BnaA Mtthcal BidUnm (1988) Vol 44, No 3, pp 547-561 in leprae

M. and Downloaded from https://academic.oup.com/bmb/article/44/3/547/283569 by guest on 28 September 2021 other pathogenic mycobacteria

P R Wheeler C Ratledge Department of Biochemistry, Untvernty of Hull, Hull

Pathogenic mycobacteria have complex lipoidal cell walls. Most of them secrete further lipids which appear as a layer around intracellular organisms. This lipoidal exterior may protect mycobacteria inside macrophages from attempts that those host cells make to kill them Such protection could be especially important in M leprae which unusually lacks catalase, an important 'self-defence' enzyme. Intracellular mycobacteria must obtain key nutrients from the host. The role of mycobactm and exochelm in acquiring iron, the carbon and nitrogen sources—including metabolic intermediates—used, and control of biosynthetic pathways are discussed. M. tuberculosis is capable of synthesismg all its macromolecules but M. leprae depends on the host for purines (precursors of nucleic acids), and maybe other intermediates Pathogenic mycobacteria grow slowly, and the possibilities that permeability of the envelope to nutrients, catabolic or anabolic (particularly DNA, RNA synthesis) reactions are limiting to growth are considered. Some characteristic activities may represent targets for antimycobactenal agents.

Although it is a considerable over-simplification, it could be asserted that most mycobacteria are no more than Escherichia coli wrapped up in a fur coat. Metabolic processes in mycobacteria, for 548 TUBERCULOSIS AND the most part, are therefore the same, in broad outline as have been elucidated in the more amenable . Thus it is the few activities which are characteristically mycobacterial and the differ- ences between pathogenic mycobacteria and more amenable mi- crobes, that we discuss in this article.

In contrast to workers using E. coli, those of us who have Downloaded from https://academic.oup.com/bmb/article/44/3/547/283569 by guest on 28 September 2021 investigated the metabolism of mycobacteria do so with the dual disadvantages of having no adequately worked-out system for genetic manipulation of the mycobacteria and having to deal with cultures which, at best, are much more slow-growing than E. coli. At worst, some mycobacteria grow only with extreme difficulty (e.g. M. lepraemurium, M. ) while M. leprae does not grow in culture at all. Researches into the metabolism of M. tuberculosis and M. leprae follow two different approaches. With M. tuberculosis, much rele- vant research can be carried out using saprophytic mycobacteria as model systems in the first instance and observations can then be extended to organisms, such as BCG or even M. avium, before carrying out key experiments with virulent strains of M. tuberculo- sis. In such work, the emphasis has usually been on identifying those unusual aspects of metabolism in mycobacteria particularly in relation to their capabilities as pathogens. With researches into M. leprae, the quest is for that quirk of metabolism which renders the organism incapable of growing in laboratory medium. It is therefore necessary with this organism to establish that each pathway of metabolism is present and functional for surely more than one must be defective to account for its inability to grow in axenic culture. Microbial biochemists who study mycobacteria do so with specific objectives in mind. Perhaps though the greatest, and as yet unsolved, problem is to explain why even metabolically competent mycobacteria grow so much more slowly than most other bacteria and why it is that even within the mycobacteria there is such a wide variation in growth rates. In this short review, we have concentrated on those aspects of mycobacterial metabolism which are either unique to mycobac- teria or which may help to throw some light on their outstanding problems.

STRUCTURE To understand mycobacteria one must begin by an understanding of the basic cell structure as it is here, within the envelope of the METABOLISM IN PATHOGENIC MYCOBACTER1A 549 bacterium, that lies the greatest difference with all other bacteria. The mycobacterial cell envelope is not only thicker than others but it is uniquely lipophilic. It is from this external barrier that arise the principal characteristics of the organism: acid-fastness, aggre- gation of cells, resistance to many bactericidal agents including

lytic enzymes produced by invaded host cells and possibly impen- Downloaded from https://academic.oup.com/bmb/article/44/3/547/283569 by guest on 28 September 2021 etrability of some nutrients and even . Various models of the mycobacterial envelope have been pro- posed which attempt to show the orientation of the major lipid components which abound in the cell wall. The model shown in Figure 1 is based on that proposed by Minnikin1 but modified (see Draper P, In: Proceedings of the Indo-UK Symposium on Leprosy: JALMA, Agra. April 1986; p. 155) to allow for the

Phthiocerol dimycocerosate Cord factor

SulphoJipid

Mycolic acid

Arabinogalactan Peptidoglycan

Plasma membrane

Cytoplasm Fig. 1 Model of the mycobactenal cell envelope (membrane and wall). This is based on the model of Minnikin1 modified according to Draper's suggestion (sec text) and thus allows for the size and branched nature of the arabinogalactan estenfied with mycohc acids on its branches. The diagram shows two dimensions; the arabinogalactan and peptidoglycan form a network of branched chains in the third dimension. The topography of the wall is speculative; complex hpids are shown interacting with arabinogalactan-bound mycohc acid chains, though the nature of such interactions has not been shown experimentally. Acyl moieties of phthiocerol dimycocerosate and sulpholipid are drawn short and could be underlapping acyl moieties of mycohc acids Carbohydrate moieties in glycohpids are shown with hatched lines. 550 TUBERCULOSIS AND LEPROSY relative sizes of the arabinogalactan (Mr ~ 30 000) and mycolate (Air ~ 1200) moieties and the branched nature of the arabino- galactan in the cell wall. The envelope is composed of a phospho- lipid plasma membrane, which may contain some unusual phospho- lipids (phosphatidylinositol mannosides) not found outside mycobacteria, to which abuts the typical peptidoglycan backbone Downloaded from https://academic.oup.com/bmb/article/44/3/547/283569 by guest on 28 September 2021 of a Gram-positive bacterial cell.1 Beyond the peptidoglycan lies a branched polymer of arabinogalactan, (arasgal2),, which is linked to the peptidoglycan via a phosphodiester bridge. On to the arabinogalactan matrix are attached the long chain (C60-C86) fatty acids, termed mycolic acids, which may also be linked to sugars such as trehalose giving molecules originally termed 'cord factor' (6,6'-dimycolyltrehalose). Sulpholipids,2 also based on trehalose, occur as do long chain wax esters of phthiocerol dimycocerosates.1 Numerous other complex molecules have been isolated from the walls and some may be associated with viru- lence of the organism—though precise correlations have yet to be made—others are more certainly involved with the antigenic reactions of the mycobacteria. One such molecule apparently unique to M. leprae is a phenolic glycolipid based on the phthiocerol dimycocerosate molecule.3 Many of these mole- cules—cord factor, sulpholipids, phthiocerol esters—appear to be readily extractable from the cell wall and thus probably occur as discrete, free entities, maybe in outer layers outside the cell wall. Others form part of larger macromolecular structures; some of the mycobacterial lipids (e.g. Wax D) detected in ageing, autolysing cultures during early studies are now recognised as breakdown products of much larger molecules. Almost nothing is known about the biosynthesis of complex, mycobacterial lipids. Some intermediates in the biochemical pathways have been elucidated with a fair degree of certainty,1 but enzymes in the pathways have yet to be shown. In M. tuberculosis, a wall-associated polypeptide of glutamic acid occurs linked to the peptidoglycan backbone.4 As this may not occur in avirulent strains or in BCG, it may have some role in virulence—though how a somewhat inocuous polymer could act in this way is far from clear. The peptidoglycan of M. leprae also has an unusual feature; the L-alanine moiety of the peptido part of the molecule appears to be completely replaced by glycine5 though no metabolic or structural significance has been suggested as arising from this difference as, in essence, a CH3 group is being replaced by an H atom. METABOLISM IN PATHOGENIC MYCOBACTERIA 551

NUTRITION OF INTRACELLULAR MYCOBACTERIA The nutritional requirements of M. tuberculosis are quite simple as the organism grows, albeit slowly, on minimal culture media but does not grow appreciably quicker on more complex media. Thus, all cell components can be synthesised from a supply of organic Downloaded from https://academic.oup.com/bmb/article/44/3/547/283569 by guest on 28 September 2021 + carbon (glycerol, glucose, etc.), inorganic nitrogen (NH4 ) and 6 2+ 2 + 3 3 + the usual inorganic elements (Mg , SO4 ~, K , PO4 ", Fe and other trace elements). However, it is difficult to elucidate the requirements of M. leprae as this organism only grows inside specific hosts (e.g. man and the ) and thus will have available to it a complex array of host-derived nutrients. Which ones it uses can only be guessed at, but more importantly this disguises which metabolites M. leprae may be incapable of pro- ducing for itself. The nutrition of M. tuberculosis however must be considerably different when it is growing within a host tissue than when it is growing in culture media. Most microbes prefer to assimilate pre- assembled molecules—amino acids, purines, pyrimidines and fatty acids—and if these are presented to the cells, as would happen in vivo, pathways for the biosynthesis of these molecules may then become repressed (i.e. 'switched off' at the gene level). In contrast, in minimal media, the biosynthetic pathways need to be expressed so an examination of the nutrition of mycobacteria grown in vitro may not always be a very useful guide to their nutrition in the host. Little is known about which substrates intracellular mycobac- teria use. A wide range of carbon and nitrogen sources can be assimilated and metabolised by suspensions of either M. leprae1 or M. tuberculosis.8 But inside host cells, mycobacteria must compete with the metabolic processes of the host for nutrients (Fig. 2) and, in extreme circumstances, parasitised host cells may catabolise substrates to prevent growth of the intracellular parasite.7 Very little is understood how mycobacteria compete for nutrients but it is becoming apparent that some substrates—for instance pyrimi- dines and aspartic acid—are more rapidly incorporated into the cellular components of M. leprae when the bacteria are inside macrophages9 or even in the presence of macrophage extracts, than simply in suspension (V Sritharan, 1988, PhD thesis, Univer- sity of Hull). An interesting fragment of information is that the transport system for tryptophan in M. tuberculosis H37R, has a greater affinity for tryptophan when the bacteria are grown in a host rather than in batch-culture.10 This may help the bacteria to Rrtty acids) [Glycecol Glucose

Fig. 2 Nutrition of mtracellular mycobacteria The lntracellular mycobactenum must compete with the host for pools of intermediates (shown in ovals). Mycobacteria] products shown are

cxochelin, for abstracting iron from host-denved iron and phosphatase, needed to break down nucleondes to molecules which the bacteria can take up 2021 September 28 on guest by https://academic.oup.com/bmb/article/44/3/547/283569 from Downloaded METABOLISM IN PATHOGENIC MYCOBACTERIA 553

compete for intracellular tryptophan. Most of the substrates which isolated M. tuberculosis and M. leprae utilise are also utilised by many bacteria (e.g. E. colt, M. smegmatis), but two unusual abilities (reviewed in Ref. 11) have been reported in M. leprae. One is the utilisation of the carbon source 6-phosphogluconate

which is formed (though probably also catabolised) rapidly in Downloaded from https://academic.oup.com/bmb/article/44/3/547/283569 by guest on 28 September 2021 activated macrophages. The other is a DOPA (dihydroxyphenyl- alanine) oxidising activity, but whether this is of metabolic sig- nificance, or an artifact of non-specific oxidation is not known. As stated above, M. tuberculosis does not appear to require any particular nutrients from the host, though it may obtain nutrients which are intermediates in metabolic pathways. M. leprae, how- ever, has not yet been grown in a cell-free medium and it may be that it requires substrates from the host that it cannot make for itself. Strong evidence now suggests that M. leprae cannot syn- thesise the purine ring for itself and thus has a requirement for preformed purines.12 Adenine nucleotides appeared to be the most readily available source of the purine ring in host cells and are readily used by M. leprae organisms, being hydrolysed first and taken up as adenosine (Fig. 2). Many other intracellular parasites have lost their ability to synthesise purines13 and studies on other parasites may point to other possible dependencies in M. leprae, such as perhaps a requirement for pyrimidines or fatty acids (see Ref. 13). This needs considerable further research and is compli- cated by the possibility that biosynthesis of such intermediates may well be repressed* in mycobacteria grown in vivo. For instance, incorporation of [14C]acetate and [14C]pyruvate into lipids does not occur in M. leprae, and phosphotransacetylase cannot reliably be detected in its extracts (Wheeler & Ratledge; work submitted for publication). These might seem to be meta- bolic deficiencies but all these activities, and indeed fatty acid biosynthesis itself, are under stringent metabolic control in myco- bacteria7 and cannot be detected when fatty acids or lipids are available, as they almost certainly are inside host cells.

SELF-PROTECTION AGAINST ATTEMPTS BY THE HOST AT INTRACELLULAR KILLING Tubercle and leprosy invade their hosts, and thus the host attempts to kill them in the ways it would any invading organism. However, both M. leprae and M. tuberculosis divide inside host macrophages, cells which are adapted to kill invading microbes. In 554 TUBERCULOSIS AND LEPROSY this section, we review how the metabolic and biochemical pro- cesses of these mycobacteria allow them to survive once they enter host cells. The consequences of phagosome-lysosome fusion—which in- cludes production of hydrolytic enzymes and lowering of intra-

cellular pH—plus the production of peroxide by macrophages Downloaded from https://academic.oup.com/bmb/article/44/3/547/283569 by guest on 28 September 2021 are important in killing invading microbes, and probably also have a role in killing both M. tuberculosis (see Ref. 14) and M. leprae.15 A wide range of microbes elaborate the enzymes catalase and superoxide dismutase to destroy the harmful peroxide and other toxic oxygen metabolites. Superoxide dismutase has been found in all mycobacteria so far studied (see Ref. 11). However, there are differences amongst mycobacteria in their possession of catalase, which breaks down peroxide. This enzyme has been detected in most mycobacteria but not M. leprae; any detectable catalase activity associated with M. leprae organisms is host- derived (see Ref. 11). M. tuberculosis normally possesses catalase, but a few strains do not and they are more susceptible to lethal effects of peroxide, and are usually less virulent than strains with catalase.14 But resistance to peroxide, and catalase activity are not well related amongst different mycobacterial species16 and both strains of M. tuberculosis lacking catalase and M. leprae are still pathogenic organisms. Therefore, either the production of toxic oxygen metabolites by the host is not alone sufficient to kill mycobacteria or else pathogenic mycobacteria have ways other than producing the above enzymes of evading toxic oxygen metabolites. An alternative method of self-protection for the mycobacterium may be wrapping itself in a lipoidal coat. Such a lipoidal coat is in addition to and outside the cell wall shown in Figure 1 and it appears around pathogenic mycobacteria in tissue sections as an electron transparent zone (ETZ). The ETZ appears only around pathogenic mycobacteria and not around saprophytes.17 It prob- ably forms as a result of interaction between the host cell and mycobacterium and contains as its mycobacterial components mainly polar lipids known as mycosides,18 in M. leprae including the phenolic glycolipid19 mentioned above in the section on 'Structure'. A diffuse outer layer than a true capsule, the ETZ probably has a role in protection. Some electron micrographs appear to show hydrolytic enzymes produced by macrophages being excluded from the cell wall of engulfed mycobacteria by the ETZ20 and extracellular bacterial lipids would be a possible site METABOLISM IN PATHOGENIC MYCOBACTERIA 555 for harmless peroxidation by oxygen metabolites14 as opposed to peroxidation at the bacterial membrane which would be lethal. In contrast to M. leprae and M. avium, M. tuberculosis orga- nisms are not very good at forming an ETZ.17 However, tubercle bacilli can inhibit the phagosome-lysosome fusion event men- tioned above by a mechanism or mechanisms not yet resolved (see Downloaded from https://academic.oup.com/bmb/article/44/3/547/283569 by guest on 28 September 2021 Ref. 14). This mechanism of self-protection does not seem to occur in M. avium and M. leprae. However, it has been suggested that M. leprae only weakly stimulates production of toxic oxygen metabolites21 though in another report, phagocytes infected by M. leprae still produce toxic oxygen metabolites22 at a level which appears sufficient to be bactericidal. Although there appears to be a contradiction here, it is clear that M. leprae does not actively inhibit production of toxic metabolites by macrophages. Finally, M. leprae also appears to survive in cells designed to kill microbes by escaping from the phagosome into the cytoplasm.

METABOLISM AND GROWTH There are a number of areas of mycobacterial metabolism which can be identified as being distinct from other bacteria. Of these, we would suggest that biosynthesis of the complex lipids of the mycobacterial envelope (see Fig. 1), iron metabolism and the integration of metabolism with growth which impinges upon the biosynthesis of RNA and DNA, are areas of potential interest. Each area offers novel opportunities for interfering with mycobac- terial metabolism in a specific manner such that new chemothera- peutic agents may be designed to inhibit these processes.

Iron metabolism The problem with iron is that in aerobic systems it exists as Fe(III), rather than Fe(II), and as such is virtually insoluble (~ 10 ~15 mol/1) at neutral pH values. This poses problems for all living cells—not just mycobacteria—as all cells require iron for their essential activities: many enzymes, cytochromes, oxygen- binding proteins, etc. require iron as an integral part of their structure and such entities are found in bacterial cells as well as animal and plant cells. Animals without iron are termed anaemic but bacteria too can be similarly depleted and suffer likewise. Deprivation of iron for micro-organisms and higher cells alike results in slow growth, poor multiplication and, in the extreme, 556 TUBERCULOSIS AND LEPROSY complete stasis and even cell death. Animals have solved the problem of iron acquisition by using ferritin as a ubiquitous iron storage compound and transferrin for movement of iron. Other iron-binding proteins, such as lactoferrin or ovaferrin occur in specialised fluids, secretions or tissues or systems. Bacteria, if they are pathogenic, must then be able to acquire iron from such Downloaded from https://academic.oup.com/bmb/article/44/3/547/283569 by guest on 28 September 2021 sources as otherwise they would be unable to grow in vivo. The ability to acquire iron is therefore considered as a key virulence factor23 though the ability to aquire iron from mammalian iron-proteins does not in itself confer pathogenicity upon a micro-organism; non-pathogens have similar mechanisms of iron acquisition to pathogens. Virulence is a multi-factorial process of which iron acquisition from host fluids and tissues is a key part. The process of iron acquisition in mycobacteria is distinct from processes described in other bacteria24 in that two molecules, instead of just one, are known which have the ability to chelate iron. One group of molecules are termed exochelins which serve to capture iron from molecules such as ferritin or solubilise iron from Fe(OH)3 or ferric phosphate if in the gut (or in the environment). These exochelins occur extracellularly and are similar in function, though not in structure, to the wider group of bacterial iron chelating compounds—the . The other group of iron binding compounds are the mycobactins which occur intracellu- larly and serve as a store of iron should iron become suddenly available to the cells. Mycobacteria, like all other pathogens, probably do not grow steadily when in vivo but go through a series of 'feasts and famines'. During the 'famine' modes, the cell accentuates its various molecules involved in nutrient acquisition so that examin- ation of mycobacteria grown in the laboratory under iron deficient conditions shows considerably elevated (x 1000-fold) amounts of both exochelin and mycobactin. Sudden presentation of iron— even insoluble iron—quickly stimulates the exochelin-mediated uptake system but the number of iron acceptors (apoenzymes, porphyrins) are quickly saturated with iron and the cell must therefore store the excess iron as ferric mycobactin until the cell machinery becomes modified to take advantage of the altered nutritional status of the cell. It usually takes about 24 h for a fast- growing mycobacterium to change its internal enzyme activity to match the new nutritional status. The process of iron acquisition is shown in Figure 3. Exochelins and mycobactins have been found in most mycobacteria exam- METABOLISM IN PATHOGENIC MYCOBACTERIA 557

Environment/ Membrane Mycobactenal host cell cytoplasm

e"(NADH) Fe (ID-mycoboctin sahcylate low mol wt -»( ISTOREl Downloaded from https://academic.oup.com/bmb/article/44/3/547/283569 by guest on 28 September 2021 soluable overflow^ V . . t/ \% Fe compounds • mycobactin* Fe (II)- salicylate • NAD* Fe(IE) FetOD-exochelin -e"(NADP) transport •— salicylate process FetiE)Vr_ exochelm <- •cni)V_ > Fed)-salicylate • NAD*

Fig. 3 Mechanism of iron uptake into mycobactena. The exochelin mediated process is usually active in saprophytes and by facilitated diffusion m pathogenic or slow growing mycobactena.

ined.25 In M. paratuberculosis, the causative organism of Johne's disease in cattle, and recently Crohn's disease, mycobactin is absent (though it appears that the genes for its synthesis are present but inoperative) and either it or exochelin must be added to laboratory culture media to achieve growth.25 In M. leprae, the position is obscure as mycobactins and exochelins cannot be recovered from in vivo grown mycobacteria. However, exochelins isolated from certain mycobacteria have been shown to stimulate iron uptake into suspensions of M. leprae recovered from arma- dillos26 thus suggesting that this organism probably uses the same system for iron uptake as other mycobacteria although M. leprae may conceivably have to rely on commensal mycobacteria to furnish the exochelins for this to occur. Recent work27 has demonstrated the presence of four or five envelope proteins in several mycobacteria whose presence is considerably increased in cells grown under iron deprivation. These proteins may possibly act as receptor sites for iron uptake. Some of the proteins have now been detected in mycobacteria, including M. leprae, recovered from infected animals. The infer- ence is therefore that, at least during some stage during the in vivo development of a mycobacterial , the bacteria become deprived of iron and respond by increasing the synthesis of the iron chelating molecules as well as the putative receptor proteins. It is only the presence of these proteins which is detected not the 558 TUBERCULOSIS AND LEPROSY exochelins or mycobactins, though they have been searched for. Similar events have been recorded with other recovered from infected tissues.23 Both the biosynthesis of the exochelins and mycobactins as well as their functions are potential targets for anti-mycobacterial agents. Some attempt has been made to use metal-analogues of the Downloaded from https://academic.oup.com/bmb/article/44/3/547/283569 by guest on 28 September 2021 exochelins to block iron acquisition though without success (see Ref. 25). Interestingly it has been suggested that PAS (p-amino- salicylate, the well-known anti-tubercular drug of the 1950s, 60s and 70s) possibly owes its success to inhibiting mycobactin function rather than acting as an anti-folate compound.23

Why pathogenic mycobacteria grow slowly There are several possible reasons why mycobacteria grow so slowly. The thick lipoidal cell wall may retard the passage of nutrients into the bacterial cells. However, since cell walls in different strains of mycobacteria are similar, this cannot explain why M. tuberculosis grows so much more slowly than M. phlei (Table 1). Alternatively, once energy substrates enter the myco- bactenal cell, differences in their metabolism may result in differ- ences in growth rate. Finally, it could be that growth is limited by the rate at which the mycobacterial cell can synthesise macro- molecules, e.g. , DNA or RNA. The time it takes to synthesise nucleic acid molecules (DNA, RNA), and therefore also the 'step-time', the time it takes to

Table 1 DNA and RNA synthesis Organism

Parameter M. tuberculosis' M. smegmatts E coli Ref

Growth rate(h~')b 0.04 0.33 0.73 29 C-penod (time to replicate 620-660 105-110 55-58 29 DNA ; mm) Time to transcribe 7.6 NDe -0.8 28 16S + 23S + 5S rRNA genes (mm) Step time (to advance RNA 100-250 ND 10-13 28 chain by one nucleotide, ras) Number of rRNA operons 1 2 7 30

Nous 'Growth rate deduced for M Uprtu m mice u bm medn used to grow bacteria for ttudy of DNA lynthens "not doae METABOLISM IN PATHOGENIC MYCOBACTERIA 559 increase nucleic acid chain length by one nucleotide can be deduced by stopping initiation of the synthesis of new nucleic acid chains using metabolic inhibitors and so observing synthesis only of chains already started. This has been done for RNA28 and 29 DNA synthesis in M. tuberculosis HJ-JR, and M. smegmatis (see

Table 1). When compared with E. coli, there is a clear relationship Downloaded from https://academic.oup.com/bmb/article/44/3/547/283569 by guest on 28 September 2021 between rates of DNA and RNA synthesis and growth rate. A further parameter which appears to be related to growth rate is the actual number of RNA genes in relation to the size of the genome (see Table 1), claimed to be surprisingly few in mycobacteria.30 Energy metabolism, as mentioned above, could be limiting to growth just as easily as the anabolic reactions of which nucleic acid synthesis are examples. Both M. leprae and M. tuberculosis are capable of oxidative metabolism of carbon sources and appear to have the mechanism for oxidative phosphorylation (see Refs. 6,11). However, it is becoming increasingly apparent that myco- bacteria can switch to microaerophilic growth,31 resulting in a slower rate of catabolism as shown in M. tuberculosis during submerged growth.32 This may also occur, when oxygen tension is low, during growth of mycobacteria in host tissue. Few direct comparisons of rates of catabolic activity have been made; even those shown in Table 2 include M. phlei grown in static batch culture but M. lepraemurium and M. leprae grown in vivo. Specific activities of the enzymes for carbohydrate catabolism are generally similar in extracts from M. phlei, M. smegmatis and M. tuberculosis although M. phlei elaborates more cytochromes than M. tuberculo- sis (reviewed in Refs. 6,11). Most of these activities are around ten times lower in M. leprae, though this may reflect the high morbidity (~80 to 90%) of cells in suspensions of leprosy bacilli rather than its slow growth rate.11 From the foregoing, it is clear that rates of nucleic acid synthesis are related to the growth rates of mycobacteria, but it is quite possible that rates of carbohydrate catabolism are also related.

Table 2 Rates of catabolism of carbon sources to CO2 Organism

Carbon source (l|iCi) Af phlei M. lepraemurium M. leprae

[U-'*C]glycerol 150000* 51000 7000 [l,4-1''C]succinate 360000 26000 1200

"not published, other results %ct Ref 11 560 TUBERCULOSIS AND LEPROSY

Furthermore, these metabolic activities could be low as a conse- quence of slow growth, rather than being limiting to growth. There is a need to perform experiments to determine directly which activity is actually most important in limiting mycobacterial growth. Very simply, the rationale is to use inhibitors of different

metabolic activities which act proportionately to their concentra- Downloaded from https://academic.oup.com/bmb/article/44/3/547/283569 by guest on 28 September 2021 tion. Thus, if they inhibit growth at any concentration (i.e. not with threshold effect) there can be no excess of the activity being inhibited over that which is required for the rate of growth observed and the activity is therefore important in limiting growth. The extent of any conclusions from such experiments is difficult to predict, but at least it should be possible to ascertain whether catabolism or anabolism limits growth of mycobacteria and thus perhaps answer the major question in the field of metabolism in mycobacteria.

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