© INsrtror PASrEua/ELsevm~ Paris 1991

Res. J14icrobioL

1991, 142, 405-4]0

Further stereochemical studies of phthiocerol and phenol phthioceroi in inycobacteria M. Daff6 Centre de Recherche de Biochimie et de G~n#tique cellulaires du CNRS, and Univer~t~ Paul Subatier, 118, route de Narbonne, 31062 Toulouse Cedex (France)

SUMMARY

Mycobacterium tuberculosis, M. marinum and some other pathogenic species elaborate waxes A, based on a long-chain #-diol (phthiocerol and companion compounds) and polymethyl-branched fatty acids. The atereochemical studies conducted on waxes A showed that those of M. tuberculosis, M. leprae and M. kansasfi differ from waxes A isolated from M. marinum and M. ulcerans by the absolute configuration of the methyl-branched chiral centres occurring in both the longchain/3-diols and the tatty acyls. Furthermore, the two mycobacterial groups also differ in the stereochemistry of the/~-diol chiral csntres. Key-words : Mycobacterium, Phthiocerol; Stereochemistry.

INTRODUCTION

The "waxiness" of mycobacteria has been long recognized and evidently derives froiaa the high lipid content of the bacilli (Goren and Brennan, 1979). More recent str,_dies have enabled the identification of the main components of the solid fractions isolated by solvent extraction from various species of mycobacteria and have shown that the presence of true waxes is restricted to waxes A (Asselineau and Asselineau, 1984) which consist of phthiocerols esterified by two moles of branchedchain fatty acid.~ (fig. I). In addition to diacyl phthiocerols, waxes A also contain two companion compounds, di~,.cyl phthiodiolone and phthiotriol (AsseSubmitted July 7, 1990, accepted November 25, 1990.

lineau, 1954; Demarteau-Ginsburg et al., 1953) (fig. 1). These compounds isolated from Mycobacteriura tuberculosis contain a threo~-diol grouping (Maskens et al., 1966), the absolute configuration of both centres 9 and i l being R (Welby-Gieusse and Tocanne, 1970), and that of the two other chirai centres of phthiocerol A of M. tuberculosis have been determined as 3R, 4S (Maskens and Polgar, 1973). Phthiocerol and companion compounds were originally found in the saponification products of M. tuberculosis (Asselineau, t954; Demarteau-Ginsburg et aL, 1953) and seemed, until recently, to be restricted to the tubercle bacillus. Comparative studies conducted on representative mycobacterial spe-

406

M. DAFFI~

cies allowed the characterization of waxes A in some other mycobacterial species (Duff6 and Lan6elle, 1988; Minnikin et aL, 1985) as well as the occurrence of related phenolic compounds (fig. 1) (Duff6 and Lan6elle, 1988).

(CIPT 14-09001) were used. The origin and growth of mycobacterial strains on Sauton's medium have been previously described (Daff6 et al., 1983; Daff~ and Lan6elle, 1988), M. leprae was obtained from organs of experimentally infected armadillos. The organs were kindly supplied by the Banque de M. leprcte, Institut Pasteur, Paris).

We previously reported the occurrence of d e x t r o r o t a t o r y polymetbyl-branched fatty acids in the waxes A of M, ulcerans and M, marinum (Daff~ and Lan6elle, 1988; Daff6 et al., 1984), whereas laevorotatory fatty acids occur the other waxes-A-containing mycobacterial species. Since the chiral centres of the polymethyl-branched fatty acids arc provided by propionate units (Gastambide-Odier et aL, 1963a, 1966; Yano and Kusunose, 1966), at least ~.wo different enzymatic systems for the incorporation of propionate in branched-chain fatty acids work in myeobacteria, one for dextrorotatory fatty acids (S configuration and the other one for laevorotatory fatty acids (R configuration) (Asselineau, 1982).

Lipid extraction and purification

By using labelled acetate and propionate, it was shown that phthiocerol probably results from the elongation of long-chain fatty acids by acetate units, followed by the incorporation of propionate (Gastambide-Odier et aL, 1963b). Therefore, [n such a hypothesis, the absolute configuration of the methyl-branched chiral centre occurring in phthiocerol and companion c o m p o u n d s may change according to bacterial origin ( e . g . M . marlhum or M. tuberculosis. The present study shows that this expected process occurs and that a difference in the stereochemistry exists in ~-diol ehiral ¢entres.

MATERIALS AND METHODS Strains and culture conditions

in the present study M. tuberculosis (CIPT 14-0010059), M. kansasii (CIPT 14-011002), M. marinum (CIPT 14-0120001) and M. ulceraas CIP'I" = Co[lcclionInstitut P~steur Tuberculose.

Cells were left in CHCIs/CH3OH (1/1 v/v) for 7 days at room temperature to kill them, and then were extracted twice with CHCI3/Cl-lsOH (2/1 v/v) for 2 days. No degradation of the lipids occurred under such conditions (Daff6 et aL, 1988a). The crude extract was chromatographed on a "Florisil" (60-100-mesh) column and elutions were performed with CHCI3 and CHCIs containing 5o70, 10% and 50°7o CH3OH. The first chloroform ¢lutions containing phthiocerol diesters were dried and reehromatographed on a "Florisil" column. Elutions were performed with petroleum ether (b.p. 50 °C) followed by 2%, 5%, 10g0 and 50go diethyl ether in petroleum ether. The final purification of individual phthioeerol diesters was obtained by preparative thin-layer chromatography (TLC) on laboratory-made ulates (.0.3-ram thick) using petroleum ether/diethyl ether (9/1 v/v, 3 times) as developing solvent (Daff6 and Lan6eile, 1988). The lipid spots were visualized by spraying with rhodamine B solution (0.01% in 0.25M monosodi¢ phosphate). The glycolipid-containing fractions, visualized by spraying with an anthrone solution (0.2% in H2SO4) followed by heating, were purified as previously described (Daff~ et aL, 1987, t988a). Hydrolyses

Acid hydrolysis of phenolic mycosides was performed with 2M HC1 at I10 °C for 2 h, and the resulting phenol phthiocerol diesters and related compounds were isolated as previously reported (Daff6 et al.. 1987, 1988a; Daff6 and Lant~elle, 1988). Phthiocerol. phenol phthiocerol and related compounds were obtained by saponification of the corresponding purified lipids in methoxyethanol containing 5% KOH (w/v) and 12% H20 (v/v) at 110 °C for 2-3 h (Daff6 et aL. 1983). Such conditions ensure complete hydrolysis of sterically hindered ester groups (polymethyl-branched fatty esters) in a few hours. Long chain s-branched L TLC - thin-layerchromatography.

S T E R E O C H E M I S T R Y OF M Y C O B A C T E R I A L M Y C O L I C ACIDS I1

CH3-(CH2) m-

~H-

tO

9

Cn 2-

~H-{CH2) 4- qH- ~H- CH 2-

OH

4

3

2

1

CR 3

CH30CH3

OH

CII3-(CH2]m-CH-CH2-CH-fCH2) 4-CH- CH-CH3 I t ~CH3 OH OH CH3

Xl

CH3-(Cn21-9n-CH2-~n-tCn2~-~H-~-CH2-CHJ OH

OH

CH 3

CH3-fCH2) m- CH-CH2-~H-(CH2)4-~H- ~H-C~2-Oo 3 ~H OH CH$ OH IV

u~(cn

)_-CH-CH~-Cn-~CH ).-CH- cu-cn2-c, a

2,,,bH

"b.

2~Su3oc.j

V

407

fatty esters (mycolates) saponified under the same conditions showed no epimerization products (Daff6 et aL, 1988b). In contrast, the chiral centre at the ¢¢ position of a keto group in phthiodiolone or phenolphthiodiolone and m ketomyeolate was epimerized under the alkaline conditions used, as expected from the literature (Asselineau et al., 1970; Welby-Gieusse and Tocanne, 1970). In order to obtain the epimerization of the latter chiral centre without hydrolysis of the ester groups, diacyl phthiodiolone and phenolphthiodiolone were treated with the alkaline reagent at room temperature for 30 rain and then extracted with diethyl ether. Fatty acids and alcohols obtained by saponification were extracted by diethyl ether and diazomethane (in diethyl ether) was added to convert free fatty acids into their methyl ester derivalives. Further separations were made by TLC (Daff6 and Lan~elle, 1988) and the compounds were identified hy mass spectrometry (Daffd et at.., 1984). Reduction of waxes A

OH~(CH2)m-CH-CHz-~H-(CH2)4-~H',~e OH OH CH 3 ~ 3H-CH3 H VI

no-~"%r¢cseJm-CH-en2-fu-Cc%)4-fH -6-cn2-cn 3 x.=J bH on CHz 0

Waxes A isolated from M . tuberculosis and M. marinum were treated with LiAIH4 as previously described to yield long-chain alcohols and phthiocerols (Daff6 et al., 1984). Optical rotations were determined in CHCI 3 soiution 1.1 g/100 mi) with a "Perkin-Elmer" automatic polarimeter model 141 at ),=589 nrn.

VII

RESULTS A N D DISCUSSION

c.3-¢C.2h-¢C.2-¢i.~- coon CH3 VIII

CH3-(CH2)n-~CH2-~H~-CH20H CH 3 IM

Fig. 1. Formulae of mycobactefial phthiocerol, phenol phthiocerol, related compounds and poIymethyl-branehed fatty acids. I - phthiocerol A; II = phthioeerol B; 111- phthiodiolone A; IV = phthiotriol A; V phenol phthiocerol A; Vl=pheno] phthiocerol B; VII = phenol phthiodiolone; VIII =polymethyl-branched fatty acids; IX = polymethyl-branehed fatty alcohols. I - I V : m=14-22; V-Vll : m=16-22; VII! : n - 1620; p = 3-4.

O n the basis o f the optical r o t a t i o n o f the diacyl phthiocerols and related phenol phthiocerols, two groups o f m y c o b a c t e r i a can be distinguished (table I), M . u l c e r a n s and M. m a r i n u m synthesize d e x t r o r o t a t o r y waxes A a n d related phenolic c o m p o u n d s whereas all the o t h e r m y c o b a c t e r i a l species elaborate corresponding l a e v o r o t a t o r y substances in agreem e n t with the nature o f the fatty acids occurring in these c o m p o u n d s (Daff~ et al.. 1984; Daff6 and Landelle, 1988) and reflecting their t a x o n o m i c status discussed elsewhere (Daff~ and Lan6elle, 1988). A l t h o u g h no detailed studies h a v e been made concerning the stereochemistry of p h e n o l p h t h i o c e r o l derivatives, the similar values f o u n d for the optical r o t a t i o n of diacyt

408

M. DAFFI~

Table !, Molecular rotation ([MID) in degrees of diacyl phthiocerol and companion compounds (waxes A)

and of diacyl phenol phthiocerol and related substances. Diesters of compounds

1

M. tuberculosis M. kansasii, M. leprae M. marinum

M. ulcerans

-107_+2 + 59

!II

-74±5

+ 83

H I t° )

-19±1

+ 31

AM

V

-55

-130_+9 + 50

+ 52

VII

VII e*)

AM

+86

+33

+53

For compounds refer to figure I. (*1 Alter epimeri2ation. ~M = [Mlt~ of the native compound compound - [M]t~ of the epimerized compound.

phthioeerols (waxes A) and phenol phthiocerols (table I) isolated from the same mycobacterial species strongly suggest that the asymetric centres of the phenolphthioeerol and companion compounds have the same configurations as those of the phthiocerol family. To investigate the absolute configuration of the methyl-branched chiral centre occurring in the phthiocerol and companion compounds, all the waxes were examined. The contribution of this asymetric centre was deduced from the difference between the molecular rotations of diacyl phthiodiolone and phenol phthiodiolone before and after epimerization of the methyl-branched chiral centre (AM, table I). The absolute values of AM were found to be very similar but the contributions were reversed according to the mycobacterial species examined. Consequently, the methyl-branched asymetric centre, occurring in the phthiodioloae and phenol phthiodiolone of M. ulcerans, may have a 4R absolute configuration (L series) whereas the opposite stereoehemistry 45 (/9 series) has been established for M. tuberculosis (Maskens and Polgar, 1973). Therefore, it is attractive to postulate that the same enzymatic system would work for the incorporation of propionic units in both phthiocerols and polymethyl-branched fatty acids occurring in waxes, A leading to chiral centres of the L series in M, u!cerans and M. marinum (Daff6 et aL, 1984; Daff6 and

Lan~elle, 1988) on the one hand and the D series in the other species (Daff~ et al., 1984; Duff6 and Lan~elle, 1988). Based on the molecular rotation values of epimerized diacyl phthiodiolon¢ and phenol phthiodiolone (table I), it is apparent that the stereochemistry of the ~-diol chiral centres has not been entirely reversed during the biosynthetic process; such a phenomenon might lead to the reverse but similar absolute values of the molecular rotations of diacyl phthiocerol, phenol phthiocerol and companion compounds. In addition, the molecular rotation of phthiocerols isolated from M. tuberculosis was found to be significantly different from that determined for the corresponding compounds from M. marinum (table II), suggesting erythro stereochemistry for the B-diol chiral centres of waxes A isolated from M. ulcerans and M. marinum since the threo stereochemistry has already established for M. tuberculosis (Maskens et al., 1966). To establish the stereochemistry of the i~-dioi chiral eentres, phthiodiolone and phenol phthiodiolone were isolated from the corresponding diacyl derivatives of M. ulceruns. Epimerization occurring during the isolation of both compounds (see experimental procedures) led to the formation of two ketone epimeres at position 4 in equimolar amounts in both cases (Welby-Gieusse and Tocanne, 1970); consequently the molecular

STEREOCHEMISTRY OF MYCOBACTERIAL MYCOLIC ACIDS

409

Table II. Molecular rotation ([M]t~) in degrees of phthiocerols and companion compounds, fatty acids and fatty alcohols isolated from II4. tuberculosis, M. mar[hum and M. ulcerans.

M. tuberculosis M . mar[hum M. ulcerans

1 + 11

Ill

- 18 - 9

- 14 - 26

Compounds VII

VIIi

IX

- 35

-36 + 33 + 33

+ 27 - 26 - 26

For compounds, refer to figure 1. r o t a t i o n values represent the sole c o n t r i b u t i o n o f the ~-diol chiral centres. T a b l e II shows that the m o l e c u l a r rotation o f p h t h i o d i o l o n e isolated f r o m M . t u b e r c u l o s i s is d i f f e r e n t but not the reverse o f that determined for the c o r r e s p o n d i n g c o m p o u n d s y n t h e t i z e d by M . u l c e r a n s , w h i c h s u p p o r t s the e r y t h r o stereoehemistry o f the/3-diol system in the latter species, Incidentally, since the c o m p l e t i o n o f this w o r k , Besra e t al. (1989, 1990) have also s h o w n the e r y t h r o stereochemistry o f the diol unit o c c u r r i n g in p h t h i o e e r o l and phthiod i o l o n e o f M . m a r [ h u m a n d M . u l c e r a n s by i n f r a r e d and p r o t o n spectroscopy. T h e c o m b i n e d results o f these studies now c o n f i r m s t h a t t h e s t e r e o c h e m i s t r y o f the /3-diol s y s t e m c h a n g e s a c c o r d i n g to t h e m y c o b a c t e r i a l species e x a m i n e d . This in itself is an i m p o r t a n t o b s e r v a t i o n reflecting the d i f f e r e n c e in e n z y m a t i c s y s t e m s e x i s t i n g between the t w o m y c o b a c t e r i a l groups, and m a y h e l p in t h e u n d e r s t a ~ , d i n g o f t h e biosynthesis o f p h t h i o c e r o l and related c o m pounds.

Acknowledgements The author thanks Dr. M.A. Lan~elle for providing bacterial cultures, Dr. C. Asselinean for helpful discus~ sions and Mrs. J. Manrel for secretarial assistance.

Rdcentes recherehes sur les phtioc~rols et les p h 6 n o l - p h t i o c & o l s des mycobactdries Mycobacterium tuberculosis, M. mar[hum ainsi que d'autres mycobact~ries pathog~nes 6laborent

des cires A constitu6es de 0-diol ~ longue cha/ne (appel6s phtioc6rol el companions) ¢t d'acidcs gras polym&hyl ram[rids. Les 6tudes st6r6ochimiques mea6es sur les circa A montrent que ccux de M. tuberculosis, M. leprae el M. kansasii diffdraient de ceux 61abordes par M. mar[hum et M. ulcerans par la configuration des centres asym&riques portant Ies ramifications m6thyle aussi bien darts les phtioccrols quc dans les acides gras. De plus, lea deux groupes de mycobact6ries different 6galement par la st6r6ochimie du groupe #-diol. Mots-clds Stdrdochimie.

: Mycobacterium,

Phtiocerol;

References Asselineau, C., Tocanne, G & Tocanne, J.F. ([970), St~r~ochimie des acides mycoliques. Bull. Soc. Chim., 4, 1455-1459. Asselineau. C. & Asselineau, J. {1984), Waxes, mycosides and related compounds, in "The Myeobaeteria, a source book" (G.P. Kubica and L.G. Wayne) part A (pp. 345-359). Marcd Dekker, Inc., New York. Asselineau, J. (1954), Sur quelques substances h 60 atomes de earbone isol~s des lip[des de souches humaines de Mycobacterium tuberculosis. Bull. Soc. Chirn., 21, 108-112. Assellneau, J. (1982), Branched-chain fatty acids of mycobaeteria. Indian J. Chest. Dis., 24, 143-157. B~ra, G.S., Mallet, A.I., Minnikin, D.E. & Ridell, M. (1989), New members of the phthiocerot and phenolphthiocerol families from Mycobacrerium marinum. J. Chem. Soc., Chem. Commun., 1451-1452. Besra, G.S., Minnikin, D.E., Sharif, A. & Stanford, J.L. (1990), Characteristic new members of the phthiocerol and phenol phthiocerol families from Mycobacterium ulcerans. FEMS Microb~oL Letters, 66, 11-14. Daffe, M., Landelle, M.A., Asselineau, C., LevyFr~bault, V. & David, H . L {1983), InfarCt taxono~aique des acides g.ras de~ mycobact~des: proposition d'une m6thode d'analyse. Ann. MictobioL /inst. Pasteur), 13413, 241 256.

410

M. DAFF~

Daff6, M., Lan6elle, M.A., Roussel, .1. & Asselineau, C. (1984), Lipldes sp~ifiques de Mycobacterium ulcerans. Ann. MicrobioL (Inst. Pastet~r), 135A, 191 ~201. DaffY, M., Lacave, C,, La~a~elle, M.A. & Landelle, G. (1987). Structure of lilt major Iriglycosyl phenol phthioceroI of Mycobacterium tuberculosis (strain canetli). Europ. J. Biochem., 167, 155-160. DaffE, M. & Lan6elle, M.A. (1988), Distribution of phthioceroi dicster, phenolic mycosides and related compounds ia mycobacteria. J. gen. MierobioL, i 34, 2049-2055. DaffY, M., Lan6elle, M.A., Lacave, C. & Lan~lle, G. (1988a), MouoglycosyIdiacyl0henol-phthiocerol of Mycobaclerium tuberculosis and Mycobacterium boris. Biochim. biopkys. Acta. (Amst.), 958, 443-449. DaffY, M., LanEeIle, M.A. & Valero-Guillen, P.L. (1988b), Tetraenoic and pentaenoic mycolic acids from Mycobacterium tho#mopheos. Structure, tax. onomic and biosynthetic implications. Europ. Z Biodtem., 177, 339-3,~1. Demarteau-Ginsburg, H., Ginsburg. A & Lederer, E, (1953), Sur trois nouvelles substances naturelles apparent~es au phthtoc~rol. Bioehim. biophys. Acta. (Amst.), 12, 587-588. Gastambide-Odier, M., Delaum6ny, J.M. & Kuntzel, H, (1966), Biosynth~se des acides phti~noi'ques; incorporation d'acide propionique. Biochim. biophys. Acta. (Amst.), 125, 33-42.

Gastambide-Odier, M., De]aumEny, J.M. & Lederer, E. (1963a), BiosynthEsede l'acide C~2-mycoc~rosique: incorporation d'aeid¢ propionique. Biochim. biophys. Acat (Amst.), 70, 670~678. Gastambide-Odier, M . DelaumEny, J.M. & Lederer, E. (1963b), Biosynthesis of phthiocerol : incorporation of methionine and proplonic acid. Chem. and lnd., 1285-1286. Goren, M.B. & Brennan, F.J. (1979), Mycobacterial lipids: chemistry and biologic activities, in "tubereulosi~" (6.P. Youmans) (pp. 64-193). W.B. Saunders Co. Ltd., Easlbourne. Maskens, K. & Polgar, N. (1973), Absolute configuration of the -CH(O-Me)-CH-Me-syslem in the phfltia~-~ois. J. Chem. Soc., 1909-1912. Maskens, K., Minnikin, D.E. & Potgar, N. (1966). Studies relating 1o phthiocerol. - V1. Stereochemical studies. J, Chem. Soc., 2113-2115. Minnikin, D,E., Dobson, G., Goodf¢llow, M., Magnusson, M. & Ride]l, M. (1985), Distribution of some mycobacterial waxes based on the phthiocerol family. J. gen. MicrobioL, 131, 1375-1381. Welby-Gieusse, M. & Tocanne, J.F. (1970), Configuration absolue du phthioc6rol A, du phthiotrol A e t de la phthiodiolone A. Tetrahedton, 26, 2873 -2882. Yano, I. & Kusunos~, M. (1966), Propionate incorporation into mycocerosic acids by resting cells of Mycobacterium tuberculosis vat. boris. BL..him. biophys. Acta (Amst.), 116, 593-596.

Further stereochemical studies of phthiocerol and phenol phthiocerol in mycobacteria.

Mycobacterium tuberculosis, M. marinum and some other pathogenic species elaborate waxes A, based on a long-chain beta-diol (phthiocerol and companion...
350KB Sizes 0 Downloads 0 Views