JOURNAL OF BACTERIOLOGY, Nov. 1980, p. 814-8220021-9193/80/11-0814/09$02.00/0

Vol. 144, No. 2

Peptidoglycolipid Nature of the Superficial Cell Wall Sheathof Smooth-Colony-Forming Mycobacteria

WILLIAM W. BARROW,'t BETH P. ULLOM,': AND PATRICK J. BRENNAN' 2§*National Jewish Hospital and Research Center, Denver, Colorado 80206,1 and Department ofMicrobiology

and Immunology, University of Colorado School ofMedicine, Denver, Colorado 802622

The most superficial cell wall layer present in smooth-colony-forming myco-bacteria was isolated from serovar 20 of the Mycobacterium avium-Mycobacte-rium intracellulare-Mycobacterium scrofulaceum (MAIS) serocomplex and ex-amined chemically and by electron microscopy. Most (70 to 80%) of the fibrillarmaterial consisted of an array of serologically active, acetylated C-mycosidicpeptidoglycolipids with the basic structure

fatty acyl-D-Phe-D-aThr-D- Ala-L-alaninol-O-(3-4-di-O-Me-rhamnose)

0

6-d-talose-[rhamnose-2-O-Me-fucose-2-O -Me-rhamnose]but in which the location of acetyl groups and the arrangement of monosaccha-rides have not been defined. Apparently, all serovars within the MAIS complexare characterized by structurally related superficies in which the monoglycosyl-lipopeptide portion is invariable but the oligosaccharide attachment is peculiar toeach serovar. These unique inert structures may be an important factor inshielding the pathogen within phagolysosomes from lysosomal enzymes.

The ability of certain pathogenic mycobacte-ria, such as Mycobacterium tuberculosis, M. le-praemurium, and M. leprae, to persist in gran-ulomatous lesions appears to be directly relatedto their capacity to survive in the host's macro-phages (21). M. lepraemurium, in particular, isable to endure and multiply within the macro-phage, even after fusion of the phagosome hastaken place (7). This attribute has been corre-lated to the presence of a protective sheath,which apparently surrounds these mycobacteriain vivo. Electron microscopic examination oftissue infected with M. lepraemurium (6, 8, 11,12) and M. leprae (14, 26, 27) produced evidenceof an electron-transparent zone surrounding thebacillus in situ. The suggestion that these zones

Rees isolated the material constituting this zonefrom in vivo-cultivated M. lepraemurium andfound it to be a collection of "parallel fibrilswreathed longitudinally around the bacteria"(12). These fibrils were found to contain keyamino acids and 6-deoxyhexoses previouslyfound in "C-mycosides" (12). Draper later iso-lated similar fibrillar material from an in vitro-cultivated strain of Mycobacterium avium andpresented evidence that it too consisted of C-mycosides (10). Thereafter, Kim and colleaguesdemonstrated the presence of these fibrillarstructures surrounding another strain of Myco-bacterium, Mycobacterium sp. NQ (16).

Until recently the basic structure of C-myco-sides had been given as (18, 25):

fatty acyl-D-Phe-(D-alloThr-D-Ala)1,2L-alaninol-0-6-deoxysugar

0

6-deoxysugarrepresent a protective capsule was made origi-nally by Chapman et al. (8) and subsequently byother researchers (11, 12). Later, Draper and

t Permanent address: Department of Microbiology andImmunology, Texas College of Osteopathic Medicine, FortWorth, TX 76107.

f Permanent address: Department of Internal Medicine,Southwestern Medical School, Dallas, TX 75235.

§ Permanent address: Department of Microbiology, Colo-rado State University, Fort Collins, CO 80523.

However, studies by Brennan and Goren (4) andBrennan et al. (5) have demonstrated that indi-vidual serovars of the M. avium-M. intracellu-lare-M. scrofulaceum (MAIS) complex containtwo classes of C-mycosides, which were calledapolar C-mycosidic peptidoglycolipids (PGLs)and polar C-mycosidic PGLs. The apolar classofC-mycosides is essentially similar to the abovestructure; however, the second class differs in

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SUPERFICIAL CELL WALL OF SMOOTH MYCOBACTERIA 815

that it has an oligosaccharide attached to theD-allo-threonine instead of the single 6-deoxy-hexose. Furthermore, it has been suggested thatthe oligosaccharides in the polar PGLs fromserovars differ in composition and contain theantigenic determinants which allow for serolog-ical specificity within the MAIS complex (4).With the knowledge that these type-specific

surface antigens are basically C-mycoside incomposition, it was important to see whetherthey were synonymous with the fibrils reportedby Draper (10, 12). If this were the case, thenthe determination of their structure could proveto be a valuable tool in the understanding ofmycobacterial survival within phagolysosomes.Knowledge of the PGL structure would also bebeneficial for devising a method of interruptingtheir synthesis, thus depleting the mycobacteriaof their outer sheath.This report is concerned with the isolation

and structural analysis of the superficial fibrillarmaterial which is produced by certain smooth-colony mycobacteria.

MATERIALS AND METHODSGrowth of mycobacteria and isolation of the

superficial cell wall. Mycobacterium sp. NQ (3) wasgrown in 7H11 medium as static cultures or by shaking(4). L-[methyl-3H]methionine (13.11 Ci/mol; 0.5 ,uCi/ml of medium) was added to shaking flasks in earlylog phase (6 days). Shaken cells were harvested inearly stationary phase (3 weeks), whereas static cul-tures were allowed to grow for 7 weeks before har-vesting.

Superficial cell wall was released from cells by agentler variation of previously described procedures(16), designed to cause minimal cell rupture. Cellswere harvested by centrifugation at 6,000 x g for 10min at 4°C and suspended, without washing, in phos-phate-buffered saline. The suspension (1 ml of packedcells per 30 ml of phosphate-buffered saline) wasblended in a Vortex mixer with intermittent chillingin ice for 3 min and centrifuged at 6,000 x g for 10 minto remove whole cells. The supernatant was againcentrifuged at 10,000 x g for 15 min and finally at30,000 x g for 1 h. This 30,000 x g pellet was thenwashed with 0.05% Tween 80 in phosphate-bufferedsaline buffer using a combination of low-speed centrif-ugation (10,000 x g, 10 min) to remove cells and high-speed centrifugation (30,000 x g, 1 h) to collect theouter cell wall fraction. The washing procedure wasrepeated with distilled water. Resulting material wasthen lyophilized for further analysis. Spent mediumwas also examined as a source of extracellular fibrillarstructures. It was subjected to the same centrifugationsteps described for the cells blended in a Vortex mixer.

Cellular fractions were directly mounted on Form-var carbon-coated grids, treated with 2% glutaralde-hyde for 30 min, washed with double distilled water,and stained with 1.5% phosphotungstic acid. Gridswere examined in a Hitachi model HU 12 transmissionelectron microscope operating at 75 kV.

Extraction and purification of lipids from cells

and superficial cell wall. Lipids were extracted fromlyophilized whole cells with CHCl3-CH30H (2:1) at50°C for 18 h (4). To extract lipids from the superficialcell wall structures, 3.5 mg of the 30,000 x g pellet wasdissolved in 6 ml of CHC13-CH30H (2:1), and water (1ml) was added. The suspension was mixed, and thelipids in the lower chloroform phase were analyzed.

Pure preparations of the native polar PGLs wereobtained by fractionating total lipid extracts on acolumn of DEAE-cellulose (acetate) (4). Columnswere successively irrigated with CHC13-CH30H-H20(9:1:0.1; 8:2:0.2; 7:3:0.3; etc). Pure polar PGLs appearedin the 7:3:0.3 and 6:4:0.4 eluates. Alternatively, toobtain a fraction containing both polar and apolardeacetylated PGLs, total lipid was treated with 0.2 NNaOH in CHC13-CH3OH (4) and applied to a silicicacid column which was eluted with CHC13 to removefatty acids followed by 30% CH30H in CHC13 to obtaina preparation containing PGLs. The PGLs from the30,000 x g pellet after deacetylation were also sub-jected to similar elution steps with a small column (6by 0.5 cm) of silicic acid-Celite.Chromatography. Solvents used for silica gel and

cellulose chromatography were: A, CHC13-CH30H-H20 (60:12:1); B, CHC13-CH30H-H20 (90:10:0.6); C,CHCl3-CH30H (98:2); D, 1-butanol-ethanol-water (4:1:5, upper phase); E, 1-butanol-pyridine-water (6:4:3),F, 1-butanol-pyridine-water (10:3:3); G, 2-propanol-NH40H (2:1); H, hexane-ether-acetic acid (90:10:3).PGLs on silica gel thin-layer chromatographic

(TLC) plates were located with orcinol-H2SO4; sugarswon cellulose TLC plates were located with aniline-oxalate; sugar alcohols and oligosaccharides on paperchromatograms were visualized with a AgNO3-NaOHdip reagent; acetohydroxamate on cellulose TLCplates was located with acidic FeCl3. Use of thesereagents has been described (4).

Gas-liquid chromatography (GLC) was conductedon a Hewlett Packard 5710A gas chromatograph cou-pled to a Hewlett Packard integrator, model 3380A.Alditol acetates were separated on 6-ft (ca. 1.8 m)columns of 3% SP-2340 or 10% SP-2401 on 100-120Supelcoport, or on 3% OV-225 or 3% ECNSS-M on100-120 Gas-Chrom Q. Operating conditions are de-scribed in the figure legends.

Analytical procedures. Lipid preparations con-taining native PGLs were dissolved in CHC13-CH30Hand treated with an equal volume of 0.2 N methanolicNaOH at 37°C for 30 min (4). This treatment servedto deacetylate the PGLs and saponify orthodox glyc-erides. That acetyl groups only were released from thePGLs by this treatment was demonstrated as de-scribed previously (4), i.e., by treating the pure polarPGLs with alkaline hydroxylamine and recognizingacetohydroxamate as the sole product by TLC insolvent G. Allo-threonine-linked carbohydrates werereleased from the purified polar PGLs by the followingmodification of our previously described reductive al-kali-catalyzed f-elimination reaction (4). Deacetylatedpolar PGLs were dispersed by sonication, dissolved bygentle heating in 50% aqueous ethanol containing 0.4M NaOH and 1.2 M NaBH4, and maintained at 600Cfor 24 h (for analysis of the linkage sugar, NaB3H4 wasemployed). Excess borohydride was destroyed with 2M CH3COOH, and boric acid was removed as methylborate. The residue was partitioned between CHCl3-

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816 BARROW, ULLOM, AND BRENNAN

CH30H and water. The bottom chloroform phasecontained the residual alkali-degraded PGLs (4). Thedried aqueous phase, containing the reduced oligosac-charide, was applied to a column of Sephadex G-25followed by a column of Sephadex G-15. Carbohydratein the eluted fractions was monitored by the phenol-H2SO4 assay (13).

Intact and deacetylated polar PGLs were hydro-lyzed with Kiliani solution (10 ml of concentrated HCI,55 ml of water, 35 ml of glacial CH3COOH [15]) for 7h in a steam bath. The hydrolysate was extracted withhexane, neutralized with a stream of N2, and passedthrough a small column of Dowex 50 H+ in methanol.Alternatively, the PGLs were methanolyzed with 2.2M HCI in CH30H at 840C for 48 h. The reducedoligosaccharide was hydrolyzed with 1.3 or 2.6 MCF3COOH at steam temperature (940C) for 2 to 6 h.Preparation of 6-deoxyhexitol acetates was carried outaccording to SteUlner et al. (24). The intact reducedoligosaccharide was acetylated with acetic anhydride-pyridine (1:1) at room temperature overnight.

Serology was conducted by previously describedprocedures (4). The rabbit antiserum to M. intracel-lulare serovar 20 had a titer of 1:160. All other proce-dures have been described (4).

Isolation of the sugar component of the "lipidcore." The deacetylated polar PGL from cells labeledwith [methyl-3H]methionine (19,000 cpm/mg of lipid)was subjected to alkali-catalyzed fl-elimination. Thealkali-degraded PGL remaining after elimination ofthe oligosaccharide was hydrolyzed with Kiliani re-agent. The neutral hydrolysate was applied to sheetsof Whatman no. 1 filter paper and chromatographedin solvent D. Only one reducing sugar (Rrha = 1.90)was obvious. This was eluted from the paper andapplied to a column of Sephadex G-10 (105 by 1 cm).A single peak was obtained in which carbohydrate andradioactivity coincided. Recovery of the pure mono-saccharide was 2.4 mg (33,900 cpm) from about 5.6 mgof carbohydrate in the alkali-degraded PGL.Mycobacterium sp. NQ was a gift from L. Barksdale,

New York University School of Medicine. Reagentsfor electron microscopy were obtained from ElectronMicroscopy Sciences, Fort Washington, Pa. L-[methyl-3H]methionine was purchased from ICN Pharmaceu-ticals Inc., Irvine, Calif. NaB3H4 (specific activity, 5Ci/mmol) was obtained from Amersham Corp., Ar-lington Heights, Ill. The synthesis of 2-0-Me-fucosewill be described at a later date. The origin of theother 6-deoxyhexoses has been described (4). The SPproducts for GLC were obtained from Supelco Inc.,Bellefonte, Pa., whereas ECNSS-M and OV-225 werepurchased from Applied Science Laboratories, StateCollege, Pa.

RESULTSCharacterization of Mycobacterium sp.

NQ. The particular organism used was chosenbecause Barksdale and colleagues had exten-sively used it to demonstrate the existence incertain mycobacteria of an outermost cell walllayer composed of long parallel filaments (2, 16).Seroagglutination (22) of Mycobacterium sp.NQ, employing antisera raised against the 31

serovars, showed a 4+ response against serovar20 antisera, a 2+ response against serovar 41antisera, and no activity against any of the otherantisera. The type-specific PGLs from Mycobac-terium sp. NQ (see below) were identical tothose from a stock culture of M. intracellulareMAIS serovar 20 in solvent A and in the otherTLC solvent systems described previously (5).On this basis we conclude that Mycobacteriumsp. NQ is serovar 20.Electron microscopy of the external fi-

brillar layer. Examination ofthe washed 30,000x g pellet by electron microscopy (Fig. 1) re-vealed strands of parallel fibers. Individual fibersmeasured about 4 nm in diameter and appar-ently were transparent since underlying fiberswere visible. Although fibrillar material wasfound in both static subsurface and shaken cul-tures, the best source was static cultures. How-ever, surface (pellicle)-grown cells, which havea predilection for rough morphology, are devoidof fibrils.As discerned by transmission electron micros-

copy, the 30,000 x g pellet contained mostly cell-free fibrillar material. Occasionally, cells werealso observed in this pellet with fibrils lyingparallel to the long axis or arranged diagonallyor transversally, much as described by Draper(10). Treatment of the 30,000 x g pellets with0.05% Tween and subsequent centrifugationwere successful in removing visible cells withonly cell-free fibrillar material remaining. Al-though recovery of fibrils (0.57% of cell mass)was comparable to that reported by others(0.66%, reference 10), it hardly represents thefull cellular complement since fibrils were seenin earlier centrifugal fractions. It appears that,during its isolation, the superficial cell wall spon-taneously disaggregated into small fibrils and toits monomeric PGLs, which then appeared in allcentrifugal fractions. Evidence for this phenom-enon came from the observation that when30,000 x g fractions were allowed to stand inbuffer for prolonged periods, the yield of fibrillarmaterial was markedly dinished. Moreover,we have successfully regenerated microscopi-cally discernible fibrils by mounting emulsionsof pure polar PGLs on Formvar carbon-coatedgrids.Composition of the fibrillar layer. Analy-

ses on a preparation of the fibrils are summa-rized in Table 1. Of this material, 95% is lipid. Ofthis, about 12% is neutral lipid, some of whichmay be residual Tween 80 from the washingstep. The majority (88%) of the lipids wereeluted from silicic acid with 30% CH30H inCHC13. Mild alkali treatment of this eluate, todestroy possible phosphoglycerides, followed byfurther silicic acid chromatography, showed that

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SUPERFICIAL CELL WALL OF SMOOTH MYCOBACTERIA 817

G.

FIG. 1. Electron micrograph of the Tween 80-washed 30,000 x g pellet, negatively stained with 1.5%phosphotungstic acid. (A) Lower magnification. (B) Higher magnification.

about 78% of the eluted lipids were alkali-stable,deacetylated PGLs.TLC in solvent A (Fig. 2) showed that the

patterns of PGLs from both the fibrillar fractionand whole cells were similar; both contained fouridentical major polar PGLs (I-IV) and an un-determined number of barely discernible apolarPGLs at the top of the plate. Moreover, whenthe PGL-containing preparations from bothsources were treated with 0.2 N NaOH, oneidentical deacetylated polar PGL was obtained(Fig. 2), indicating that the four polar PGLsdiffered one from the other simply in the numberof acetyl groups at the hydroxyl positions. Fromcarbohydrate determinations on polar and apo-lar PGLs eluted from preparative plates, it wasdetermined that about 70% of the total PGLsare of the polar variety.Structures of polar PGLs. The polar PGLs

are the predominant lipid class in the isolated

fibrils and hence were the objects of more de-tailed chemical and serological analyses; the de-acetylated polar PGL, obtained from the pureintact PGLs of whole cells, was used. Carbohy-drate content was the same whether estimatedas total carbohydrate (13) or 6-deoxyhexose (9)and amounted to 2.60 ,umol of rhamnose equiv-alents per mg of PGL. Fatty acids were esti-mated gravimetrically at 1.11 ,umol of palniticacid equivalent per mg of PGL. Colorimetricestimation of the peptide was unsatisfactory;however, GLC of the amino compounds as theirN-trifluoroacetyl-n-butyl esters (4) showed onlyphenylalanine, threonine, alanine, and alaninolin equimolar quantities. These data are similarto those obtained previously and suggest a tetra-peptide-based polar PGL (4). The percent fig-ures for carbohydrate, (43%), lipid (28.6%), andpeptide (28.5% by difference) are reasonablyclose to the theoretical figures (49%, 27%, 24%,

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818 BARROW, ULLOM, AND BRENNAN

TABLE 1. Analysis of the fibrillar fraction (30,000 xg pellet) from serovar 20

fig/mg of agmO g/mg ofComponent dry frac- lipidb pola

tion' pd lipid'Protein 40Total lipid 950Neutral lipids 120Polar lipids 880PGLs 770Phosphoglycerides 220

a Protein was estimated by a modified Lowry pro-cedure (19). Total lipid was estimated gravimetricallyas the material in the lower phase of a biphasic ex-tractant.

b Neutral lipid was that eluted by CHC13 when lipidsfrom the 30,000 x g pellet were applied to a column(45 by 5 mm) of silicic acid-Celite (2:1). Polar lipidswere subsequently eluted with CHC13-CH3OH (7:3).'To estimate the amounts of PGLs and phospholip-

ids, the polar lipids from the previous column weretreated with 0.2 N NaOH and applied to a new column.The fatty acids, eluted with CHC13, were an indicatorof phosphoglyceride content. The subsequent CHC13-CH30H (7:3) eluate contained the deacetylated PGLs.

Neutral Lipids

Apolar-PGLs

Pnlar-PGL

°s7- IIt

:m ~ ~II wunknown

K

respectively) for a 3-hydroxy, mono-unsatu-rated, C32 fatty-acylated, tetrapeptide-basedPGL with a tetrasaccharide attachment (see be-low). The fatty acids of the deacetylated PGLfrom serovar 20 were identified only insofar asTLC in solvent H and GLC-mass spectrometryas described previously (4) showed an assort-ment of C32, C3, and C36, 3-hydroxy unsaturatedfatty acids, as well as orthodox C16 and C18saturated fatty acids.The constituent sugars in the polar PGL from

serovar 20 were analyzed. Five sugars were pres-ent in the polar PGL hydrolysate, as shown byGLC of the alditol acetates (Fig. 3) and celluloseTLC (solvent D) of the reducing sugars. Threeof the sugars, 3,4-di-O-Me-rhamnose (Rrha =1.8), 6-deoxytalose (Rrha = 1.2), and rhamnose,were readily identified on cellulose thin-layerplates by comparison with the authentic stand-ards. The mobilities of the two remaining sugars(Rrha = 1.3 and 1.5) were indicative of mono-O-Me-6-deoxyhexoses (4). They were identified as2-O-Me-rhamnose and 2-O-Me-fucose by co-chromatography of their alditol acetates withthose of authentic sugars on SP-2340 andECNSS-M and by cellulose TLC in solvent D.To determine which of these sugars were as-

sociated with the lipid core and which consti-tuted the oligosaccharide, the acetyl-free polar

cLCO)z0cn

cc

w

I--wa-

Deacetylated-Polar PGLs

- origin

A B CFIG. 2. TLC in solvent A of: (A) intact polar PGL

preparation from MAIS serovar 20; (B) deacetylatedpolar PGLs from serovar 20; (C) lipid extract from30,000 x gpellet. Plate was sprayed with 0.1% orcinolin 40% H2SO4 and heated (4).

5 10 15 20 25 30

TIME (MIN)FIG. 3. GLC of the alditol acetates derived from

the polar PGLs. The deacetylated polar PGLs (200pg) were hydrolyzed with Kiliani reagent for 7 h at94°C. Preparation and acetylation ofthe 6-deoxyhex-itols are described in the text. GLC was conducted ona 6-ft (ca. 1.8-m) coiled column of3% SP-2340 on 100-120 Supelcoport at 175°C with N2 flow rate of 60 ml/min.

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SUPERFICIAL CELL WALL OF SMOOTH MYCOBACTERIA 819

PGL was subjected to alkali-catalyzed reductivecleavage, and the products were partitioned be-tween CHC13-CH30H and water. The alkali-de-graded PGL in the CHC13 phase was shown tobe chromatographically similar to the lipid like-wise derived from serovar 9 (Fig. 4). Previouslywe concluded that the alkali-degraded PGL fromserovar 9 had the structure

fatty acyl-CO-NH-Phe-CO-NH-2-butenoic-CO-

NH-Ala-CO-NH-alaninol-O-(3,4-di-O-Me-Rha)in which the allo-threonine residue was con-verted to the 2-amino-2-butenoic acid substitu-ent during the fl-elimination reaction. The chro-matographic resemblance between the alkali-de-graded PGLs from serovars 9 and 20 suggestssimilar structures. To determine whether theconstituent sugar in the product from serovar 20was in fact 3,4-di-O-Me-rhamnose, the 3H-sugar

I..

associated with the alkali-degraded PGL grownin the presence of [methyl-3H]methionine wasisolated as described above. Cellulose TLC andpaper chromatography (solvent D) showed thatradioactivity and reactivity to aniline oxalatecoincided with and corresponded exactly to au-thentic 3,4-di-O-Me-rhamnose. Moreover, thesugar in question, as the alditol acetate, cochro-matographed with the diacetate of 3,4-di-O-Me-rhamnitol on SP-2340, SP-2401, ECNSS-M, andOV-225. Finally, a portion of the sugar was de-methylated with BC13 (1), and GLC of the re-duced acetylated product showed that rhamnosewas the only unmethylated 6-deoxyhexose pro-duced.The water-soluble products from the,f-elimi-

nation reaction were applied to a column ofSephadex G-25, followed by Sephadex G-15 (Fig.5). Chromatography of the resulting pure re-duced oligosaccharide is-shown in Fig. 6. Oddly,on paper chromatography (Fig. 6A), it had mo-bility similar to that of rhamnitol and ran muchfaster than the disaccharides, maltose and tre-halose. On TLC the chromatographic propertieswere somewhat more typical of an oligosaccha-ride (Fig. 6B). Moreover, when the reduced oli-gosaccharide was fully acetylated and comparedwith octaacetyl trehalose, it displayed chromato-graphic properties highly indicative of an oligo-saccharide (Fig. 6C). Presumably, in partitionchromatography the lipophilic nature of the in-herent sugars lends to the reduced oligosaccha-ride a mobilty far in excess of conventional hex-ose-containing ogligosaccharides. Only when the

-i

cn

00)

wC-)z

0C')

ORIGIN

1 2FIG. 4. TLC of the alkali-degraded lipid remain-

ing after alkaline borohydride cleavage of the puredeacetylated polar PGL from serovar 9 (lane 1) andserovar 20 (lane 2). Plate was chromatographed insolvent B, sprayed with 0.1% orcinol in 40% H2SO4,and heated.

100 200 150 200

EFFLUENT (ml)

FIG. 5. Purification of the reduced oligosaccha-ride. The aqueous phase resulting from alkaline re-ductive cleavage of the pure acetyl-free polar PGL(205 mg) was applied to Sephadex G-25 (105 by 1 cm)in water (A). Mixed fractions were applied to Seph-adex G-15 (105 by 1 cm) (B). About 15 mg of purereduced oligosaccharide was recovered.

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820 BARROW, ULLOM, AND BRENNAN

A. B.

r-OLIGO _ ' ..-RHfA-OL RHA - f

MALT - I _-

C.

I - Ac-TREH

Ac-r-OLIGO - *

TREH - I I -MALT

FIG. 6. Chromatography of the reduced oligosaccharide. (A) Paper chromatography of reduced oligosac-charide in solvent E. Spots were detected with the AgNO3-NaOH dip reagent. (B) Silica gel TLC of reducedoligosaccharide in solvent F. Plate was sprayed with 0.1% orcinol in 40% H2SO4. (C) Silica gel TLC of theacetylated reduced oligosaccharide in solvent C. Plate was sprayed with orcinol-H2SO4. MALT, maltose;RHA, rhamnose; RHA-OL, rhamnitol; TREH, trehalose; Ac-TREH, octa-acetyltrehalose; Ac-r-OLIGO,acetylated reduced oligosaccharide.

oligosaccharide was fully acetylated and sub-jected to absorption chromatography did theeffect of its size manifest itself.

Figure 7 shows the sugar composition of thepure reduced oligosaccharide obtained from theG-15 Sephadex column. The individual sugars,2-O-Me-fucose, 2-O-Me-rhamnose, rhamnose,and 6-deoxytalose, were present in the relativeproportions of 1.28:0.83:1.05:1, respectively, sug-gesting a tetrasaccharide. Incidentally, the ab-sence of 3,4-di-O-Me-rhamnose in the oligosac-charide further substantiates the evidence thatthis sugar is inherent to the core lipopeptideregion.To determine which of the four sugars present

occupied the alditol terminus, the reduced oli-gosaccharide was hydrolyzed with CF3COOH,and the products were chromatographed on cel-lulose TLC in solvent D and stained with anilineoxalate. Three reducing sugars were thus shown,2-O-Me-fucose, rhamnose, and 2-O-Me-rham-nose. 6-Deoxytalose was absent, evidently be-cause it was present as its alcohol. Some of thehydrolysate was also acetylated without furtherreduction, and the retention time ofthe productswas compared with those of the fully reducedand acetylated sugars by GLC on OV-225(1600C). The only peak from the two prepara-tions which cochromatographed was due to 6-deoxytalitol. Finally, 3H-reduced oligosaccha-ride, obtained from an alkaline reductive cleav-age reaction containing NaB3H4, was hydrolyzedand subjected to radiochromatography in sol-vent D. When the products were scanned forradioactivity, only one radioactive peak was ev-ident which corresponded to 6-deoxytalitol.Serology on the polar PGLs. The four na-

tive polar PGLs from cells of serovar 20 werepurified free of one another by a combination ofcolumn chromatography and TLC and reactedagainst rabbit antisera raised against the parentorganism. The activity of polar PGL II is shown

wz0.wm ~~~~~~~~~~L.

w C4J cow

5 10 15 20 25 30

TIME (min)FIG. 7. GLC of the alditol acetates derived from

the reduced oligosaccharide. The pure preparation(150 pg of carbohydrate) from Sephadex G-15 washydrolyzed for 2 h at 94°C with 2.6 M CF3COOH,reduced, and acetylated. GLC operating conditionsare described in the legend to Fig. 3.

in Fig. 8. Likewise, polarPGL I, III, IV, a mixtureof the native polar PGLs from the 30,000 x gsuperficial cell wall fraction, and the fully de-acetylated polar PGL were active, and all gavea single line of identity on Ouchterlony gels.Thus, it was shown that the polar PGLs, besidesbeing the major constituents of the extemalsheath, are also the typing antigens of serotype20, and the presence of acetyl functions seemsto have little bearing on serological activity.

DISCUSSIONResults from this investigation substantiate

evidence for the presence of a superficial sheath

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SUPERFICIAL CELL WALL OF SMOOTH MYCOBACTERIA 821

1

2

POLAR-PGL-I[

3FIG. 8. Agar gel immunodiffusion (Ouchterlony) ofpure polar PGL II isolated from serovar 20. The pure

lipid was sonicated in phosphate-buffered saline (1 mg/50 gil) and added in amounts of 100, 200, and 400 ugto wells 1, 2, and 3, respectively. To the center well was added 20 ,ul of rabbit antiserum to serovar 20 with atiter of 1:160. Gels were developed in a moist sealed atmosphere at room temperature.

surrounding certain nontuberculous (atypical)mycobacteria. Moreover, analysis of this super-ficial material has revealed that the structuralmatrix consists largely of polar PGLs, related tothose reported earlier by Brennan and Goren (4)for another member of the MAIS serocomplex.Also, the PGLs from serovar 20 were demon-strated to have activity towards rabbit antise-rum raised against the homologous organism.On the basis of previous work (4, 5), it seemsmost likely that this activity and the polar PGLsresponsible for it are entirely specific for serovar20.At this time it is difficult to define the exact

architecture of this extracellular component; theapparent instability of the filamentous struc-tures accentuates the difficulties. It appears fromthe results reported herein and those revealedby others (10, 12, 16) that the fibrillar networkis superficial and covers the entire cell. It almostcertainly corresponds to the most superficial, Li,layer, succinctly described by Barksdale andKim (2) as "filaments, tapes, or ribbons" whenrevealed by negative-staining techniques. How-ever, as substantial amounts of PGLs can beobtained from shaken cultures which containvery few observable filaments, it appears that atleast some of this component is more rigidlyattached to the cell. This view is enhanced bythe fact that ample quantities of the PGLs canbe chemically extracted from cells which havebeen stripped of the outer sheath by mechanicalmeans. It is entirely possible that the PGLsconstitute at least two major surface regions: aninner, more rigidly attached, "micro" capsuleand an outer, less firmly attached, "macro" cap-sule. The outer region is probably the one whichconstitutes the electron-transparent zone de-scribed in earlier reports (6, 11, 14). Thus, it islikely that as the cell grows in a static mode, itproduces more of these antigenic PGLs whichcombine to form a superficial sheath. An inter-esting possibility, which has not been examined,

is that the outer sheath is composed of theantigenic polar PGLs, whereas the inactive apo-lar PGLs are mostly confined to the inner region.The overall structures of the antigenic PGLs

from serovar 20 are consistent with those alreadyidentified in serovar 9 (4) and other serovars (P.J. Brennan, H. Mayer, G. 0. Aspinall, J. E. NamShin, unpublished data). Differences are basedon the number and types of sugars attached tothe threonine in the peptide portion. In serovar20 these sugars are apparently present as a tet-rasaccharide with 6-deoxytalose as the linkagesugar. The other three sugars contained withinthe oligosaccharide have been identified as 2-0-Me-fucose, 2-0-Me-rhamnose, and rhamnose,but their order is not yet known. However, inthe PGLs from serovars 8, 9, and 25, rhamnoseis always penultimate to the reducing end of theoligosaccharide (P. J. Brennan, G. 0. Aspinall,and G. R. Gray, unpublished data). In addition,a characteristic of all of these oligosaccharides isthat some or all of the free hydroxyl groups areacetylated.

Since M. intracellulare serovar 20 is a mem-ber of the MAIS serocomplex, it is reasonable topropose that all serovars are endowed with asheath of exquisitely specific polar PGLs. In-deed, this principle may apply to all smooth-colony mycobacteria, since we have recentlyshown that species (e.g., Mycobacterium kan-sasii) devoid of the C-mycosidic PGL antigenscontain yet other undefined antigenic peptido-glycolipids (P. J. Brennan, unpublished data).Moreover, MAIS organisms and M. lepraemu-rium are antigenically related (17, 23), andDraper and Rees (12) have presented evidenceshowing that the capsular material isolated fromM. lepraemurium contains C-mycosides. Al-though the presence of C-mycosides in M. lepraehas not been examined, it has been suggestedthat the electron-transparent zone which sur-rounds M. leprae in vivo is lipid (20). Thus, thefindings in this report may be inferentially im-

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822 BARROW, ULLOM, AND BRENNAN

portant in understanding the pathogenicity ofnot only all nontuberculous smooth-colony my-co. acteria, but also of M. leprae.

ACKNOWLEDGMENTSThis investigation was supported by contract AI-02079

from the United States-Japan Medical Sciences Program andby grant AI-14687, both administered by the National Insti-tutes of Health. W.W.B. is the beneficiary of a postdoctoralfellowship from the Heiser Fellowship Program for Researchin Leprosy.

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