodies have indicated that phase Ispecificity resides in phenol-water extracts of whole cells,whereas phase II specificity resides in an epitope associatedwith a 29,500-dalton surface protein of phase II whole cells(25). In the present study, we were not successful in ourattempts to remove protein determinants from the PG-PCwithout destroying their immunogenicity. In another study(3), we have shown that proteins can be removed from thePG-PC and that the saccharide portion is sensitive to lyso-zyme hydrolysis.

ACKNOWLEDGMENTSThe assistance provided by the Medical Illustrations Department

(USAMRIID) is gratefully appreciated.

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