Microbiostatic effect of murine-activated macrophages for Toxoplasma gondii. Role for synthesis of inorganic nitrogen oxides from L-arginine

Recent studies show the importance of a single amino acid, L-arginine, as a necessary substrate for activated macrophage-mediated cytotoxic activity for tumor target cells and microbiostatic function for Cryptococcus neoformans. The present studies were carried out to determine the role of the L-arginine-dependent macrophage effector function on the microbiostatic effects of activated macrophages on the obligate intracellular protozoan, Toxoplasma gondii. A guanidino methylated derivative of L-arginine, NGmonomethyl-L-arginine (NGMMA), a competitive inhibitor of the L-arginine-dependent effector pathway, virtually abolished the normally potent microbiostatic effect of macrophages for Toxoplasma gondii after activation of the macrophages in vitro by IFN-gamma and LPS or in vivo by i.p. injection of killed Corynebacterium parvum. Addition of supplemental L-arginine to the culture medium overcame the capacity of NGMMA to block activated macrophage-mediated microbiostasis of Toxoplasma. The ability of NGMMA to inhibit the microbiostatic capacity of activated macrophages for Toxoplasma gondii correlated with almost total inhibition of synthesis of nitrite, nitrate, and L-citrulline from L-arginine. Therefore, as is the case for tumor target cells and C. neoformans, the synthesis of inorganic nitrogen oxides from a terminal guanidino nitrogen atom of L-arginine appears to be essential for murine cytotoxic activated macrophage mediated microbiostatic capacity for T. gondii.     

L-arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells

L-Arginine is required for expression of the activated macrophage cytotoxic effector mechanism that causes inhibition of mitochondrial respiration, aconitase activity, and DNA synthesis in tumor target cells. This effector mechanism is active in the presence of L-arginine even when the cocultivation medium lacks all other amino acids and serum. Cytotoxic activated macrophage-induced inhibition of mitochondrial respiration in target cells is proportional to the concentration of L-arginine in the medium. L-Arginine must be present during the cocultivation period. Pretreatment of cytotoxic activated macrophages with L-arginine or posttreatment of the target cells after cocultivation is not effective. D-Arginine does not substitute for L-arginine and at high concentrations is a competitive inhibitor of the L-arginine-dependent effector mechanism. Other analogues that could not replace L-arginine include agmatine, argininic acid, arginine hydroxamate, and tosyl-L-arginine methyl ester. L-homoarginine, however, can effectively substitute for L-arginine. NG-monomethyl-L-arginine is a potent competitive inhibitor of this effector mechanism. High concentrations of lipopolysaccharide do not reverse inhibition of the L-arginine-dependent effector mechanism by NG-monomethyl-L-arginine. However, inhibition of the effector mechanism by NG-monomethyl-L-arginine can be overridden by increasing the concentration of L-arginine in the culture medium. We compared NGNG-dimethyl-L-arginine and NGN1G-dimethyl-L-arginine with NG-monomethyl-L-arginine as inhibitors of the L-arginine-dependent effector mechanism. The results show that the inhibitory effect of these guanidino methylated derivatives of L-arginine is highly determined by structure. Guanidine is a weak competitive inhibitor of the L-arginine-dependent effector mechanism. The requirement for L-arginine does not appear to be for protein synthesis, creatine biosynthesis, polyamine biosynthesis, or ADP ribosylation reactions. Bacterial lipopolysaccharide is effective as a second signal only when the cocultivation medium contains L-arginine, and this strict L-arginine dependency is not overridden by increasing the concentration of lipopolysaccharide. Bovine liver arginase, by competing for L-arginine in the cocultivation medium, inhibits the L-arginine-dependent activated macrophage cytotoxic effector mechanism.     

Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells

Previous studies show that cytotoxic activated macrophages cause a reproducible pattern of metabolic inhibition in viable tumor target cells. This includes inhibition of DNA synthesis, two oxidoreductases of the mitochondrial electron transport chain (NADH: ubiquinone oxidoreductase and succinate: ubiquinone oxidoreductase), and the citric acid cycle enzyme aconitase. This pattern of metabolic inhibition is induced by a cytotoxic activated macrophage associated biochemical pathway with L-arginine deimination activity that synthesizes L-citrulline from L-arginine and oxygenated nitrogen derivatives from the imino nitrogen removed from the guanido group of L-arginine. Here we report that macrophages activated in vivo by infection with bacillus Calmette-Guérin or in vitro by murine rIFN-gamma or murine IFN-alpha/beta (in the presence of the second signal LPS in all cases) develop inhibition of aconitase and the same two oxidoreductases of the mitochondrial electron transport chain as was documented earlier in target cells of cytotoxic activated macrophages. In addition, this pattern of metabolic inhibition which develops in cytotoxic activated macrophages is caused by the L-arginine-dependent effector mechanism. Inhibition of mitochondrial respiration by effectors of the L-arginine-dependent cytotoxicity system results in a compensatory increase in activity of the glycolytic pathway. We speculate that the pattern of metabolic inhibition induced in cytotoxic activated macrophages by the L-arginine-dependent effector system causes changes in the macrophage intracellular environment that increases resistance to certain facultative and obligate intracellular pathogens.     

Activated macrophages destroy intracellular Leishmania major amastigotes by an L-arginine-dependent killing mechanism

Macrophages infected with amastigotes of Leishmania major and treated with IFN-gamma in vitro develop potent antimicrobial activities that eliminate the intracellular parasite. This antileishmanial activity was suppressed in a dose dependent fashion by NG-monomethyl-L-arginine (NGMMLA), a competitive inhibitor of nitrite, nitrate, nitric oxide and L-citrulline synthesis from L-arginine. Excess L-arginine added to infected macrophage cultures reversed the inhibitory effects of NGMMLA. Addition of arginase to culture media inhibited intracellular killing by IFN-gamma-treated cells. Similar effects were seen with macrophages obtained from BCG-infected C3H/HeN mice. Increased levels of nitrite, an oxidative product of the L-arginine-dependent effector mechanism, was measured in cultures of infected IFN gamma-treated macrophages as well as infected BCG-activated macrophages. Nitrite production correlated with development of antileishmanial activity. Nitrite production and microbicidal activity both decreased when in vivo or in vitro-activated macrophages were cultured in the presence of either arginase or NGMMLA. Nitric oxide synthesized from a terminal guanidino nitrogen atom of L-arginine and a precursor of the nitrite measured, may disrupt Fe-dependent enzymatic pathways vital to the survival of amastigotes within macrophages.     

Regulation of macrophage physiology by L-arginine: role of the oxidative L-arginine deiminase pathway

The L-arginine content of the extracellular fluid in sites of predominant macrophage infiltration is reduced below plasma levels due to the activity of macrophage-derived arginase. Investigation of the effects of altered L-arginine availability on macrophage physiology reveals that culture of rat peritoneal macrophages in media containing L-arginine in the concentrations present in inflammatory lesions (less than 0.1 mM) enhances activation-associated functions. In contrast, culture in the higher L-arginine concentrations found in standard tissue culture media (0.4 to 1.2 mM) suppresses most macrophage functions (superoxide production, phagocytosis, and protein synthesis). An exception is the tumor cytotoxicity of Corynebacterium parvum-elicited macrophages which is enhanced by culture in supraphysiologic concentrations of L-arginine. Work reported here investigated the mechanisms for these L-arginine-dependent effects and, more specifically, the role of the recently described oxidative L-arginine deiminase pathway in the regulation of macrophage physiology. Overnight culture of resident or C. parvum-elicited peritoneal macrophages in media containing increasing concentrations of L-arginine (6 microM to 1 mM) resulted in: inhibition of electron transport chain activity (resident and C. parvum-elicited macrophages), increased lactate production (resident macrophages), and decreased ATP content (resident and C. parvum-elicited macrophages). In line with these findings, viability was markedly decreased after 2 days of culture when the initial L-arginine concentration was greater than or equal to 0.1 mM. As shown before, increasing media concentrations of L-arginine were associated with suppression of superoxide production and cytotoxicity in resident macrophages, and with reduced superoxide production and increased cytotoxicity in C. parvum-elicited macrophages. All L-arginine-dependent metabolic and functional alterations, as well as the loss of viability, were prevented by NG-monomethyl-L-arginine, a specific inhibitor of the oxidative L-arginine deiminase pathway. These results demonstrate that flux of L-arginine through the oxidative L-arginine deiminase pathway results in the inhibition of oxidative metabolism in rat macrophages. This metabolic inhibition may, through alterations in the macrophage high energy phosphate stores, mediate the suppression of cell functions and result ultimately in cell death.     

Factors influencing the in vitro growth of Mycobacterium leprae: effect of oxygen.

In our efforts to evaluate factors responsible for in vitro growth of Mycobacterium leprae in DH medium and also to improve the system, effects of oxygen tension on in vitro growth of M. leprae were determined. This was achieved by varying the ratio between DH medium and free air space above the medium in the culture tubes. Growth-competent M. phlei (ATCC 11758) could tolerate all the oxygen it can get in the medium. On the other hand, M. leprae seemed to be of microaerophilic nature. In vivo-grown M. leprae cells were more sensitive than their counterparts that were adapted to in vitro environment. In vivo-grown cells grew better when 70% of the space in culture tube was occupied by DH medium. These in vitro-adapted cells gave optimum growth in subcultures when the air spaces in the culture tubes were 40-50%. The role of oxygen tensions in the development of lesions in leprosy patients and armadillos has been discussed.     

Superoxide-induced deimination of arginine in hematopoietic cells

Murine bone marrow cells can produce citrulline directly from L-arginine without intermediate ornithine. An L-arginine-dependent biochemical pathway synthesizing L-citrulline and nitrate, coupled to an effector mechanism has also been recently demonstrated in murine cytotoxic activated macrophages. We show herein that L-citrulline synthesis in murine bone marrow cells can be induced by the generation of superoxide. It can take place in an arginine-free medium, suggesting the implication of a superoxide-dependent peptidyl arginine deiminase.     

Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages

We examined the cytolytic mechanisms of activated macrophages by using proteose peptone- or thioglycollate broth-induced mouse peritoneal macrophages or mouse macrophage hybridomas as effector cells, L.P3 cells, a clone of L929 cells, and P815 cells as target cells, and IFN-gamma and LPS as activators. It was determined that TNF is the main cytolytic molecule against L.P3 cells from the following results: 1) activated macrophages can produce TNF; 2) TNF shows cytotoxic activity against L.P3 cells; 3) the addition of anti-TNF antibody inhibited most of the cytolytic activity of activated macrophages against L.P3 cells. On the other hand, it was concluded that the main cytolytic mechanism against P815 cells is the production of NO2-/NO3- from L-arginine, from the following results: 1) activated macrophages can produce NO2-; 2) NaNO2 shows high cytotoxic activity against P815 cells; 3) the depletion of L-arginine from the medium inhibited most of the cytolytic activity of activated macrophages against P815 cells and NO2- production by activated macrophages. In this study, however, cytostatic effects of L-arginine-dependent effector mechanism were not studied. Thus, these results show that activated macrophages can express at least two cytolytic mechanisms independently, namely, the one that appears to be mediated by the L-arginine-dependent effector mechanism and the second that appears to be mediated directly by TNF. Furthermore, it was demonstrated that TNF and L-arginine-dependent NO2- production act synergistically as killing mechanisms of activated macrophages. These mechanisms can explain the cytolytic activity of activated macrophages against a variety of target cells.     

An in vitro model for the lepromatous leprosy granuloma: fate of Mycobacterium leprae from target macrophages after interaction with normal and activated effector macrophages

The lepromatous leprosy granuloma is a dynamic entity requiring a steady influx of macrophages (Mphi) for its maintenance. We have developed an in vitro model to study the fate of Mycobacterium leprae in a LL lesion, with and without immunotherapeutic intervention. Target cells, consisting of granuloma Mphi harvested from the footpads of M. leprae-infected athymic nu/nu mice, were cocultured with normal or IFN-gamma-activated (ACT) effector Mphi. The bacilli were recovered and assessed for viability by radiorespirometry. M. leprae recovered from target Mphi possessed high metabolic activity, indicating a viable state in this uncultivable organism. M. leprae recovered from target Mphi incubated with normal effector Mphi exhibited significantly higher metabolism. In contrast, bacilli recovered from target Mphi cocultured with ACT effector Mphi displayed a markedly decreased metabolic activity. Inhibition by ACT Mphi required an E:T ratio of at least 5:1, a coculture incubation period of 3-5 days, and the production of reactive nitrogen intermediates, but not reactive oxygen intermediates. Neither IFN-gamma nor TNF-alpha were required during the cocultivation period. However, cell-to-cell contact between the target and effector Mphi was necessary for augmentation of M. leprae metabolism by normal effector Mphi as well as for inhibition of M. leprae by ACT effector Mphi. Conventional fluorescence microscopy and confocal fluorescence microscopy revealed that the bacilli from the target Mphi were acquired by the effector Mphi. Thus, the state of Mphi infiltrating the granuloma may markedly affect the viability of M. leprae residing in Mphi in the lepromatous lesion.     

L-arginine independent macrophage tumor cytotoxicity

We have investigated the role of L-arginine in macrophage tumor cytotoxicity in coculture. L929, EMT-6, MCA-26, and P815 targets were all susceptible to cytolysis by activated macrophages when cocultured in medium containing L-arginine. When cocultured in arginine-free medium, these targets displayed comparable or even higher levels of lysis. L1210 targets were lytically resistant under either condition. However, 59Fe release from this target did reflect strong dependence on the presence of arginine. The structural analogue, NG-monomethyl-L-arginine, was an effective inhibitor of iron-release from L1210 targets cocultured with activated macrophages, whereas it had minimal inhibitory effects on release of 51Cr from cocultured L929 cells. These results suggest that the L-arginine requiring cytotoxic pathway of activated macrophage is independent of major effector mechanisms involved in tumor cell lysis.     

Tissue-specific down-regulation of RIPK 2 in Mycobacterium leprae-infected nu/nu mice

RIPK 2 is adapter molecule in the signal pathway involved in Toll-like receptors. However, there has been no reported association between receptor-interacting serine/threonine kinase 2 (RIPK 2) expression and the infectious diseases involving mycobacterial infection. This study found that its expression was down-regulated in the footpads and skin but was up-regulated in the liver of Mycobacterium leprae-infected nu/nu mice compared with those of the M. leprae non-infected nu/nu mice. It was observed that the interlukin-12p40 and interferon-gamma genes involved in the susceptibility of M. leprae were down-regulated in the skin but were up-regulated in the liver. Overall, this suggests that regulation of RIPK 2 expression is tissue-specifically associated with M. leprae infection.     

Effect of L-arginine on the retention of macrophage tumoricidal activity

It has been reported that the tumoricidal activity of macrophages (M phi) depends on L-arginine and that L-arginine metabolites such as reactive nitrogen intermediates alter M phi physical capacities. The aim of this report is to investigate the dose-related effect of L-arginine on the expression and retention of M phi tumoricidal activity. Cytotoxicity of M phi activated by IFN-gamma plus LPS was detected in the presence of about 0.1 mM or more of L-arginine. This paralleled the NO2- production in the presence, but not in the absence, of L-arginine. On the other hand, activated M phi were destined to die and lost their tumoricidal activity with time in the presence of 0.3 mM or more L-arginine. They retained, however, considerable activity in the absence or presence of 0.15 mM L-arginine. This retention of M phi cytotoxicity was longer when M phi were preactivated by 100 ng/ml than 10 ng/ml of LPS in combination with IFN-gamma. Addition of indomethacin, an inhibitor of prostaglandin production, did not prevent the decay of M phi cytotoxicity but rather facilitated it even in the absence of L-arginine. Regardless of indomethacin, consecutive stimulation with LPS or LPS plus IFN-gamma during culture was effective in maintaining the tumoricidal activity at a high level. In addition, we found that M phi which had lost tumoricidal activity during culture in L-arginine deficient medium could be reactivated by LPS to attack tumor target cells.     

Effect of trifluoperazine on in vitro ATP synthesis by Mycobacterium leprae

The effect of trifluoperazine (TFP), a calmodulin antagonist, was investigated on in vitro ATP levels of human derived Mycobacterium leprae . M. leprae were obtained from biopsies from multi-bacillary forms of leprosy and were incubated in a modified Dubos medium system which supports limited in vitro synthesis of M. leprae . This incubation was carried out in the absence and presence of different concentrations of trifluoperazine. Samples for estimation of bacillary ATP levels were taken at day 0 and at 14 days of incubation. TFP inhibited ATP levels in M. leprae and this inhibitory effect was marginal at 2.5 μg ml⁻¹ (35% inhibition), highly significant at 5 μg ml⁻¹ (87% inhibition) and almost total at 10 μg ml⁻¹ (98.5% inhibition). This compound appears to have potential as an anti-leprotic drug and also as a broad spectrum anti-mycobacterial agent in view of its anti-tubercular activity reported earlier.     

Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway

The growth and survival of intracellular parasites depends on the availability of extracellular nutrients. Deprivation of nutrients viz glucose or amino acid alters redox balance in mammalian cells as well as some lower organisms. To further understand the relationship, the mechanistic role of L-arginine in regulation of redox mediated survival of Leishmania donovani promastigotes was investigated. L-arginine deprivation from the culture medium was found to inhibit cell growth, reduce proliferation and increase L-arginine uptake. Relative expression of enzymes, involved in L-arginine metabolism, which leads to polyamine and trypanothione biosynthesis, were downregulated causing decreased production of polyamines in L-arginine deprived parasites and cell death. The resultant increase in reactive oxygen species (ROS), due to L-arginine deprivation, correlated with increased NADP+/NADPH ratio, decreased superoxide dismutase (SOD) level, increased lipid peroxidation and reduced thiol content. A deficiency of L-arginine triggered phosphatidyl serine externalization, a change in mitochondrial membrane potential, release of intracellular calcium and cytochrome-c. This finally led to DNA damage in Leishmania promastigotes. In summary, the growth and survival of Leishmania depends on the availability of extracellular L-arginine. In its absence the parasite undergoes ROS mediated, caspase-independent apoptosis-like cell death. Therefore, L-arginine metabolism pathway could be a probable target for controlling the growth of Leishmania parasites and disease pathogenesis.     

Measurement of ATP generation and decay in Mycobacterium leprae in vitro

The intracellular ATP content of Mycobacterium leprae isolated from armadillo tissue was approximately 1.5 X 10(-16) g per bacillus. During in vitro incubation of bacilli at 4 degrees C, 33 degrees C or 37 degrees C there was an exponential decrease in ATP content, the rate depending on the medium and the temperature. M. leprae incorporated phosphate into ATP and into other nucleotide materials during in vitro incubation.     

Alterations of ribonucleotide reductase activity following induction of the nitrite-generating pathway in adenocarcinoma cells

The murine adenocarcinoma cell line TA 3 synthesized nitrite from L-arginine upon stimulation with gamma-interferon (IFN-gamma) associated with tumor necrosis factor (TNF), and/or bacterial lipopolysaccharide (LPS), but not with IFN-gamma, TNF, or LPS added separately. Induction of the NO2(-)-generating activity caused an inhibition of DNA synthesis in TA 3 cells. This inhibition was prevented by the L-arginine analog N omega-nitro-L-arginine, which inhibited under the same conditions nitrite production by TA 3 cells. The TA 3 M2 subclone, selected for enhanced ribonucleotide reductase activity, was found to be less sensitive than the wild phenotype TA 3 WT to the cytostatic activity mediated by the NO2(-)-generating system. Cytosolic preparations from TA 3 M2 cells treated for 24 or 48 h with IFN-gamma, TNF, and LPS exhibited a reduced ribonucleotide reductase activity, compared to untreated control cells. No reduction in ribonucleotide reductase activity was observed when N omega-nitro-L-arginine was added to treated cells. Addition of L-arginine, NADPH, and tetrahydrobiopterin into cytosolic extracts from 24-h treated TA 3 M2 cells triggered the synthesis of metabolic products from the NO2(-)-generating pathway. This resulted in a dramatic inhibition of the residual ribonucleotide reductase activity present in the extracts. The inhibition was reversed by NG-monomethyl-L-arginine, another specific inhibitor of the NO2(-)-generating activity. No L-arginine-dependent inhibition of ribonucleotide reductase activity was observed using extracts from untreated cells that did not express NO2(-)-generating activity. These results demonstrate that, in an acellular preparation, molecules derived from the NO2(-)-generating pathway exert an inhibitory effect on the ribonucleotide reductase enzyme. This negative action might explain the inhibition of DNA synthesis induced in adenocarcinoma cells by the NO2(-)-generating pathway.     

Radiometric Measurement of Metabolic Activity of Mycobacterium lepraemurium

A sensitive and nondestructive radiometric method has been applied to the detection of metabolism of Mycobacterium lepraemurium, as a model for the study of the metabolism and substrate requirements of M. leprae. The method is based on the measurement of the (14)CO(2) produced through the bacterial conversion of [U-(14)C]acetate or [U-(14)C]glycerol by 7 x 10(9) bacteria suspended in 10 ml of either a simple buffer system (K-36) or a complex medium (NC-5). Metabolism of the bacilli was easily detected within 3 days after inoculation and was measured daily. NC-5 medium supported metabolism of M. lepraemurium for several weeks longer than the simple K-36 buffer. The radiometric technique shows promise as a rapid and efficient system for evaluating the metabolism of mycobacteria without introducing any changes in the physiologic state of the organisms, studying their metabolic pathways, determining conditions potentially favorable for multiplication of these organisms in vitro, and studying their susceptibility to inhibition by drugs.     

Growth of Mycobacterium leprae under low oxygen tension

Despite numerous attempts, Mycobacterium leprae has yet to be cultivated in vitro. This organism has been considered as microaerophilic. The effects of various known gas mixtures on the in vitro growth of M. leprae were investigated. A gas mixture containing 2.5% O2 and 10% CO2 was found to be more favourable for the growth of this mycobacterium on artificial medium. Growth was evaluated by three parameters namely cell counts, bacterial ATP and DNA. An optimal growth of M. leprae, as determined by all three parameters, on both liquid and solid media was obtained between 18 and 24 weeks of incubation under optimal gas mixture. Solid medium which contained egg-yolk was relatively more beneficial for in vitro growth than the liquid medium. The cultivated bacilli exhibited some important characteristics specific for M. leprae, including growth in mouse foot-pads. The bacilli gradually lost their power of adaptation to grow on artificial media and did not show any ATP or DNA after about 36 weeks of incubation.     

Macrophage cytotoxicity against Entamoeba histolytica trophozoites is mediated by nitric oxide from L-arginine.

The killing of Entamoeba histolytica trophozoites by phagocytes involves oxidative and nonoxidative mediators. In this study, we determine whether L-arginine-derived nitric oxide (NO) is involved in the killing of E. histolytica trophozoites by activated murine macrophages in vitro. Elicited peritoneal and bone marrow-derived macrophages activated with IFN-gamma alone or with IFN-gamma and LPS killed 62 to 73% of amebae, concomitant with increased levels of nitrate (NO2). Depletion of L-arginine by addition of arginase to culture medium abrogated macrophage amebicidal activity. NG-monomethyl L-arginine, an L-arginine analog, competitively inhibited NO2 release and amebicidal activity in a dose-dependent fashion, without affecting H2O2 production; however, the addition of excess L-arginine competitively restored macrophage amebicidal effects. In culture, sodium nitrite and sodium nitroprusside were cytotoxic to E. histolytica and this was reversed by the addition of myoglobin. Exogenously added FeSO4 prevented macrophage cytotoxicity. Addition of superoxide dismutase, a scavenger of O2-, partially inhibited amebicidal activity, without influencing NO2 production. Untreated and LPS-exposed macrophages produced high levels of H2O2 independent from NO2 production and amebicidal effects. However, the addition of catalase, a scavenger of H2O2, inhibited both amebicidal activity and NO2 production by activated macrophages. Our results demonstrate that NO is the major cytotoxic molecule released by activated macrophages for the in vitro cytotoxicity of E. histolytica and that O2- and H2O2 may be cofactors for the NO effector molecule.     

Factors influencing the in vitro growth of Mycobacterium leprae: effect of sulfhydryl compounds

In an attempt to determine the factors that influence the in vitro growth of Mycobacterium leprae in DH medium, the effects of sulfhydryl compounds were studied. Growth of M. leprae was monitored using two biochemical indicators. Only the sulfhydryl compounds, in reduced form, containing carboxyl group could support the growth of M. leprae. Higher cell yields were obtained when these sulfhydryl compounds were supplemented with dithiothreitol, presumable to keep the monothiols in reduced state during long incubation periods. Ascorbic acid could not replace dithiothreitol for this purpose. It is suggested that these carboxylated sulfhydryl compounds play a role in the metabolic activity of M. leprae along with maintaining low redox potential of the medium.