STRUCTURE AND FUNCTIONS O f THE MYCOBACTERIAL CELL ENVELOPE h~gh resistance to ~-iactam anti0iotics in Mycobacterium chetonei. (in press). Kirst, H.A. & Sides, G.D. 11989L New directions for macrolide antibiotics: structural modifications and in vitro activity. Antimicrob. Agents a, Chemother., 33, 1413-1418. Minnikin, D.E. (1982), Lipids: Complex lipids, their chemistry, biosynthesis, and roles, in "The biology of the Mycobacteria" (C. Ratledge & J. Stanford) vol. I (pp. 95-184), Academic Press, London, New York. Mizuguchi, Y., Udou, T. & Yamada, T. (|983L Mechanism of antibiotic resistance in Mycobaeterium intrucellulare. Microbiol. t m m u n o L , 27,

425-4.3 t. Mizuguchi, Y., Ogawa, M. & Udou, T. (1985), Morphological changes induced by B-lactam antibiotics in Mycobacterium ovium-intracellulare complex. Antimicrob. Agents a. Chemother., 27, 541-547. Naik, S. & Rack, R. (1989), In vitro activities of several new macrolide antibiotics against Mycobacterium avium complex. Antimicrob. Agents a. Chemother., 33, 1614-1616. Nikaido, H. (1976), Outer membrane of Salmonella typhimu-

rium. Transmembrane diffusion of some hydrophobic substaoces. Biochim, biophys. Aeta lAmst.), 433, 118-132. Nikaido, H. & Normark, S. {1987), Sensitivity of Escherichia coil to various 3-1actams is determined by the interplay of outer membrane permeability and degradation by periplasmic /~lactamases: a quanlitative predictive treatment. Mol. Microbial., 1, 29-36. Nikaido, H. & Rosenberg, E.Y. (1981), Effect of solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coll. J. gen. PIrysiol., 77, 121-135. Nikaidn, H. & Vaara, M. (1955), Molecular basis of bacterial outer membrane permeability. Mtcrootot. Key., 49, i-32. Nikaido, H., Rosenberg, E.Y. & Foulds, J. (1983), Porin channels in Escherichia cull: studies with ~-lactams in intact cells. J. Bact., 153, 232-240. Ramasesh, N., Krahenbuhl, J . L & Hastings, R.C. (1989), In vitro effects of antimicrobial agents on Mycobacterium leprae in mouse peritoneal macrophages. A ntimicrob, Agents a. Chemother., 33, 657-662. Rastogi, N., Gob, K.S. & David, H.L. (1990), Enhancement of

443

drug susceptibility of Mycobacterium avium by inhibhors of cell envelope synthesis. Antimicrob. Agents ¢. Chemother., 34, 759-764. Rastogi, N., Mnreau, B., Capmau, M.L., Goh, K.S. & David, H.L. (1988), Antibacterial action of amphipathic derivatives of ioniazid against the Mycobacterium avium complex. ZbL Bakt, Mikrobiot. Hyg., A.268, 456-462. Stahl. D,A, & Urbance, J.W. (1990), The division between fast- and slow-growing species corresponding to natural relationshigs among the mycobacteria. J. Boer., 172, 116-t24. Stein, W.D. (1967), The inurement of molecules across cell membranes. Academic Press, New York, London. "~Valln~:c, R.J. Jr, Daiovisio, J.~,. & Pankey, G.A. 0979), Disk diffusion testing of susceptibility of Mycobacterium fortuiturn and Mycobacterium chelonei to antibacterial agents. A ntimicrob. Agents a. Chemother., 16, 6tl-614. Zimmermann, W. & Rosselet, A. (1977), The function of the outer membrane of £schericMa coli as a permeability barrier to 3-1actam antibiotics. Antimicrab. Agents a. Chemother., 12, 368-372.

Structure-to-function relationship of mycobaeterial cell envelope components K. T a k a y a m a a n d A . K . D a t t a

Mycobacteriology Research Laborazory, William S. Middleton Memorial Veterans Hospital, Madison, W I 53705 (USA) and Department o f Bacteriology, College o f Agricultural and Life Sciences, University o f Wisconsin, Madison, W f 53706 ¢USA)

Cytokine induction by myeobacterial cell envelope components We have attempted to write aboi!t the st.-fractures of cell enve-

lope components of mycobacteria as they relate to the activation or suppression of the host's i m m u n e s y s t e m , n a m e l y the induction of the various cytokines by the macrcL~ha2es

This is a reasonable approach (Collins, 1990). These cytokines can be either positive or negative e f f - e t o r s o f a mycobacterial infection. In this regard, we have naxv e s t a b l i s h e d the fine

444

7th FORUM IN MICROIHOLOG Y

with TNF, they observed growth inhibition. They also reported that both IL4 and IL6 activate these macrophages, whereas Denis and Gregg (1990) reported that IL6 actually increases the growth of M. avium in human macrophages. Douvas and Crowle (1985)reported that IFNgamma enhanced the growth of macrophage-associat ed mycobacte4ia. However, when it came to the structure-to-function aspect of the cell surface components, there simply wasn't enough material in the literature for us to write a good discussion. We found the work of Silva and Faccioli (1988) rather interesting in ~t.ttvat~ 1~11~, I , r l ~[drll.~ I l l | [ l U L l . I r a , tlii~ respect. They examined the induction of TNF in mouse by intracellular multiplication of trehalose 6,6'-dimycolate (TDM) Mycobacterium avium complex. When mice infected with in situ. It appears that we are Mycobacteri,zm boris BCG were just beginning to examine this administered anti-TNF antiaspect of the problem. The availbody. it inhibited the developability of the purified comment of granuloma in the host pound~ to be studied does not and allowed the bacilli to grow seem to be a problem. The (Kindler et al., 1989). Similar preparation of TDM (Noil and results were obtained with Bloch, 1955), cell wall skeleton Listeria.infected mice (Havetl, (CWS) (Azuma et al., 1974), 1989). Administration of TNF mycoside C (Brennan and and iL2 togeH~er to mice Goren, 1979; Hunter and Bren. infected with M. avium complex nan, 1981), lipoarabinomannan decreased the number of organ(LAM) (Hunter et aL, 1986) and isms in the organs of the host the recently discovered cell wall (Bermudez e~ al., 1988). And or surface-associated proteins finally, the monocytes isolated (Hunter et al., 1989) have all from patients with chronic tuberbeen described. The arabinoculosis had depressed production ga~.actan-mycolate (AG-M) can of TNF as compared to the be obtained by the mild acid monocytes from control and new hydrolysis of the CWS. tuberculosis patients (Takashima The TDM from Mycobacet at., 1990). terium tuberculosis still appears Other cytokines have also to be an important compound to been reported to make macrostudy. It is a hydrophobic phages antimycobacterial but the gly¢olipid that is virtually insolupicture is not clear. Kaufmann ble in water. Thus, its biological and Fl~sch (1990) showed that activities are shown onty when IFN-gamma alone at high conpresent in oil phase of an emul¢entrations is very effective in sion or placed on hydrophobic activating the bone-marrowbeads (Retzinger et aL, 1981). derived macrophages to inhibit Although discovered early the growth of M. boris BCG, (Bloch, 1950), the mode of whereas TNF alone was not action of this "toxic" material is effective. When they combined not known. This compound small amounts of iFN-gamma nerds to he reexamined. The

structure-to-function relationship of Ihe Iipid A moiety of lipopolysaccharide of Gramnegative bacteria in the induction of cytokines by monocytes and macrophages (Qureshi et al,, 1991a; Golenboek et al., 1991; Qureshi et al., 1991). Similar studies might be done with a specific cell envelope component(s) of mycobacteria th'~t is active. We noted that within the past few years, much work has been done to determine the influence of ¥NF, IFN-gamma, 1L4 and IL6 on the activation of macrophages to become antimycobacterial. Bermudez and Young (1988) showed that TNF can

AG-M, CWS, the mycosides, and the ceil wall or surfaceassociated proteins have not been studied in this context. Wallis et aL (1990) found that, unlike the lipopolys.accharide, LAM was only slightly active in the induction of TNF by human monocytes. The cell-wall-associated proteins of Brennan and associates (Hunter et aL, 1989~ r~;%ht be active in the induction of cytokines, tn this regard, De Jong et aL (1991) found that the cell surface glycoproteins associated with the motility of Cytophaga johnsonae are powerful immunostimulants. Wallis et aL (1990) discovered three proteins in M. tuberculosis culture filtrate that induced TNF by monocytes. We wonder if any of these proteins are cell surface in origin. Our conclusion here is that the purified cell surface compounds of mycobacteria should be studied in vitro in the context of induction of cytokines by macrophages and lymphocytes. Such study should allow one to determine which compounds are important in modulating the host's immune system. This should lead to the important structure-to-function analysis of these compounds and eventually to the study of the agonist-tareceptor interaction. Elucidation of the anabolie pathway of mycolic acids

The most important cell surface lipids of the tubercle bacillus based on abundance are the mycolic acids (Kanetsuna, 1968). These C~o-Cgo, alphaalkyl, beta-hydroxy fatty acids are found in TDM and AG-M. The pathogenicity of the tubercle bacillus might be influenced, in part, by the relative rates of synthesis of these two glycolipids. Here, one must be aware that microbial pathogenesis is complex ~nd ~=drifacr~ria| (Fal;:.3w~

STRUCTURE AND FUNCTIONS OF THE MYCOBACTERIAL CELL ENVELOPE

1990). These glycolipids could modulate the immune response of the host. For example, excess TDM synthesis might lead to toxicity, whereas greater :ynthesis of cell-wail-bound A G - M might be tolerated by the host. In this way, T D M , which is known to be present in large amounts in virulent M . tuberculosis (Lederer, 1984), could be one of the pathogenicity factors. The synthesis o f mycolic acids by mycobacteria is thought to involve a complex series of reactions which includes the synthesis of normal fatty acids, elongation, Claisen-type condensation and reduction (Takayama and Qureshi, i984). Direct evidence for the existence of such a p a t h w a y has been lacking because of the absence o f a suitable system to study. However, Laeave et aL (1990) recently discovered a cell-free system f r o m M y c o b a c t e r i u m aurum that is able to synthesize mycolic acids. They then examined the distribution of radiolabel into different parts of the newly synthesized mycolic acids with labeled acetate as the precursor (Lacave et al., 1990a). This discovery enables closer examination of how mycolic acids axe synthesized. We have recently discovered a novel mycolate-containing phospholipid in Mycobacterium sraegmatis that appears to be a product of the above c o n d e n s a t i o n - r e d u c t i o n steps (Takayama et at., 1990). This lipid was extracted from cells of M. amegmatis with chloroformmethanol (2/1) and purified by silica gel column chromatography using the neutral (chloroform-methanol) and basic (chloroform-methanol-ammonium hydroxide) solvent systems in a two-step procedure. It contained a mycolyl group, a phosphate, a fatty alcohol and an unknown moiety. It was sensitive to mild acid hydrolysis. HPLC of the methylated derivative on a

Cls-bonded silica cartridge revealed two series of peaks, one smaller, eluting at 83-87%0, and the other much larger, eluting at 90-93% isopropanol in acetonitrile (fig. 1). This was consistent with the presence of short-chain monenyl and long-chain dienoyl mycolate analogs of M. smegmatis (Gray et al., 1982). Analysis of the two major FIPI.C peaks I

[2 21

:lO0

o OJ

//

/.,t

//

.//

/

dl)

//

g 75 ,~

30

.E O g o~

//¢ /

o

12

it

24 3'6 Mip-#.?s

a8

25 H

6O

Fig. I. Reverse-phase HPLC of monomethyl ester of mycolate-comaining phospholipid isolated from M. smegmaris. A Cts-bonded silica cartridge was used. the mobile phase was a linear gradient of 40-100% isopropanol in aeetonitrile at 3.0 mi/min over 60 mira The AUFS at 210 nm was set at 0.08 for 2.0 mg of sample. Peaks 1 and 2 were recto vered and analysed by californi~am plasma desorption mass spec~romo_try.

445

and 2 by californium plasma desorption mass spectrometry revelaed MNa+ ions at m / z 1888 and 1918, respectively (fig. 2). The molecular weights of these two fractions were 1865 and 189i. This was consistent with the presence of the two major dienoyl myeolates with molecular weights of 1135 and 1163 as the methyl esters (Gray et aL, 1982). The fragment ions at 1402 and 1431 might be MNa ÷ fatty alcohol. A similar oxocorynomyeolate-containing phospholipid was found in the chloroform-methanol extract of Corynehacterium diphtheriae (Takayama and Dat:a, 1989). Further characterization of these novel p h o s p h o l i p i d s is in progress. More recently, pulse-chase experimems using radiolabetled acetate have shown that the label in the mycolyl moiety of this novel phospholipid as well as trehafose 6-monomycolate {TM) in growing calls of M. smegmatis can be chased out with a large excess of untabelled acetate (Sievert and Takayama, unpublished results). This implies that these two iipids are intermediates in the synthesis of TDM and AG-M. We arc now studying the rote of this mycolate-containing phospholipid in the syw.hesis of TM, TDM and AG-M. These recent advances should enable investigators to elucidate how mycolic acids are synthesized in m,/cobaeteria as well as how they are transferred to suitable acceptots to form TDM and AG-M. Effects o f isoniazid and elhambutol on the synthesis o f m ~ e r =ell envelope components

[soniazid inhibits the synthesis o f myeolic acids in growing cells of M. tuberculosis (Winder and Collins, 1970; Takayama et al., 1972) and thi~ leads to decreases in the TDM and AG-M

446

7th I"ORU~! IN MICROtItOLOGF 10000

10000

431

B

A

8000

BOO0

1402

0000'

6000

W

P W

~ 4000'

4ooo

J W 2000 2000 1888

800

do0

1

;

12 0

IgO0 1 4 .0 0

. 1BOG .

1000

1:2010 1 4 0 0

1600

1 8 0 0 2OOB a g a o

aOO~ ~. 0 0 0 2 2 0 0 .

MIZ MIZ

Fig. 2. Partial californium plasma desorption mass speetmm of HPLC-purified monomethyl ester of mycolate-containing phospholipids (see fig. l). Samples: (A) HPLC peak 1; IB) HPLC peak 2.

contents. Tubercle bacilli low in AG-M tend to lose their structural integrity and become labile. They become susceptible to the action of autolytic enzymes and the immune system of the host. The site of action of this drug appears to be in the synthesis of the mona-unsaturated C24 and C26 fatty acid~ which are the precursors of meromycolic acids (Davidson and Tak.ayama, 1979). This ~hould be confirmed by the use of the cell-free enzyme system. The meromycalates are substrates in the Claisen-type condensation reaction for the synthesis of the 3-oxo precursor of myeolie acids (Takayama and Qureshi, 1984). Ethambutol is another antimyeobacterla! drug tha'~ affects the metabolism o f myeolic acids. It inhibits tl:c formation of arabinogalaetan, thus effectively blocking the synthesis of the cell-wall-bound AG-M

(Takayama and Kilburn, 1989). The precise mechanism of this inhibition is not known. With decreasing amounts of arabinogalactan aeceptor available, more o f the newly synthesized mycolic acids are diverted to the synthesis o f T D M (Kitburn and Takayama, 1981). The cell walls of the drug-treated mycobacteria are weakened by the decrease in the AG-M content In a point where the cells become permeable to various metabolites and then ceil destruction occurs. in conclusion, we can provisionally assign roles fat the two major cell envelope components of the tubercle bacillus. The AGM appears to be a vi~al gtruetural component, whereas the "toxic" TDM might be one of the pathogenicity factors. The work reported in t~:is paper was supported, in part, by the Medical Research Service of the Department of

vgterans Affairs and by Gr~nts GM-36054 and AI-25856 from the National Institutesof Health. Masg spectral determinations were

carried out at the Middle Atlamie Mass Spectrometry Laboratory, The Johns

Hopkins UniversitySchoolof Medicine, Baltimore, MD 21205÷ We thank Todd Sievcrt for his excellent technical a~istr.nce and gong Wang for performing the californium plasma de~rption mass spectrometry.

References Azuma, !., Ribi, E., Meyer, T. & Zbar, B. (1974), Biologically active components from mycobacte~ial cell wall, I. Isolation and composition of cell wall skeleton and e~mponent P~ ..~ ant. Cencd~ inst., 52, 95-101, Bermudez, L.E., Stevens, P., Kolonoski, P., Wit, M. & Young, L.S. 0989), Treatment of experimental disseminated mycobacterium avium complex

STRUCTURE. A N D FUNCTIONS OF THE MYCOBACTERL4L CELL E`NVELOPL" infection in mice with recombinant IL-2 and tumor necrosis factor. J. l m m u n o L , 143, 2996- 3000. Bermudez, L.E. & Young, L.:~. (1988), Recombinant tumor necrosis factor alone for in combination with interleukin-2 but not gamma-interferon is associated with killing of Mycobacterium avium complex. J. t m m u n o L . 140, 3006-3013. Bloch, H. (1950), Studies on the virulence of tubercle bacilli. Isolation and biological properties of a euns~.iluent of virulent organisms. J. exp. Med., 91.: 197-218. Brennan, P . J . & Goren, M.B. (1979), Structural studies on the type-specific antigens and lipids of the Mycobacterlum aviumMycobacterium intracellulareMycobacterium scrofuluceum serocomplex. Myeobacterium intraeellulare serotype 9. or. bioL Chem., 2.54, 4205-421 I. Collins, F.M. 11990), In vivo vs in vitro killing o f virulent Mycobacterium tuberculosis. Res. MicrobioL, 141,212-217. Davidson, L.A. & Takayama, K. (1979), Isoniazid inhibition of the synthesis of monounsaturated long-chaln fatty acids in Mycobacterium tuberculosis H37Ra. Antimicrob. Agents a. Chemott)er., !6, lfl,4.--10~. De Jong, D,M. Pate, J.L., Kirkland, T.N., Taylor, C . E . , Baker, P.J. & Takayama, K. (1991), Lipopolysaceharide-tike immunological properties of cell wall glycoproteins isolated from Cytophaga johnsonae. Infect. lmmun. (submitted). Denis, M. & Gregg, E.O. (2990), Recombinant tumor necrosis factor-alpha decreases whereas recombinant interleukin-6 increases growth of a virulent strain of Mycobacrerium a vium in human macrophages. Immunology, 7l, 139-141. Douvas, G.S. & Crowle, A.J. (1985L Gamma interferon activates human macrophagcs to become tumoricidai and teishmanicidal but enhances replication of macrophage-associaled mycobacteria. Infect. fmmun., 50, 1-8. Faikow, S. (1990), Tile " z e n " of bacterial pathogenicity, in

"Molecular basis of bacterial pathogenesis" (B.H. lglewski & V.L. Clark) X I (pp. 3-9). Academic Press, New York, London. Golenbock, D.T., Hampton, R.Y.. Qureshi, N., Takayama, K. & Raetz, C.R.H. (1991), Lipid-Alike molecules that antagonize the effects of endotoxins in human monocytes, J. clin. Invest. (in press), Gray, G.R., Wong, M.Y.H. & Danielson, S.3. (1982), The major my¢olie acids of Mycobacterium smegmatis. Prog. Lipid Res., 21, 91 107. Havell, E.A. (1989), Evidence that tumor necrosis factor has an important role in antibacterial resistance, d. lmmunof., 143, 2894--2899. Hunter, S.W. & 8rennan, P.J. (1981), A novel phenolic glycolipid from Alycobacrerium lepre possibly involved in immunogenicity and pathogenicity. J. Beet., t47, 728739. Hunter, S.W., Gaylord, H. & Brennan, P.J. (1986), Structure and antigenicity o f the phosphorylated llpopolysaccharide antigens from the leprosy and tubercle bacilli. 3'. bioL Chem., 261, 12345-12351. Hunter, S.W., McNeil, M., Modlin, R.L., Mehra, V., Bloom, B.R. F_.,,.,man, P,J. (1989)0 lsolalion and characterization of the highly immunogenic cell wallassociated protein of J~lycobecrerium lepre. J. lmmunoL, 142, 2864-2872. Kapetsuna, F. (1968, Chemical analysis of mycobacterial cell walls. Biochim. biophyy. Acta (Amst.), 1.58, 130-142. Kaufmaon, S.H.E. & Flesch, I.E.A. (1990), Antimycohacterial functions in bone-marrow-derived macrophages. Res. MicrobioL, 141, 244-248. Kilburn, .LO. & Takayama, K(1981), Effects of ethambutol on accumulation and secretion of trehalose mycolates and free mycolic acid in My¢'vbacterium smegmot,~. Anrimicrob. Agenls a. Chemother., 20, 401 404-. Kindler, V., Sappino, A . P , Grau, G.E., Piguet, P.F. & Vassalli, P. tt989L The inducing role of tumor necrosis factor in the development of bactericidal

447

granulomas during BCG infection. Cell, 56, 731-740. Lacave, C., Laneeile, M.A. & Lan~elle, G. (1990)) Mycolic acid synthesis by Mycobacterium aurum cell-free extracts, Biochim. biophys. A cla (Amst.), 1042, 315-323, Lacave, C., Quemard, A. & Lant~elle, G. il990a), Cell-free synthesis of mycolic acids in Mycobecterium aurum: radioactivity distribution in newly synthesized acids and presence of cell wall in the system. Biochtm. biophys. Acta (Amst.}, 1945, 58-68. Lederer, E. (1984), Chemistry of mycobat, teria[ cord factor and related natural and synthetic trehalose esters, in " T h e mycohacteria, A sourcebook'" (G.P. Kubica & L G . Wa)~e) (pp. 361 378). Marcel Dckker, Inc., New York. Null, H. & Bloch, H. (1955), Studies on the chemistry of the cord factor of Mycobacterium tuberculosis. J. bioL Chem., 214, 251-265. Qureshi, N., Takayama, K. & Kurtz, R. {1991}, Diphosphoryl lipid A obtained from the nontoxic Iipopolysaccharide of Rhodopseudomonas ~phaeroides is an endotoxin antagonist in mice. Infect. Immun. (in press). Qurcshi, N., Takayama, K., Meyer, K.C., Kirklaad, T.N., Bush, C.A., Chert, L., Wang, R. & Co,ter, R.J. (1991a), Chemical reduction of 3-oxo and unsaturated groups in fatty acids of diphosphoryl lipid A from the lipopolysaccharide of Rhodopseudomonas sphaeroldes. C o m p a r i s o n o f biological properties before and after reduction. J. bioL Chem. (in press). Retzinger, G.S-, Meredith, S.C., Takayama, K., Hunter, R.L. & Kezdy, F-J. (19gt), The role of surface in the biological activities of trehalose 6,6'-dimycolate. Surface properties and development of a model system. J. biol. Chem., 256, 8208 8216. Silva, C.L. & Faccioli, L.H. (1988), Tumor necrosis factor (cachcctin) mediates induction of cachexia by cord factor from mycobacteria. Infect. tmmun., 56, 3067-3071.

448

7th FORUM IN MICROBIOLOGY

Takashima, T., Uela, C., Tsuyuguchi, 1. & Kishimoto, S. (1990), Production of tumor necrosis factor alpha by monocytes from patients with pulmonary tuberculosis. Infect. lmmun., 58, 3286-3292. Takayama, K. & Dana, A.K. {1989), Isolation and characlerizalion ol" novel myeolate-eontaining $1yeolipids from Cor),nebactetium diphtheriae. Proceedings 24th US-Japan Tuberculosis Conference, San Diego, CA, Aug. 23-25, 1989, pp. 108-111. Takayama, K., Dalta, A.K., Sievert, T., Randall, R.B., Wan,,, R. & Cotter, R.J. (1990), Characterization of a novel mycolate-

eontaininj glycolipid from Mycobacterium smegmatis. Proceedings 25th US-Japan Tuberculosis Conference, Sapporo, Japan, Aug. 27-29, 1990, pp. 13-17. Takayama. K. & Kilburn, J.O. (1989), Inhibition of syntlae~is of arabinogalactan by ethambutol in Mycobacterium smegmarls. A,~i,microb, Agents a. Chemother., 33, 1493-1499. Takayama, K. & Qureshi, N. (1984), Structure and synthesis of iipids, in "The mycobacteria. A sourcebook" (G.P. Kubica & L.G. Wayne) (pp. 315-344). Marcel Dekker, Inc., New York. Takayama, K., Wang, L. & David,

H.L. (1972), Effect of isoniazid on the in vtvo myeolie acid synthesis, cell growth, and viability of Mycobacterium tuberculosis. Antimicrob. Agents a. Chemother., 2, 29-35. Wallis, R.S., Amir-'l'ahmasseb, M. & EIIner, J.J. (t990), Identification by 2-D gel electrophoresis of monocytes-activating proteins of Mycobacterium tuberculosis. Proceedings 25th US-Japan Tuberculosis Conference, Sapporo, Japan, Aug. 27-29, 1990, pp. 117-121. Winder, F.G. & Collins, P.B. (1970), Inhibition by isoniazid of synthesis of myeolic acids in Mycobacrerium tuberculosis. J. gen. Microbiok, 63, 41-4g.

Stt dies on the role of the mycobacterial cell envelope in the multiple drug resistance of atypical myeobacteria S.E. Hoffner and S.B. Svenson Division for Mycobacteriology and Department c f Vaccine Development and P, oduclion, The National Bacteriological Laboratory, S-105 21 Stockholm

INTRODUCTION A broad resistance to various antibacterial agents is a common feature of myeobaeteria. The structure and function of the mycobacterial cell envelope is of key importance in this drug resistance. In particular, the captivity of the cell envelope to z¢clude drug entrance is thought t: be one major factor in the resistance o f m y c o b a c t e r i a against several drugs (David 1981; Rastogi et al., 1981). Hence, drugs a f f e c t i n g the biosynthesis and integrity of the mycobacterial cell envelope are of special interest as, by their mode of action, they are likely to enhance the penetration of other drugs which have intrace!!u!ar

targets such as ribosomes, RNA potymerase, etc. A m o n g the most drugresistant of all mycobacteria are those belonging to the Mycobacterium avium complex. Bacteria of this complex are also becoming increasingly clinically important, as they cause severe opportunistic infections in immunesuppressed patients, e.g. in HIVinfected patients, The relevance and the clinical usefulness of resistance/susceptibility data on atypical mycobacteria obtained by conventional in vitro susceptibility tests is doubtful. We feel that the use of conventional test methods such as the resistance ratio method on LdwensteinJensen medium with a susceptible M. tuberculosis strain as

reference should be avoided in the evaluation of drug susceptibility of atypical mycobacteria. A pronounced resistance of e.g. M. avium to each of the commonly used antimycobacterial drugs is often seen when the drugs are tested one by one. Therefore, such drugs are frequently considered to be " n o n effective" and are not recommended for use. However, the oppos[ze result (a high susceptibility) is frequently found when two-drug combinations of such "non-effective" drugs are tested. Tests of combinations of drugs also seem to be more relevant, as in the ciinicel setting combinations of drugs are always used in the treatment of M. avium infections.