A novel phenolic glycolipid from Mycobacterium leprae possibly involved in immunogenicity and pathogenicity.

A phenolic glycolipid was obtained in high amounts (2% of dry weight) from Mycobacterium leprae isolated from infected armadillo liver. Infrared and nuclear magnetic resonance spectroscopy showed that it is closely related to "mycoside A" from Mycobacterium kansasii and is therefore a glycosylphenolic phthiocerol diester. The crucial difference between the two products is in the composition of the attached trisaccharide. Gas-liquid chromatography-mass spectroscopy showed that the product from M. kansasii is composed of 2,4-di-O-methylrhamnose, 2-O-methylrhamnose, and 2-O-methylfucose, whereas that from M. leprae contains 2,3-di-O-methylrhamnose, 3-O-methylrhamnose, and 3,6-di-O-methylglucose. The distinct composition of the oligosaccharide segment of the glycolipid from M. leprae may make it useful for the chemical and serological differentiation of this organism from other mycobacteria. Surprisingly large quantities (2.2 mg/g of dry liver) of the glycolipid were also found in infected liver residue freed of M. leprae, suggesting that it may be responsible for the electron-transparent "foam" surrounding the organism in infected lepromatous tissue.     

Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus Calmette-Guérin. An in vitro comparison of leprosy vaccine components

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.     

Subcellular localization of Mycobacterium leprae-specific phenolic glycolipid (PGL-I) antigen in human leprosy lesions and in M. leprae isolated from armadillo liver

Phenolic glycolipid (PGL-I), an antigen specific to Mycobacterium leprae, was localized subcellularly in M. leprae residing in human skin, in M. leprae isolated from armadillo liver ('isolated M. leprae') and outside M. leprae in human lepromatous skin. For a quantitative localization of PGL-I sites, specimens, including skin segments stored for 6 years in glutaraldehyde, were embedded in hydrophilic Lowicryl (K4M) resin for ultrathin sectioning. Ultracryosections and Araldite sections of comparable specimens were used for comparison of localization results. A monoclonal antibody (F 47-21-3) directed to antigenic oligosaccharide of PGL-I was employed as primary antibody in immunogold labelling of ultrathin sections. K4M-immunogold methods gave very satisfactory quantitative gold-labelling of PGL-I. The localization of PGL-I by this method partially corresponded with sites detectable in both ultracryosections and the qualititatively superior Araldite sections, but new sites were also localized. Cell walls in human M. leprae and in isolated M. leprae possessed many PGL-I sites, particularly in dividing organisms. PGL-I or its antigenic oligosaccharide was also found, to a lesser extent, in the bacterial cytoplasm. Capsules discernible around part of isolated M. leprae cells displayed heavy PGL-I labelling, sometimes clearly confined to a zone distant from the cell wall. Extrabacterial PGL-I in M. leprae-infected human skin was encountered (1) in phagolysosomes and cytoplasm proper of dermal macrophages containing M. leprae, and (2) intra- and extracellularly in epidermal areas where basal cells harboured M. leprae in untreated multibacillary patients.     

Immunogold labeling method for Mycobacterium leprae-specific phenolic glycolipid in glutaraldehyde-osmium-fixed and Araldite-embedded leprosy lesions

Phenolic glycolipid (PGL)-I, a Mycobacterium leprae-specific antigen currently used for serodiagnosis of preclinical leprosy, has thus far not been localized subcellularly in leprosy bacilli and their host cells. In this study, we developed an immunogold-labeling technique for qualitative identification of PGL-I sites in glutaraldehyde-osmium-fixed and Araldite-embedded M. leprae and host macrophages in human skin biopsies. Such "hard-fixed," plastic-embedded skin and nerve biopsies from patients with varying cell-mediated immunity to leprosy are amply available worldwide. Our method involves etching of plastic sections with H2O2, incubation with swine serum to eliminate nonspecific labeling, and long (22 hr) incubation at room temperature with monoclonal antibodies to PGL-I. Gold labeling was seen predominantly on cell walls of M. leprae, in vacuolar spaces of bacillated phagolysosomes, and occasionally on the cytoplasm and cell membrane of M. leprae. Host macrophage cytoplasm was labeled very infrequently. This technique allows studies on possibly persisting antigenic PGL-I in multibacillary leprosy patients during or after multidrug therapy. The method may also prove useful for subcellular localization of specific bacterial lipids in other mycobacterial diseases, including tuberculosis.     

Structural elucidation of the major phenolic glycolipid from Mycobacterium kansasii. II. Presence of a novel dideoxyhexose

A novel O-methyl-2,6-dideoxyhexose was isolated from the major phenolic glycolipid (previously called mycoside A) of Mycobacterium kansasii. Its molecular weight (162) was determined by gas chromatography-mass spectrometry analysis (chemical ionization with ammonia as reactant gas) of its underivatized reducing form. The methoxyl group was located by electron impact-mass spectrometry of its alditol acetate. The configuration was established by 1H NMR of its peracetylated derivative. The structure 2,6-dideoxy-4-O-methyl-arabino-hexopyranose is proposed for this new sugar. Evidence is also presented that the phenolic glycolipid previously called mycoside A is an antigen of M. kansasii since it reacts with rabbit antisera raised against whole M. kansasii.     

Human phagocyte oxidative burst activation by BCG, M. leprae, and atypical mycobacteria: defective activation by M. leprae is not reversed by interferon gamma

The activation of the phagocyte oxidative respiratory burst by various mycobacteria was evaluated in an in vitro assay, by measuring the chemiluminescence, associated to the release of oxidizing species, generated by normal human whole blood phagocytes. All mycobacterium species, except Mycobacterium leprae, induced a significant chemiluminescence response. The strongest stimulus was provided by BCG, followed by M. triviale, M. chelonei, and M. fortuitum. M. kansasii, M. intracellulare, and M. lepraemurium elicited a weak response, although higher than that triggered by M. leprae. Both polymorphonuclear and mononuclear cells contributed to the whole blood cell chemiluminescence stimulated by mycobacteria, mononuclear cells being more efficient on a per cell basis. Phagocyte activation by recombinant interferon gamma did not improve M. leprae ability to trigger a significant chemiluminescence response. The failure of M. leprae and of some atypical mycobacteria to stimulate a strong phagocyte oxidative respiratory burst may have some relevance to their pathogenicity.     

Structure of the major triglycosyl phenol-phthiocerol of Mycobacterium tuberculosis (strain Canetti)

Phenol-phthiocerol glycolipids have been found previously in Mycobacterium leprae, M. kansasii, M. bovis and M. marinum, but not in M. tuberculosis. A search for glycolipids in this latter species showed that the Canetti strains of M. tuberculosis synthesize a major triglycosyl phenol-phthiocerol, accompanied by minor amounts of other glycolipids with a similar aglycone moiety. The triglycoside moiety has the following structure: 2,3,4-tri-O-methyl L-fucopyranosyl(alpha 1----3)L-rhamnopyranosyl(alpha 1----3)2-O-methyl L-rhamnopyranosyl(alpha 1-. The aglycone moiety consists in phenol-phthiocerol (two homologs). Its two secondary alcohol functions are esterified by mycocerosic acids (homologs with 26-32 carbon atoms and with 2-4 methyl branches). The proposed structure differs on several points from the M. leprae glycolipids, but presents some analogy with the major glycolipid of M. kansasii. A minor monoglycosyl phenol-phthiocerol was also studied. Its overall structure is very similar to that of M. bovis, with 2-O-methyl rhamnose as sugar moiety.     

Inhibition of macrophage priming by sulfatide from Mycobacterium tuberculosis

Sulfatide from the outer surface of Mycobacterium tuberculosis blocked priming in cultured human monocytes. Monocytes were primed in vitro with either lipopolysaccharide (LPS) or interferon-gamma. Primed monocytes released increased amounts of superoxide anion (O2-) when stimulated with formyl-methionyl-leucyl-phenylalanine or with phorbol myristate acetate. Primed monocytes also showed increased phagocytosis of sheep erythrocytes and increased release of interleukin 1. When primed monocytes were treated with 10 micrograms/ml of sulfatide, these enhanced functions, characteristic of primed monocytes, returned to levels found in unprimed monocytes. (With respect to these functions and others, monocytes or macrophages primed in vitro by exposure to LPS or interferon-gamma resemble macrophages activated in vivo by infection. In vivo, activated macrophages provide non-specific resistance to infection). Inhibition of priming by sulfatide could be detected within 10 min, but maximum effect of sulfatide required 3 to 5 hr. Sulfatide had no effect on O2- release, if it was added after the cells had been stimulated by PMA, suggesting that sulfatide did not inhibit enzymes involved in formation of O2-, but rather that sulfatide inhibited priming. Increasing the amounts of LPS or interferon-gamma did not counteract the effects of sulfatide. Sulfatide did cause monocytes to release some prostaglandin E2 (less than 1 nM), but the amount was not sufficient to inhibit monocyte functions. The effect of sulfatide was not blocked by indomethacin. Other sulfated compounds and other products of mycobacteria did not produce the sulfatide effect. We conclude that M. tuberculosis has on its outer surface a chemical that directly interferes with monocyte priming. In vivo, M. tuberculosis might use sulfatide to block macrophage activation and thereby resist being killed by macrophages.     

DNA hybridization analysis of mycobacterial DNA using the 18-kDa protein gene of Mycobacterium leprae

Abstract DNA hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae , demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae , ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to posses DNA sequences homologous to the 18-kDa protein gene of M. leprae . RFLP analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare . The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae , and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous sequences in various mycobacteria did not affect the specificity of a previously described PCR test for detection of M. leprae , based on the M. leprae 18-kDa protein gene.     

Production and characterization of peptide mimotopes of phenolic glycolipid-I of Mycobacterium leprae

Phenolic glycolipid-I (PGL-I), a Mycobacterium leprae-specific antigen, has been widely used for the serodiagnosis of leprosy and has been implicated in the pathogenesis of leprosy. In an effort to produce an alternate antigen of PGL-I, the mimotope peptides of PGL-I, W(T/R)LGPY(V/M), were obtained using a monoclonal antibody, III603.8, specific to PGL-I by a phage library. The biotin-labeled predominant mimotope peptide of PGLP1, WTLGPYV, bound to III603.8 in a dose-dependent manner in an immunoassay. However, PGLP1 did not bind to anti-PGL-I antibodies in the serum samples from leprosy patients that were reactive to PGL-I. Although the mimotope peptide of WTLGPYV was not effective as an alternate antigen of PGL-I for the serodiagnosis of leprosy, but it would be of interest to know how the mimotope peptides mimic the role of PGL-I antigen in the pathogenesis of M. leprae infection.     

GM-CSF-mediated T-cell activation by macrophages infected with recombinant BCG that secretes major membrane protein-II of Mycobacterium leprae

The potential of Mycobacterium bovis Bacillus Calmette–Guerin (BCG) needs to be augmented to efficiently activate CD4⁺ T cells through macrophages. Mycobacterium leprae -derived recombinant major membrane protein (MMP)-II induced GM-CSF production from macrophages. A recombinant BCG-SM that secretes MMP-II more efficiently produced GM-CSF and activated interferon (IFN)-γ-producing CD4⁺ T cells than did vector control BCG when infected with macrophages. The T-cell activation by BCG-SM was dependent on the GM-CSF production by macrophages. Interleukin (IL)-10 production by macrophages stimulated with M. leprae was inhibited in a GM-CSF-dependent manner when the precursor monocytes were infected with BCG-SM. BCG inducing GM-CSF production was effective in macrophage-mediated T-cell activation partially through IL-10 inhibition.     

A role for CD40-CD40 ligand interactions in the generation of type 1 cytokine responses in human leprosy

The interaction of CD40 ligand (CD40L) expressed by activated T cells with CD40 on macrophages has been shown to be a potent stimulus for the production of IL-12, an obligate signal for generation of Th1 cytokine responses. The expression and interaction of CD40 and CD40L were investigated in human infectious disease using leprosy as a model. CD40 and CD40L mRNA and surface protein expression were predominant in skin lesions of resistant tuberculoid patients compared with the highly susceptible lepromatous group. IL-12 release from PBMC of tuberculoid patients stimulated with Mycobacterium leprae was partially inhibited by mAbs to CD40 or CD40L, correlating with Ag-induced up-regulation of CD40L on T cells. Cognate recognition of M. leprae Ag by a T cell clone derived from a tuberculoid lesion in the context of monocyte APC resulted in CD40L-CD40-dependent production of IL-12. In contrast, M. leprae-induced IL-12 production by PBMC from lepromatous patients was not dependent on CD40L-CD40 ligation, nor was CD40L up-regulated by M. leprae. Furthermore, IL-10, a cytokine predominant in lepromatous lesions, blocked the IFN-gamma up-regulation of CD40 on monocytes. These data suggest that T cell activation in situ by M. leprae in tuberculoid leprosy leads to local up-regulation of CD40L, which stimulates CD40-dependent induction of IL-12 in monocytes. The CD40-CD40L interaction, which is not evident in lepromatous leprosy, probably participates in the cell-mediated immune response to microbial pathogens.     

Mycobacterium lepraemurium activates macrophages but fails to trigger release of superoxide anion

Mycobacterium lepraemurium failed to stimulate a normal respiratory burst when presented to mouse peritoneal or bone marrow macrophages. By comparison, Mycobacterium bovis (strain Bacillus Calmette-Guerin) or Saccharomyces cerevisiae, as expected, stimulated macrophages to release a large amount of superoxide anion (O2-). M. lepraemurium did not interfere with the response to yeast when both microbes were added together to macrophages. The low release of O2- induced by M. lepraemurium was not due to failure of M. lepraemurium to activate or prime macrophages, because exposure of macrophages to M. lepraemurium caused the expected enhancement of O2- release when the macrophages were stimulated by PMA. Similarly, macrophages taken from mice infected with M. lepraemurium were activated, as indicated by high PMA-stimulated O2- release. Macrophages primed in vitro by exposure to Escherichia coli LPS for 24 h did show a moderate O2- response when stimulated by M. lepraemurium, but macrophages primed by exposure to IFN-gamma muramyl dipeptide, or M. lepraemurium showed a weak response when subsequently challenged with M. lepraemurium. The priming effect of M. lepraemurium or LPS decreased substantially after macrophages were cultured in fresh medium for 24 h. Heat killing or opsonization of M. lepraemurium caused the M. lepraemurium to stimulate a high amount of O2- release from LPS-primed macrophages, but heat killing or opsonization of M. lepraemurium had no effect on release of O2- from unprimed macrophages. The results suggest that M. lepraemurium is taken into macrophages by a mechanism that bypasses the FcR and other receptors that are capable of triggering the production of O2-.     

Identification of trehalose dimycolate (cord factor) in Mycobacterium leprae

Glycolipids of Mycobacterium leprae obtained from armadillo tissue nodules infected with the bacteria were analyzed. Mass spectrometric analysis of the glycolipids indicated the presence of trehalose 6,6'-dimycolate (TDM) together with trehalose 6-monomycolate (TMM) and phenolic glycolipid-I (PGL-I). The analysis showed that M. leprae-derived TDM and TMM possessed both alpha- and keto-mycolates centering at C78 in the former and at C81 or 83 in the latter subclasses, respectively. For the first time, MALDI-TOF mass analyses showed the presence of TDM in M. leprae.     

Molecular and immunological analysis of a fibronectin-binding protein antigen secreted by Mycobacterium leprae

By screening a Mycobacterium leprae lambda gt11 genomic DNA library with leprosy-patient sera we have previously identified 50 recombinant clones that expressed novel M. leprae antigens (Sathish et al., 1990). In this study, we show by DNA sequencing and immunoblot analysis that three of these clones express a M. leprae homologue of the fibronectin-binding antigen 85 complex of mycobacteria. The complete gene was characterized and it encodes a 327-amino-acid polypeptide, consisting of a consensus signal sequence of 38 amino acids followed by a mature protein of 289 amino acids. This is the first sequence of a member of the M. leprae antigen 85 complex, and Southern blotting analysis indicated the presence of multiple genes of the 85 complex in the genome of M. leprae. The amino acid sequence displays 75-85% sequence identity with components of the antigen 85 complex from M. tuberculosis, M. bovis BCG and M. kansasii. Furthermore, antibodies to the antigen 85 complex of M. tuberculosis and M. bovis BCG reacted with two fusion proteins containing the amino acid regions 55-266 and 266-327 of the M. leprae protein. The M. leprae 30/31 kDa protein induces strong humoral and cellular responses, as judged by Western blot analysis with patient sera and proliferation of T cells derived from healthy individuals and leprosy patients. Amino acid regions 55-266 and 265-327 both were shown to bind to fibronectin, indicating the presence of at least two fibronectin-binding sites on the M. leprae protein. These data indicate that this 30/31 kDa protein is not only important in the immune response against M. leprae, but may also have a biological role in the interaction of this bacillus with the human host.     

The 16S rRNA nucleotide sequence of Mycobacterium leprae: phylogenetic position and development of DNA probes

The almost complete 16S rRNA sequence from Mycobacterium leprae was determined by direct sequencing of the chromosomal gene amplified by the polymerase chain reaction. The primary sequence revealed an insertion of 12 nucleotides at the 5' end of the 16S rRNA gene, which consists of an A-T stretch and appears to be unique for M. leprae. Within the mycobacteria M. leprae branches off with a group of slow-growing species comprising M. scrofulaceum, M. kansasii, M. szulgai, M. malmoense, M. intracellulare and M. avium. A systematic comparison of the nucleotide sequence resulted in the characterization of oligonucleotide probes which are highly specific for M. leprae. The probes hybridized exclusively to 16S rRNA nucleic acids from M. leprae, but not to nucleic acids from 20 cultivable fast- and slow-growing mycobacteria.     

Axenic incorporation of [U-14C]palmitic acid into the phenolic glycolipid-I of Mycobacterium leprae

Abstract Incorporation of [U-¹⁴C]palmitic acid ([¹⁴C]PA) into the specific phenolic glycolipid-I (PGL-I) of freshly harvested, nude mouse-derived Mycobacterium leprae was investigated in an axenic modified Dubos medium. Incorporation was approximately linear for 10–14 days at pH 7.2, 33°C. No incorporation of radiolabeled phenol, acetate, tyrosine, phenylalanine, bicarbonate, proprionate or UDP-glucose was detected. Procedures known to remove residual host tissue did not diminish the rate of [¹⁴C]PA incorporation, indicating that bacterial metabolism was being measured. The antileprosy compounds, rifampicin and dapsone, significantly reduced incorporation of the label. The ability to quantitate PGL-I synthesis in the extracellular bacillus should facilitate a better understanding of the optimum conditions for metabolism in M. leprae .     

Generation of leukotrienes by human monocytes pretreated with cytochalasin B and stimulated with formyl-methionyl-leucyl-phenylalanine

The N-formylated tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) initiated the generation of immunoreactive C-6 sulfidopeptide leukotrienes and of leukotriene B4 (LTB4) in a dose-dependent manner from monolayers of human monocytes pretreated for 10 min with 5 micrograms/ml of cytochalasin B. The EC50 for the immunoreactive C-6 sulfidopeptide leukotrienes was 10(-8) M FMLP and for immunoreactive LTB4 was 5 X 10(-8) M FMLP. The maximal response to FMLP occurred within 10 min, and the sum of the two classes of leukotrienes generated was about 1/6 that obtained from monocytes stimulated with calcium ionophore A23187. The requirement for cytochalasin B in order for FMLP, but not the calcium ionophore, to stimulate leukotriene generation is compatible with the ability of cytochalasin B to augment in other cells certain stimulus-specific transmembrane responses that are not dependent on the integrity of the cytoskeleton. Resolution by reverse phase high performance liquid chromatography of the products released from monocytes pretreated with cytochalasin B and stimulated with FMLP or calcium ionophore yielded a single peak of immunoreactive LTB4 eluting at the same retention time as the synthetic standard; immunoreactive C-6 sulfidopeptide leukotrienes eluted at the retention times of leukotriene C4 (LTC4) and leukotriene D4 (LTD4). [3H]LTB4 was not metabolically altered by monocytes pretreated with cytochalasin B and activated with FMLP in comparison with cells treated with buffer alone, whereas [3H]LTC4 was partially converted to [3H]LTD4. The leukotriene-generating response of monolayers of human monocytes pretreated with cytochalasin B to FMLP is receptor-mediated, as indicated by the inactivity of the structural analog N-acetyl-methionyl-leucyl-phenylalanine and by the capacity of the FMLP receptor antagonist carbobenzoxyphenylalanyl-methionine to inhibit the agonist action of FMLP in a dose-response fashion.     

Mycobacterium leprae inhibits dendritic cell activation and maturation

Leprosy presents with a clinical spectrum of skin lesions that span from strong Th1-mediated cellular immunity and control of bacillary growth at one pole to poor Ag-specific T cell immunity with extensive bacillary load and Th2 cytokine-expressing lesions at the other. To understand how the immune response to Mycobacterium leprae is regulated, human dendritic cells (DC), potent inducers of adaptive immune responses, exposed to M. leprae, Mycobacterium tuberculosis (Mtb), and Mycobacterium bovis bacillus Calmette-Guerin (BCG) were studied for their ability to be activated and to prime T cell proliferation. In contrast with Mtb and BCG, M. leprae did not induce DC activation/maturation as measured by the expression of selected surface markers and proinflammatory cytokine production. In MLR, T cells did not proliferate in response to M. leprae-stimulated DC. Interestingly, M. leprae-exposed MLR cells secreted increased Th2 cytokines as well as similar Th1 cytokine levels as compared with Mtb- and BCG-exposed cells. Gene expression analysis revealed a reduction in levels of mRNA of DC activation and maturation markers following exposure to M. leprae. Our data suggest that M. leprae does not induce and probably suppresses in vitro DC maturation/activation, whereas Mtb and BCG are stimulatory.     

*NO dissociation represents the rate limiting step for O2-mediated oxidation of ferrous nitrosylated Mycobacterium leprae truncated hemoglobin O

Mycobacterium leprae truncated hemoglobin O (trHbO) protects from nitrosative stress and sustains mycobacterial respiration. Here, kinetics of M. leprae trHbO(II)-NO denitrosylation and of O(2)-mediated oxidation of M. leprae trHbO(II)-NO are reported. Values of the first-order rate constant for *NO dissociation from M. leprae trHbO(II)-NO (k(off)) and of the first-order rate constant for O(2)-mediated oxidation of M. leprae trHbO(II)-NO (h) are 1.3 x 10(-4) s(-1) and 1.2 x 10(-4) s(-1), respectively. The coincidence of values of k(off) and h suggests that O(2)-mediated oxidation of M. leprae trHbO(II)-NO occurs with a reaction mechanism in which *NO, that is initially bound to heme(II), is displaced by O(2) but may stay trapped in a protein cavity(ies) close to heme(II). Next, M. leprae trHbO(II)-O(2) reacts with *NO giving the transient Fe(III)-OONO species preceding the formation of the final product M. leprae trHbO(III). *NO dissociation from heme(II)-NO represents the rate limiting step for O(2)-mediated oxidation of M. leprae trHbO(II)-NO.