INFCTION AND IMUNrTY, Ja. 974, p. 142-149Copyright 0 1974 American Society for Microbiology

Vol. 9, No. 1Printed in U.S.A.

Lipids of Putative Relevance to Virulence in Mycobacteriumtuberculosis: Correlation of Virulence with Elaboration of

Sulfatides and Strongly Acidic Lipids'MAYER B. GOREN, OLGA BROKL, AND WERNER B. SCHAEFER

National Jewish Hospital and Research Center and Department of Microbiology, University of Colorado Schoolof Medicine, Denver, Colorado 80206

Received for publication 18 June 1973

From examination of some 40 patient strains of Mycobacterium tuberculosis, astatistically very significant correlation (Spearman's rho) can be drawn betweenthe root index of virulence for the guinea pig (D. A. Mitchison) and the ability ofthe individual strains to elaborate strongly acidic lipids (SAL) in culture. Theseinclude both sulfatides (SL) and phospholipids (PL). Since essentially all, ifindeed not all, of the virulent and only few attenuated strains are prolific inelaborating SAL, this criterion may be a necessary requirement for the expressionof virulence in M. tuberculosis. Tested by chi-square, this premise is seen to bestatistically and pragmatically highly significant. We speculate that SL maycontribute to the pathogenesis of tuberculosis because of a demonstrable activitydirected against host liver mitochondrial membranes (manuscript in prepara-tion) and its synergistic potentiation of the specific toxicity of trehalosedimycolate (cord factor). The activity may also be expressed against phagosomaland lysosomal membranes within macrophages. Because of their strongly anioniccharacter, SL and PL may interact with cationic sites on lysosomal hydrolaseswith resultant immobilization and/or inactivation of the enzymes. By a similarmechanism, these ionic lipids may alter the activity of bactericidal basic pro-teins, previously recognized in the lysosomal armamentarium. Since a minorbut significant fraction of demonstrably attenuated strains is neverthelessprolific in SL or SAL elaboration, this facility alone is evidently not a sufficientcriterion for expression of virulence.

Specific substances have been unequivocallyassociated with expression of virulence or toxic-ity in a variety of pathogenic microorganisms.However, demonstration of similarly endowedproducts elaborated by Mycobacterium tuber-culosis has not been as dramatically convincing.Kochan et al. suggest that certain bacterialproducts may be considered as virulence factorsif they enable survival of the parasite in thehost; and for M. tuberculosis they offer as anactive candidate in that role the mycobactins(30) which function to overcome serum tuber-culostasis by "capturing" Fe from transferrinand supplying it to the tubercle bacilli (22).Attempts have been made to relate the viru-

lence of different strains of M. tuberculosis tocertain biochemical properties of the organism.Mitchison (27) quantitated the susceptibility ofa series of strains to low levels of hydrogen

'Dedicated to Professor Edgar Lederer on the occasionof his sixty-fifth birthday.

peroxide, the data leading to an apparent divi-sion of the microbial population into twogroups. A root index of virulence of the individ-ual strains for the guinea pig was also estab-lished on the basis of the extent of diseaseproduced after 6 weeks by intramuscular inocu-lation of a 1-mg dose of bacilli. For some dozenmembers in a series of isoniazid-sensitive In-dian and British strains, a highly significantassociation was found between low virulenceand high susceptibility to peroxide (see alsoColeman and Middlebrook [6]).Within the domain of mycobacterial lipids,

persistent efforts have been dedicated to anavid search for substances which might becorrelatable with pathogenicity. The search ledto the discovery of the very toxic glycolipid,designated "cord factor" (6,6'-dimycoloyl tre-halose) (see for example 5, 20, 21, 23, 29),whereas in a parallel effort Middlebrook et al.discovered the sulfolipids (sulfatides, SL) ofvirulent M. tuberculosis var. hominis (24).

142

on Decem

ber 15, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

LIPIDS RELEVANT TO M. TUBERCULOSIS VIRULENCE

These are a group of several multiacylatedtrehalose sulfate derivatives (11-13) with whichthe cytochemical neutral red test for virulence(7) is very likely often associated. Unlike cordfactor, these sulfatides do not contain mycolicacids, nor do they by themselves exhibit thetype of toxocity characteristic of cord factor.However, evidence in our laboratory (unpub-lished data) indicates that several apparentlynontoxic trehalose glycolipids elaborated by M.tuberculosis, and especially SL, may act syner-gistically with cord factor to enhance the toxic-ity of the latter substance. We are thus led tothe inference that such trehalose glycolipidsmay be teleonomically advantageous in promot-ing virulence of the pathogen. The fact thatcord factor is elaborated by nonpathogenicstrains as well (4), however, remains a curiousobservation.

In an extension of the sulfatide study, Gan-gadharam et al. examined the Mitchison seriesof British and Indian strains for SL elaborationin culture (9). They also re-established relativeorders of virulence for these strains throughaerogenic infection of groups of guinea pigs (8)and obtained data generally in accord withthose of Mitchison. The results led to a convinc-ing correlation between sulfolipid content andorder of infectivity for the guinea pig. It wasstressed, however, that these results did notestablish "a direct relationship between thegeneral biological property of virulence of tuber-cle bacilli on the one hand and ability ... tosynthesize the sulpholipid fraction on theother" (9).Our earlier studies (11) in isolation and puri-

fication of the various sulfatides led us toanticipate that the elaborate SL recoveryscheme employed by Gangadharam et al. (9, 17,24) might often fail to separate and distinguishthe sulfatides from contaminant phospholipidsor perhaps from relatively polar carboxylicacids. (The present studies show this inferenceto be correct, particularly where these contami-nants are present as the major anionic lipids orwhere the sulfatides themselves are present asrelatively minor components of the lipid ex-tracts.) Accordingly, we sought to test thepreceding results by re-examining the availablemembers of the series of British and Indianstrains, employing the more selective chromato-graphic separation processes of our earlierinvestigations. The present studies, after exten-sion to some 40 strains of M. tuberculosis,confirm a correlation between SL elaborationand virulence index which statistically appearsto be highly significant. They also show thatprolific sulfatide elaboration alone is neither a

necessary nor a sufficient requirement for ex-pression of virulence. Other factors are unques-tionably involved.

MATERIALS AND METHODS

Bacterial cultures. Ten of the 12 Indian andBritish strains examined in the earlier studies ofMitchison (27) and Gangadharam et al. (9) and anadditional 50 strains from Rangoon, Madras, andEast Africa were kindly supplied to us on Lowenstein-Jensen slants by D. A. Mitchison. All of these hadbeen derived from patients, and virulence indiceswere assessed by methods previously described (26,28, 31). Since their isolation, these strains have beenmaintained in liquid nitrogen so that their originalcharacteristics should be well preserved (D. A. Mitch-ison, personal communication). In our present stud-ies, bacteria were cultured and harvested, and lipidswere extracted as previously described (11).

Neutral red reactivity. Gross neutral red reactiv-ity was estimated by the method of Middlebrook et al.(24) and reference 11. Complexing of the dyestuff byacidic lipids in the hexane solution solubilized the dyeinto the organic phase; the absorbance was measuredin a Coleman Jr. spectrophotometer at 525 nm. Theneutral red absorbance (NRA units) calculated for thetotal extract was a measure of the bulk of acidic,anionic lipids present in the extract-carboxylicacids, phospholipids, and sulfatides. Purified SL-Ihad a neutral red activity of about 1.3 NRA units/mg.An approximation of strong acid neutral red activity(attributable to SL and phospholipids) which dis-criminated against the contribution of weak acidcomponents was obtained by carrying out the dyecomplexing in 2% aqueous citric acid instead of inwater. The complexing activity of purified mycobac-terial sulfatides was only slightly reduced (about 5%)under these conditions.

Precise quantitation of purified sulfatides. Puri-fied SL (50 to 200 1ug) in 4.0 ml of hexane wascontacted with 12 ml of 0.002% neutral red in 2%aqueous citric acid solution. After centrifugation, a1-ml sample of the hexane phase was diluted to 5.0 mlwith ethanolic hydrochloride (4 ml of 12 N HCl to 100ml of absolute ethanol), and the optical density wasmeasured at 540 nm in 1-cm cells in a Beckman DBspectrophotometer. The concentration of SL (ex-pressed as SL-I) was derived from a standard curve.For optical densities in the range 0.02 to 0.20, theabsorbance-concentration relation was quite linear.

Semimicro column chromatography. The princi-pal sulfatide (SL-I) and the minor products SL-I',SL-II, and SL-II' (11-13) had almost identical behav-ior in an abbreviated diethylaminoethyl (DEAE)-cel-lulose chromatography. Together, these constitutedabout 95% of the total hexane-extractable sulfolipidsof tubercle bacilli. The remainder of the sulfatides(about 5%) were considerably more polar and wereseparated from contaminant phospholipids only withdifficulty. In the present study, only the SL-I-IIfamilies were quantitated in comparing the differentstrains.

VOL. 9, 1974 143

on Decem

ber 15, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

GOREN, BROKL, AND SCHAEFER

Micro-chromatographic columns were prepared bypacking 0.15 g of conditioned DEAE-cellulose (11) inPasteur pipettes, giving columns about 5 cm inheight. A typical chromatography is summarized inTable 3. All experiments were run in duplicate andoccasionally in quadruplicate.

Quantitation of major sulfatides (SL-I-II fami-lies). The bulk eluate of step 5 (Table 3) may occasion-ally contain some phospholipid contaminants. Wherethis was significant, the sulfatides were separatedfrom these eluates by preparative silica gel thin-layerchromatography (TLC) (see Fig. 1). The SL-I-II area

was scraped, and the closely related sulfatides were

recovered as one. The SL content of an appropriatesample was determined by spectrophotometry inethanol-HCl (vide supra) and the radiolabeling was

assessed. From the specific activity, the total SLcontent of the entire step 5 DEAE-eluate was cal-culated. In the absence of preparative TLC, the SLcontent of the step 5 eluate was simply determinedcolorimetrically.

RESULTS

Rank orders of virulence. Our studies beganwith the available members of the Indian-Brit-ish series (9). Table 1 summarizes for the 10strains the order of infectivity and pathogenic-ity derived by Gangadharam et al. and com-

pares these with the Mitchison root indices ofvirulence (and ranks). Although some variancesexist, Mitchison's statistical analysis (26) indi-cated that the correlation between his rootindex of virulence order and Gangadharam'sinfectivity order are in good agreement (P <0.001). For our original comparison, we accord-ingly summed the two sets of data (rank) togenerate a composited rank order of virulence.At the top of Table 2 are found the more

TABLE 1. Virulence orders: Mitchison,Gangadharam, and composited

-dOrder ofa MitchisonOriginal

dt'Cm

Strain identifi- Path- positedcation' ogen- Infec- Root rankc

icityn tivity index

A I 2644 3 3 1.19 3 6B I 2646 1 2 1.22 1 3C 78342 4 4 1.10 4 8D 78691 9 8 0.85 7 15E 79084 8 10 0.78 8 18F 79112 6 5 0.77 9 14G 79157 7 9 0.63 10 19H 79499 2 1 1.20 2 3K 79500 5 7 0.98 5 12L 79665 10 6 0.86 6 12

a See references 8 and 9.See reference 27.

c Sum of column 4 and 6.

TABLE 2. Lipid extracts of Indian-British strains

Crude 35S incor- Neutral

Straina lipids porationb redStrain~ lipids (counts/min activity(%) x 1O-4) (total)

B 2.8 28.0 8.76H 4.3 19.0 20.6A 3.2 28.0 9.5C 3.7 47.0 11.9K 3.7 2.4 2.5L 2.4 0.3 2.7F 4.0 37.0 11.3D 3.6 15.0 7.3E 3.7 1.6 4.7G 3.8 1.7 4.0H37Rv 3.7 46.0 14.7

aIn descending order of composited virulence rank.H37Rv included for comparison.

bAll figures: per gram dry bacillary residue.

virulent (composited) strains, with the mostattenuated at the bottom.Recovery and gross characterization of

crude lipid extracts. The pertinent data re-garding lipid yield, radiolabeling, and totalneutral red activity of the crude lipid extractsare summarized in Table 2. Since radiosulfatewas always incorporated into the medium, arough comparison of relative amounts of poten-tial sulfatides present in the hexane crudeextracts can be derived simply from the levels ofincorporated 35S. This is not definitive, owing toincorporation of sulfur into nonsulfolipid prod-ucts, as alluded to with respect to extract E(below). The radiolabeling data from Table 2suggest the notable variation of sulfolipid elabo-ration which was, in fact, confirmed.

Sulfatide recovery. The SL separationscheme used routinely is outlined in Fig. 1.Qualitatively, the sulfatides of many of the lipidextracts were further identified by thin-layerchromatography, by infrared spectrophotome-try, and by desulfation in anhydrous ether (12),with quantitative loss of neutral red reactivityand of hexane-soluble radiosulfate.Chromatography. Data for a typical experi-

ment (extract from strain A) are reproduced inTable 3. The quantitation of the minuteamounts of sulfolipid in E, G, and L extracts isinordinately difficult. The loading and washingsteps (1 to 4 of Table 3) are accompanied byrelatively high (perhaps 30%) leakage of labeledsubstances. Neither among these fractions norin the fraction 5 (ordinarily SL-I-II eluate) couldthe normal sulfatides be identified. The con-tents were not neutral red reactive; infraredspectra did not show the characteristic SLabsorption peaks due to sulfate (825, 1,250

INFECT. IMMUNITY144

on Decem

ber 15, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

LIPIDS RELEVANT TO M. TUBERCULOSIS VIRULENCE

cm 1) or associated with trehalose (808 cm-1)(11). Instead, by TLC and radioautography,only small amounts of impure, much more-

polar, sulfur-containing substances were recog-nizable. Our conclusion that extracts E, G, andL contain sulfatides equal to less than 0.2 NRAper g of bacillary residue is in accord with thevery low gross levels of radiosulfate incorpora-tion (Table 2) and with their insignificantstrong acid NRA values.

Strong acid neutral red reactivity. For very

low levels of SL, determination of the strongacid neutral red activity of crude extracts yieldsa good estimate of the maximum possible sulfa-tide content, because the contribution of au-

thentic SL-neutral red complex to absorbance isdiminished only minimally under these condi-tions. By contrast, lipid extracts of strains E, L,and G retain only about 1 to 2% of their totalaqueous neutral red activity when the complex-

2.5 to 3 NRA units

Microcolumn DEAE-chromatography

Sulfolipid I-II eluate* - TLC identification(Step 5 of Table 3)

1Preparative TLC*

IElute SL area*

1(i) Infrared spectro-

Purified SL I-II* ) photometry(ii) Desulfation

Neutral red complex*Specific activity

Spectrophotometric SL I-IIquantitation* Radiometric assaysFIG. 1. Recovery and quantitation of sulfatides

from crude lipids.

ing is conducted in the acid solution. Whenquantities of pure SL are added to samples of E,L, or G crude extracts in hexane, the neutral redactivity due to the added SL is essentially fullyexpressed in the contacting with the dye-citricacid solution. Accordingly, the maximum SLcontents of E, L, and G extracts probably do notexceed 2% of the total neutral red activity.

Distribution of sulfatides in Indian andBritish strains. The results of the quantitativestudies described in the preceding paragraphsare given in Table 4. The table compares totaland strong acid neutral red reactivity; SLcontent is expressed as the neutral red absorb-ance equivalent and as milligrams per gram ofdry bacillary residue. Comparable data aregiven for strain H37Rv. Figure 2 is a bar graphpresentation of similar data. Table 4 also in-cludes, for comparison, the SL quantitation

TABLE 4. Neutral red-reactive lipids: quantitativedata

Neutral SL I-II recoveredared activitya

FromStrain data of

TtlStrong NRA mg angad-Total acid units mg haram(9)

~~(mg)B 8.76 7.6 6.5 5.0 5.5H 20.6 12.5 3.4 2.6 11.5A 9.5 7.2 6.2 4.8 6.0C 11.9 9.8 9.0 6.9 7.0K 2.5 0.8 0.4 0.3 1.5L 2.7 0.03 <0.06 <0.05 3.5F 11.3 8.5 8.5 6.5 5.0D 7.3 3.5 3.6 2.8 1.1E 4.7 0.05 <0.2 <0.15 <0.5G 4.0 0.06 <0.2 <0.15 0.6H37Rv 14.7 11.5 11.4 8.8

aAll values per gram of dry bacillary residue.

TABLE 3. Micro-DEAE-chromatography of crude extract lipidsa

tb ~~Vol Counts/minStep Solvent' (ml) Eluate contents; comments (X 10-3)

1 C/M/ 70/30 10 Neutral lipids; non-SL sulfur 1.082 C/M/F 70/30/5 5 Carboxylic acids; polar neutral None

lipids, occasionally PL3 M 5 Column wash4 C/M 70/30 5 Column wash None5 C/M/Am 70/30/1.5 10 SL I, I', II, II', occasional III, PL 64.56 C/M/Am 70/30/1.5 5 Traces of SL7 C/M/Am/F 70/30/1/0.6 10 Polar SL and PL 2.88 Regeneration HAc 5 ml, M to neutrality

a Charge: 3.0 NRA units of total anionic lipids; 68,000 counts/min.bAbbreviations: C, CHCl,; M, CH3OH; F, HCOOH; HAc, acetic acid; Am, 28% NH4OH; PL, phospholipids.

VOL. 9, 1974 145

on Decem

ber 15, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

GOREN, BROKL, AND SCHAEFER

2

l._

u-cn°. c12 a

Z0

4

B H A C K L F D G E

[-High -Low-

Virulence Virulence

FIG. 2. Distribution of total acidic andacidic lipids and of sulfatides in the original 1

and Indian strains.

data of Gangadharam et al. (9) adjustebacillary residue.

Statistical analysis confirmed what isfrom mere inspection of Fig. 2, i.e., thatseries, in apparent contradiction withsults of Gangadharam et al., no meacorrelation could be drawn between SL etion and virulence. For reasons that are

ered in the Discussion, the study was ex

to embrace an additional 30 strains, an,sufficient to afford a statistically more

ingful assessment. (The virulence indthese numerous examples had previousassessed and kindly transmitted to us bMitchison [personal communication].)sults are summarized in the bar graph o

which also incorporates the data fromoriginal strains. When the data in ttested by Spearman's rank order corr

the comparison is found to be statihighly significant (P < 0.001).

DISCUSSIONThe cytochemical neutral red fixatior

Dubos and Middlebrook (7) is valu,distinguishing between virulent and asstrains of tubercle bacilli. A significantbution to that activity is logically attrito the sulfatide component of envelopephospholipids also contribute (in the istrain H, the neutral red reactivity of t]extracts was found to reside largely in pilipids). The sulfatides are also endow(certain toxic qualities and significantly e

the particular toxicity of cord factorGoren; M. Kato and M. B. Goren, mani

in preparation). These properties theref(port a putative role for the sulfatides

pathogenesis of tuberculosis. In confirmation,the present studies strongly suggest that thelevel of sulfatide elaboration is, to an extent,expressed in the virulence characteristics oftubercle bacilli.The Spearman correlation, which is statisti-

cally highly significant (P < 0.001), neverthe-less afforded a value for p (0.51) which ispragmatically unexciting. Examination of Fig. 3confirms that most of the strains prolific in

sulfatide elaboration are clustered in the region-37 Rv of the chart containing the strains of higherH37Rv virulence index, with a scattering of moderate

to prolific SL elaborators distributed among theremaining strains.

strongly The deviation of our SL data from those ofOBritish Gangadharam was particularly significant re-

specting strains H, K, L, and D (Table 4).Within this small series, strain F (attenuated

d to dry but prolific in SL elaboration), strain H (highlyvirulent but of intermediate SL production),

evident and strain D (attenuated, but with SL elabora-for this tion identical with strain H) have inordinatethe re- weight in the statistical analysis. It was this

iningful consideration that first prompted our extensionalabora- of the study so as to embrace a much largerconsid- number of strains to provide a more reliable.panded pool of data for analysis. The possibility thatamount any of these cultures suffered some alteration inmean- virulence or capacity for SL production during

ices for low-temperature storage cannot, of course, be;ly been discounted. Of the variables within our control,iy D. A. SL quantitations for H and F were repeatedThe re- several times, with satisfactorily reproducibleIf Fig. 3, results. (Strain H exhibits the highest total andthe 10 strong acid neutral red reactivity of the entire

;oto are series. Most of the strong acid activity is foundelation, in a highly polar fraction which elutes onlyisticallv during the DEAE-column regeneration and

i test ofable invirulentcontri-butableM lipids;virulenthe lipidhospho-ed with-nhance(M. B.uscriptsare sup-in the

1.0

14 (H) - sulfatidetotal strong acid lipids

21

10 :a ~~~~~~~~~~(F)

8 : :

6

4 ILW2j

0~~~~~~~~~~~~~~~~3R

1.34 High Root Index of Virulence Low 0.55(Mitchison)

FIG. 3. Distribution of acidic lipids in 40 strains ofM. tuberculosis.

El Total Anionic Activity

"Strong Acid" Activity

o g3 Sulfatide Component

_0-

INFECT. IMMUNrrY146

.01

1-

0:z

H 37Rv

on Decem

ber 15, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

LIPIDS RELEVANT TO M. TUBERCULOSIS VIRULENCE

which we find attributable almost entirely to amixture of phospholipids. In the Gangadharamseparation scheme, these are recovered in theSL fraction and thus account for the inordi-nately high level of SL reported in the previouswork [9].) To make certain that the strains hadnot been inadvertently interchanged, guineapigs were aerogenically infected with these; thetests confirmed the virulence of H and theattenuation of F. Additional powerful evidencein support of these assignments is offered in thecompanion study (14).No significant correlation of SL elaboration

with order of virulence could be establishedwith the first 10 strains regardless of whichorder of virulence was compared (e.g., Mitchi-son's, Gangadharam's, or composited). How-ever, even in this limited sampling, Fig. 2 showsthat the most virulent strains among the group(B, H, A, C) all have relatively high levels ofstrongly acidic lipids (SAL), whereas the at-tenuated strains, with the exception of F, allhave much lower levels. This treatment accom-modates strain H much more convincinglyamong the virulent strains than does the SLcomparison, so that even within this smallsampling, a statistically significant correlationcan already be drawn between order of SALelaboration and composited virulence index (p= 0.69; P < 0.05). Since no virulent strain inthe series of 10 lacks comparatively high levelsof SAL, this criterion was proposed for consider-ation as a necessary, although perhaps not yetsufficient, requirement for virulence.The postulate was tested by simple chi-

square among the members of the much largerseries (where some exceptions appear to exist;for a critical treatment of some of the excep-tional strains, see companion paper [14]). Bythis statistical treatment, the distribution ofhigh-level SAL (>4 NRA) among the morevirulent strains (Mitchison index > 1) is seen tobe highly significant (P < 0.001; X = 0.79). SLdistribution in the complete series may also betested by chi-square with quite similar results.Examined by Spearman's p, correlation of orderof SAL elaboration with order of virulence indexis, however, statistically of about the samesignificance as the SL virulence index order (P< 0.001; p = 0.563). Analysis of Fig. 3 indicatesthat, in strains prolific in SAL elaboration, themajor contribution to SAL activity is mostfrequently due to sulfatide, a feature whichaccounts for the similarity in Spearman's p inthe correlation of either SL or SAL levels withthe order of virulence index (and in chi-square).The choice of virulence root index 1 as the

division between the categories "virulent" and"attenuated" was suggested to us by D. A.Mitchison (personal communication) and isfurther inferred from Mitchison's study inwhich susceptibility to dilute (0.02%) hydrogenperoxide was assessed for a number of strainsalready classified as to virulence root index (27).The results showed an abrupt discontinuity inpercent survivors at a virulence root index valueof aboug 1.0: strains of index above 1.0 showedsurvival of 48 to 95% of the population, whereasfor strains below this root index, fewer than 5%survived. Observations of our own respectingthe distribution of an indicator lipid (14) yieldfurther evidence supporting the concept of twomajor distinct populations of tubercle bacilliamong the present series of strains. The divisionby this independent criterion also appears (for-tuitously?) at the Mitchison root index of about1.Derivative conclusions and speculations. If

we entertain the thesis that strongly acidiclipids of M. tuberculosis may be a requirementfor expression of virulence, what roles might beascribable to these in contributing to pathoge-nicity, or what function may these exert inpromoting intracellular survival of the tuberclebacillus? Within the classes of the trehaloseglycolipids such as cord factor and the myco-bacterial sulfatides, well-defined biological ac-tivity (20, 21) vis-a-vis mitochondrial mem-branes has already been discerned (M. Katoand M. Goren, manuscript in preparation). Likecord factor, the sulfolipids are capable of adirect dramatic attack on mitochondrial mem-branes of liver and spleen, for example, withattendant distortion, swelling, aberration inelectron transport in the respiratory chain, anddisruption of oxidative phosphorylation. Sincesimilar phenomena are recognizable both incord factor-intoxicated or in tuberculous ani-mals (19), it may be inferred that the biologicalactivities demonstrated for the respective glyco-lipids in vitro may be expressed in this specificfashion in the pathogenesis of the disease.

It seems likely that membrane-active compo-nents may also be generated from phagocytosedtubercle bacilli, with phagosomal membranesas targets. Activity of sufficient magnitudemight result in membrane rupture with intra-cellular release of otherwise trapped virulentorganisms. Such a phenomenon seems to becontra-indicated by numerous observations ofArmstrong and D'Arcy Hart, in which at leastat early stages of macrophage infection (up to 4days) no extra-phagosomal tubercle bacilli wereseen (3). However, contributions from mem-

VOL. 9, 1974 147

on Decem

ber 15, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

GOREN, BROKL, AND SCHAEFER

brane-active and other lipids to the order ofevents leading to ultimate cell death and escapeof the bacilli cannot unequivocally be ruled out;thus, the immobilization of leukocytes whichhave ingested crude preparations of cord factor(5) or of phthienoic acids (16) may be offered insupport. As a perhaps more credible alternative,lysosomal membranes may become leaky underthe influence of appropriate mycobacterial lip-ids, with resultant detrimental intracellularrelease of lysosomal enzymes-a chemical ana-logue of an activity attributable to phagocy-tosed silica particles (2) which demonstrablypotentiate a tuberculosis infection (1).

Specific activity may also find expression inpossible alterations in the functional propertiesof lysosomal or phagosomal membranes so thatfusion phenomena whereby lysosomal contentsare believed transferred to the phagosome maybe inhibited (P. D'Arcy Hart, personal commu-nication). Hart and colleagues have offeredconvincing evidence that the incidence of suchfusion appears to be notably suppressed whenthe phagosomes contain viable tubercle bacillior M. microti (15). However, whether "thebacilli are intact because of a failure of fusion orconversely that fusion does not occur becausethe bacilli are intact and alive" remains an openquestion.

Finally, we may consider the possible interac-tion of strongly acidic lipids, with certain cat-ionic proteins apparently elaborated as part ofthe lysosomal armamentarium and for whichantibacterial activity has been demonstrated(from mouse peritoneal macrophages [10] andpolymorphonuclear leukocyte lysosomes [32]).We know that the strongly anionic mycobacte-rial lipids of the present study are vigorouslyadsorbed by cationic substances such as DEAE-cellulose. In their presumed peripheral locationon the bacillary surface, these could very likelyform stable ionic bonds with basic sites on avariety of proteins, including the lysosomalhydrolases, possibly with resultant immobiliza-tion of the proteins and/or alteration of biologi-cal activities owing to allosteric distortion oftertiary structure, processes which would ac-crue to the benefit of the pathogen. Kanai hasreported that in vivo-derived tubercle bacilli are"coated" with host acid phosphatase and that,in a model in vitro system, incubation of tuber-cle bacilli with lysosomal contents similarlycoats the organisms with lysosomal acid phos-phatase (18). These observations may be evi-dence of an immobilization process, which isstrikingly implicit in the avid adsorption of thecationic neutral red at the surface of eitherviable or killed tubercle bacilli.

Although the foregoing inferences are clearlyspeculative, many appear as potential candi-dates for definitive testing. The concepts arelargely elementary; the logistics are somewhatmore complex. We are nevertheless confrontedwith the paradoxical situation wherein severalstrains within the present series have beencharacterized which are prolific in elaborationof strongly acidic lipids, but which are unequiv-ocally attenuated. The (speculative) functionswe have postulated for these lipids in thevirulent strains (if valid) must reasonably beequally valid for the attenuated members.Clearly the solution must be sought in biochem-ical lesions which are evidently independent ofthe facility for elaboration of acidic lipids.These may be reflected in an increased suscepti-bility of the organism to SAL-resistant hostdefenses (H202? transferrin? humoral antibo-dies?) or in a weakening or even evanescence ofother presumed weapons among the pathogen'sarmaments. Thus, we reiterate that productionof strongly acidic lipids may be a necessaryalthough not yet sufficient requirement for thevirulent state in M. tuberculosis. Additionalrequired factors are probably numerous. Someare considered in the succeeding communica-tion (14).

ACKNOWLEDGMENTSThis investigation was supported by Public Health Service

grant AI-08401 from the U.S.-Japan Cooperative MedicalScience Program administered by the National Institute ofAllergy and Infectious Diseases.We thank Lynelle Brown and Evie Verderber for technical

assistance and Nadia de Stackelburg for illustrations.

LITERATURE CITED

1. Allison, A. C., and P. D'Arcy Hart. 1968. Potentiation bysilica of the growth of Mycobacterium tuberculosis inmacrophage cultures. Brit. J. Exp. Pathol. 49:465-476.

2. Allison, A. C., J. S. Harrington, and M. Birbeck. 1966. Anexamination of the cytotoxic effects of silica on macro-phages. J. Exp. Med. 124:141-153.

3. Armstrong, J. A., and P. D'Arcy Hart. 1971. Response ofcultured macrophages to Mycobacterium tuberculosis,with observations on fusion of lysosomes with phago-somes. J. Exp. Med. 134:713-740.

4. Azuma, I., T. Nagasuga, and Y. Yamamura. 1962.Studies on the toxic substances isolated from mycobac-teria. II. Toxic glycolipids of Mycobacterium smegma-tis, Mycobacterium phlei and atypical mycobacteriastrain no. 6 and no. 22. J. Biochem. 52:92-98.

5. Bloch, J. 1950. Studies on the virulence of tuberclebacilli: isolation and biological properties of a constitu-ent of virulent organisms. J. Exp. Med. 91:197-217.

6. Coleman, C. M., and G. Middlebrook. 1956. The effects ofsome sulfhydryl compounds on growth of catalase-posi-tive and catalase-negative tubercle bacilli. Amer. Rev.Tuberc. 74:42-49.

7. Dubos, R. J., and G. Middlebrook. 1948. Cytochemicalreaction of virulent tubercle bacilli. Amer. Rev. Tu-berc. 58:698-699.

8. Gangadharam, P. R. J., M. L. Cohn, C. L. Davis, and G.Middlebrook. 1963. Infectivity and pathogenicity of

148 INFECT. IMMUNITY

on Decem

ber 15, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

LIPIDS RELEVANT TO M. TUBERCULOSIS VIRULENCE

Indian and British strains of tubercle bacilli studied byaerogenic infection of guinea pigs. Amer. Rev. Resp.Dis. 87:200-205.

9. Gangadharam, P. R. J., M. L. Cohn, and G. Middle-brook. 1963. Infectivity, pathogenicity and sulpholipidfraction of some Indian and British strains of tuberclebacilli. Tubercle 44:452-455.

10. Gershon, Z., and A. L. Olitzki. 1965. Monocytin, a

protecting substance produced by murine monocytes.Proc. Soc. Exp. Biol. Med. 119:32-36.

11. Goren, M. B. 1970. Sulfolipid I of Mycobacterium tuber-culosis, strain H37Rv. I. Purification and properties.Biochim. Biophys. Acta 210:116-126.

12. Goren, M. B. 1971. Mycobacterial sulfolipids: spontane-ous desulfation. Lipids 6:40-46.

13. Goren, M. B., 0. Brokl, B. C. Das, and E. Lederer. 1971.Sulfolipid I of Mycobacterium tuberculosis, strainH37Rv. Nature of the acyl substituents. Biochemistry10:72-81.

14. Goren, M. B., 0. Brokl, and W. B. Schaefer. 1974. Lipidsof putative relevance to virulence in Mycobacteriumtuberculosis: phthiocerol dimycocerosate and the at-tenuation indicator lipid. Infect. Immunity 9:150-158.

15. Hart, P. D'Arcy, J. A. Armstrong, C. A. Brown, and P.Draper. 1972. Ultrastructural study of the behav-ior of macrophages toward parasitic mycobacteria.Infect. Immunity 5:803-807.

16. Husseini, H., and S. Elberg. 1952. Cellular reactions tophthienoic acid and related branched chain acids.Amer. Rev. Tuberc. 65:655-672.

17. Ito, F., C. M. Coleman, and G. Middlebrook. 1961.Purification of sulfolipid of virulent tubercle bacilli.Kekkaku 36:764-769 (English summary, p. 768).

18. Kanai, K. 1968. Some aspects of the lysosome-bacillusinteraction in experimental mouse tuberculosis. Ann.N.Y. Acad. Sci. 154:177-193.

19. Kato, M. 1967. Studies of a biochemical lesion in experi-mental tuberculosis in mice. VI. Effect of toxic bacte-rial constituents of tubercle bacilli on oxidative phos-phorylation in host cell. Amer. Rev. Resp. Dis.96:998-1008.

20. Kato, M. 1969. Studies of a biochemical lesion in experi-mental tuberculosis in mice. XI. Mitochondrial swell-ing induced by cord factor in vitro. Amer. Rev. Resp.

149

Dis. 100:47-53.21. Kato, M. 1970. Site II-specific inhibition of mitochondrial

oxidative phosphorylation by trehalose-6, 6' dimyco-late (cord factor) of Mycobacterium tuberculosis. Arch.Biochem. Biophys. 140:379-390.

22. Kochan, I., D. L. Cahall, and C. A. Golden. 1971.Employment of tuberculostasis in serum-agar mediumfor the study of production and activity of mycobactin.Infect. Immunity 4:130-137.

23. Lederer, E. 1961. Glycolipids of acid-fast bacteria, p.

207-238. In M. L. Wolfrom (ed.), Advances in carbohy-drate chemistry, vol. 16. Academic Press Inc., NewYork.

24. Middlebrook, G., C. M. Coleman, and W. B. Schaefer.1959. Sulfolipid from virulent tubercle bacilli. Proc.Nat. Acad. Sci. U.S.A. 45:1801-1804.

25. Middlebrook, G., R. J. Dubos, and C. H. Pierce. 1947.Virulence and morphological characteristics of mam-malian tubercle bacilli. J. Exp. Med. 86:175-183.

26. Mitchison, D. A. 1963. The infectivity and pathogenicityof tubercle bacilli. Amer. Rev. Resp. Dis. 88:267-268.

27. Mitchison, D. A. 1964. The virulence of tubercle bacillifrom patients with pulmonary tuberculosis in Indiaand other countries. Bull. Int. Union Against Tuberc.35:287-306.

28. Mitchison, D. A., J. B. Selkon, and J. Lloyd. 1963.Virulence in the guinea-pig, susceptibility to hydrogenperoxide, and catalase activity of isoniazid-sensitivetubercle bacilli from South Indian and British patients.J. Pathol. Bacteriol. 86:377-386.

29. Noll, H. 1956. The chemistry of cord factor, a toxicglycolipid of M. tuberculosis, p. 149-183. In H. Birk-hauser and H. Bloch (ed.), Advances in tuberculosisresearch, vol. 7. Karger, New York.

30. Snow, G. A. 1970. Mycobactins: iron-chelating growthfactors from mycobacteria. Bacteriol. Rev. 34:99-125.

31. Subbaiah, T. V., D. A. Mitchison, and J. B. Selkon. 1960.The susceptibility to hydrogen peroxide of Indian andBritish isoniazid-sensitive and isoniazid-resistant tu-bercle bacilli. Tubercle 41:323-333.

32. Zeya, H. I., and J. K. Spitznagel. 1966. Cationic proteinsof polymorphonuclear leukocyte lysosomes. II. Compo-sition, properties, and mechanism of antibacterialaction. J. Bacteriol. 91:755-762.

VOL. 9, 1974

on Decem

ber 15, 2020 by guesthttp://iai.asm

.org/D

ownloaded from