Molecular characterization and immunogenic function of ML1899 (LipG) of Mycobacterium leprae.

Introduction. ML1899 is conserved in all mycobacterium sp. and is a middle member of mle-ML1898 operon involved in mycolic acid modification.Aim. In the present study attempts were made to characterize ML1899 in detail.Methodology. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in Escherichia coli for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.Results. In silico analysis predicted ML1899 as a member of α/ß hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM-1 min-1 catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. K m, V max and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min-1 ml-1 and 27 U mg- 1, respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients.Conclusion. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.

Giant cells lepromatous leprosy. Diffuse dermatitis with exuberant foreign body giant cells in treated lepromatous leprosy / Células gigantes en lepra lepromatosa. Dermatitis difusa con células gigantes tipo cuerpo extraño exuberantes en lepra lepromatosa tratada

Patients with lepromatous leprosy that have received treatment for many years usually get follow up biopsies for persistent skin lesions or positive bacilloscopy even if the values are lower than in the initial bacilloscopy. We report the case of a 48-year old woman with long-standing lepromatous leprosy of 15 years of evolution, with a bacterial index of 4 in the direct smear and the initial skin biopsy. The patient was treated with multidrug therapy for 32 months although the treatment recommended by the World Health Organization (WHO) is only for 12 months. A skin biopsy was taken to determine if there was an active disease. We observed a diffuse dermal inflammation with numerous foreign body giant cells and vacuolated macrophages (Virchow´s cells). These cells contained granular acid-fast material that was also positive with immunohistochemistry for BCG. There were fragmented bacilli and the BI was 2. These cells were also strongly positive for CD68. The biopsy was interpreted as a residual form of lepromatous leprosy that did not require further multidrug therapy. We have observed similar histological profiles in several cases. The lack of clinical data makes it a histological challenge. The accumulation of lipids in these giant cells is due to bacillary destruction and fusion of vacuolated macrophages. We discuss here the role of bacillary and host lipids in the pathogenesis of lepromatous leprosy. We concluded that there was no need to extend the 12-month multidrug therapy recommended by WHO.

Los pacientes con lepra lepromatosa (LL) que han recibido tratamiento durante años, usualmente tienen seguimiento con biopsias de piel para lesiones persistentes o con baciloscopia positiva, con valores menores a los iniciales. Presentamos una mujer de 48 años con LL de 15 años de evolución, con índice bacilar (IB) 4 en el extendido directo y en la biopsia, que recibió terapia multidroga durante 32 meses, aunque el tratamiento recomendado por la Organización mundial de la salud (OMS) es de 12 meses. Se tomó una biopsia de piel para determinar si la enfermedad estaba activa. Se observó inflamación dérmica difusa con numerosas células gigantes tipo cuerpo extraño y macrófagos vacuolados (células de Virchow). Estas células, CD68 positivas, contenían material granular ácido-alcohol resistente, positivo con inmunohistoquímica para BCG. Se encontraron bacilos fragmentados y el IB fue de 2. Se interpretó como una forma residual de LL y que la paciente no requería MDT adicional. Este perfil histológico lo hemos observado en casos similares. Sin datos clínicos estas biopsias son un reto diagnóstico. La acumulación de lípidos en estas células gigantes se debe a la destrucción bacilar y a la fusión de macrófagos vacuolados. Revisamos el papel de los lípidos del bacilo y del huésped en la patogénesis de la LL. En estos casos no es necesario extender los 12 meses de MDT recomendados por la OMS. En el seguimiento de los pacientes se recomienda contar con los hallazgos clínicos, la baciloscopia, la biopsia anual de piel y los títulos IgM anti-glicolípido fenólico.

Células gigantes en lepra lepromatosa: dermatitis difusa con células gigantes exuberantes de tipo cuerpo extraño en lepra lepromatosa tratada / Giant cells lepromatous leprosy: Diffuse dermatitis with exuberant foreign body giant cells in treated lepromatous leprosy

Resumen Los pacientes con lepra lepromatosa que han recibido tratamiento durante años, usualmente requieren seguimiento con biopsias de piel para detectar lesiones persistentes o si la baciloscopia es positiva, incluso si los valores son menores que los iniciales. Se presenta el caso de una mujer de 48 años de edad con lepra lepromatosa de 15 años de evolución, índice bacilar de 4 en el extendido directo y en la biopsia, que recibió tratamiento con múltiples medicamentos durante 32 meses, aunque lo recomendado por la Organización Mundial de la Salud (OMS) es una duración de 12 meses. Se tomó una biopsia de piel para determinar si la enfermedad estaba activa. Se observó inflamación dérmica difusa con numerosas células gigantes de tipo cuerpo extraño y macrófagos vacuolados (células de Virchow). Estas células, CD68 positivas, contenían material granular ácido-alcohol resistente positivo con inmunohistoquímica para BCG. Se encontraron bacilos fragmentados y el índice bacilar fue de 2. Se interpretó como una forma residual de lepra lepromatosa y se concluyó que la paciente no requería prolongar el tratamiento con múltiples medicamentos. Este perfil histológico se ha observado en casos similares, pero sin datos clínicos estas biopsias representan un reto diagnóstico. La acumulación de lípidos en estas células gigantes se debe a la destrucción bacilar y a la fusión de macrófagos vacuolados. Se revisó el papel de los lípidos del bacilo y del huésped en la patogenia de la lepra lepromatosa. En estos casos, no es necesario extender los 12 meses de tratamiento con múltiples medicamentos recomendados por la OMS. En el seguimiento de los pacientes, se recomienda contar con los hallazgos clínicos, la baciloscopia, la biopsia anual de piel y los títulos IgM antiglucolípido fenólico.

Abstract Patients with lepromatous leprosy that have received treatment for many years usually get follow up biopsies for persistent skin lesions or positive bacilloscopy even if the values are lower than in the initial bacilloscopy. We report the case of a 48-year old woman with long-standing lepromatous leprosy of 15 years of evolution, with a bacterial index of 4 in the direct smear and the initial skin biopsy. The patient was treated with multidrug therapy for 32 months although the treatment recommended by the World Health Organization (WHO) is only for 12 months. A skin biopsy was taken to determine if there was an active disease. We observed a diffuse dermal inflammation with numerous foreign body giant cells and vacuolated macrophages (Virchow´s cells). These cells contained granular acid-fast material that was also positive with immunohistochemistry for BCG. There were fragmented bacilli and the BI was 2. These cells were also strongly positive for CD68. The biopsy was interpreted as a residual form of lepromatous leprosy that did not require further multidrug therapy. We have observed similar histological profiles in several cases. The lack of clinical data makes it a histological challenge. The accumulation of lipids in these giant cells is due to bacillary destruction and fusion of vacuolated macrophages. We discuss here the role of bacillary and host lipids in the pathogenesis of lepromatous leprosy. We concluded that there was no need to extend the 12-month multidrug therapy recommended by WHO. Clinical findings, bacilloscopy, annual skin biopsy, and anti-phenolic glycolipid-I IgM titers are recommended procedures for the follow-up of these patients.

M. leprae components induce nerve damage by complement activation: identification of lipoarabinomannan as the dominant complement activator.

Peripheral nerve damage is the hallmark of leprosy pathology but its etiology is unclear. We previously identified the membrane attack complex (MAC) of the complement system as a key determinant of post-traumatic nerve damage and demonstrated that its inhibition is neuroprotective. Here, we determined the contribution of the MAC to nerve damage caused by Mycobacterium leprae and its components in mouse. Furthermore, we studied the association between MAC and the key M. leprae component lipoarabinomannan (LAM) in nerve biopsies of leprosy patients. Intraneural injections of M. leprae sonicate induced MAC deposition and pathological changes in the mouse nerve, whereas MAC inhibition preserved myelin and axons. Complement activation occurred mainly via the lectin pathway and the principal activator was LAM. In leprosy nerves, the extent of LAM and MAC immunoreactivity was robust and significantly higher in multibacillary compared to paucibacillary donors (p = 0.01 and p = 0.001, respectively), with a highly significant association between LAM and MAC in the diseased samples (r = 0.9601, p = 0.0001). Further, MAC co-localized with LAM on axons, pointing to a role for this M. leprae antigen in complement activation and nerve damage in leprosy. Our findings demonstrate that MAC contributes to nerve damage in a model of M. leprae-induced nerve injury and its inhibition is neuroprotective. In addition, our data identified LAM as the key pathogen associated molecule that activates complement and causes nerve damage. Taken together our data imply an important role of complement in nerve damage in leprosy and may inform the development of novel therapeutics for patients.

Heparin-binding hemagglutinin (HBHA) of Mycobacterium leprae is expressed during infection and enhances bacterial adherence to epithelial cells.

A heparin-binding hemagglutinin (HBHA) expressed on the surface of Mycobacterium tuberculosis is an antigenic protein that has been implicated in bacterial adherence to epithelial cells and systemic dissemination. In this study, the potential role of the Mycobacterium leprae HBHA (ML-HBHA) homologue in leprosy was investigated. Initially, the in vivo expression of HBHA and its association with the M. leprae cell envelope was confirmed by immunoblotting and proteomic analysis. Mycobacterium leprae recombinant HBHA (rML-HBHA) bound to a heparin-Sepharose column, and its capacity to act as an adhesin was demonstrated in experiments showing that the exogenous addition of the protein to latex beads or to M. leprae cells promotes a dramatic increase in association with epithelial cells. Finally, serum anti-HBHA immunoglobulin G levels were investigated in individuals infected with M. leprae. Altogether, our data indicate that HBHA is recognized during the course of bacterial infection in humans and may play a role in leprosy pathogenesis.

Functional characterization of a small heat shock protein from Mycobacterium leprae.

BACKGROUND: Small heat shock proteins are ubiquitous family of stress proteins, having a role in virulence and survival of the pathogen. M. leprae, the causative agent of leprosy is an uncultivable organism in defined media, hence the biology and function of proteins were examined by cloning M. leprae genes in heterologous hosts. The study on sHsp18 was carried out as the knowledge about the functions of this major immunodominant antigen of M. leprae is scanty. RESULTS: The gene encoding Mycobacterium leprae small heat shock protein (sHsp18) was amplified from biopsy material of leprosy patients, and cloned and expressed in E. coli. The localization and in vitro characterization of the protein are detailed in this report. Data show that major portion of the protein is localized in the outer membrane of E. coli. The purified sHsp18 functions as an efficient chaperone as shown by their ability to prevent thermal inactivation of restriction enzymes SmaI and NdeI. Physical interaction of the chaperone with target protein is also demonstrated. Size exclusion chromatography of purified protein shows that the protein can form multimeric complexes under in vitro conditions as is demonstrated for several small heat shock proteins. CONCLUSION: The small heat shock protein sHsp18 of M. leprae is a chaperone and shows several properties associated with other small heat shock proteins. Membrane association and in vitro chaperone function of sHsp18 shows that the protein may play a role in the virulence and survival of M. leprae in infected host.

[Current advances in leprosy research activities].

Due to the advent of multi-drug therapy (MDT) recommended by the WHO, for the treatment of leprosy, presently, leprosy is regarded as a "curable disease". The number of new cases in Japan is relatively very low, due to which the disease is likely to be neglected, but on scientific grounds, there is a necessity to perform in depth studies. Leprosy caused by M. leprae is still unclear on various aspects including transmission, immunology, nerve damage etc. Here we introduce the recent advances in the field of basic leprosy research.

Studies of lipoproteins of Mycobacterium leprae.

The deciphering of the genomic sequence of Mycobacterium leprae has made possible to predict the possible lipoproteins. The consensus sequence at the N-terminal region of the protein, including the cysteine residue to which the lipid moiety gets attached, provides a clue to the search. As such, more than 20 putative lipoproteins have been identified from Mycobacterium leprae genomic sequence. Lipoprotein LpK (Accession no. ML0603) which encodes for 371 amino acid precursor protein, was identified. Expression of the protein, in Escherichia coli revealed a 33 kD protein, and metabolic labeling experiments proved that the protein was lipidated. The purified lipoprotein was found to induce production of IL-12 in human peripheral blood monocytes which may imply that M. leprae LpK is involved in protective immunity against leprosy. Pursuit of such lipoproteins may reveal insights into the pathogenesis of the disease.

Soroprevalencia para hanseniase em areas endemicas do estado do Para / Soroprevalence for leprosy in endemic areas of the state of Para

Analisou-se a prevalencia de anticoepos IgM utilizando teste sorologico e antigeno especifico Glicolipidio Fenolico I (PGL-I) do Mycobacterium leprae, por meio do teste rapido ML Dipstick em 1611 individuos sadios categorizados em contatos de pacientes hansenicos intradomiciliares e comunitarios, residentes em cinco municipios endemicos para hanseniase. Realizou-se estudo tranversal pela analise do banco de dados do laboratorio de hanseniase, Instituto Evandro Chagas. O estudo estendeu-se de março a dezembro de 1998, com reavaliaçoes anuais ate dezembro de 2002, no qual, os municipios selecionados-Curinopolis, Eldorado do Carajas, Xinguara, Rondon do Para e Dom Eliseu - apresentaram taxas de prevalencia em hanseniase maiores que 20 em cada 10.000 hab. e detecçao de casos novos maiores que quatro em cada 10.000 hab. A idade variou de 2 a 90 anos, devido ao fato de que a doença em areas de alta endemicidade possibilita o diagnostico de crianças. Estudou-se 957 mulheres (957/1611-59.4 por cento), 654 homens (654/1611-40.6 por cento) e observou-se predominancia de soropositividade entre as mulheres na faixa etaria entre 21 e 50 anos, 62.81 por cento contra 53.97 por cento nos homens. A analise foi realizada nos bancos de dados D-BASE, EPI-INFO 6.4 e 2002, BIOESTAT 3.0 e SINAM, existentes no Instituto Evandro Chagas e Secretaria Estadual de Saude do Para...

A tetramer-octamer equilibrium in Mycobacterium leprae and Escherichia coli RuvA by analytical ultracentrifugation.

In the context of the bacterial RuvABC system, RuvA protein binds to and is involved in the subsequent processing of a four-way DNA structure called Holliday junction that is formed during homologous recombination. Four crystal structures of RuvA from Escherichia coli (EcoRuvA) showed that it was tetrameric, while neutron scattering and two other crystal structures for RuvA from Mycobacterium leprae (MleRuvA) and EcoRuvA showed that it was an octamer. To clarify this discrepancy, sedimentation equilibrium experiments by analytical ultracentrifugation were carried out and the results showed that MleRuvA existed as a tetramer-octamer equilibrium between 0.2-0.5 mg/ml in 0.1 M NaCl with a dissociation constant of 4 muM, and is octameric at higher concentrations. The same experiments in 0.3 M NaCl showed that MleRuvA is a tetramer up to 3.5 mg/ml, indicating that salt bridges are involved in octamer formation. Sedimentation equilibrium experiments with EcoRuvA showed that it was tetrameric at low concentration in both salt buffers but the protein was insoluble at high-protein concentrations in 0.1 M NaCl. It is concluded that free RuvA exists in an equilibrium between tetrameric and octameric forms in the typical concentration range and buffer found in bacterial cells.

Structure of Mycobacterium tuberculosis chaperonin-10 at 3.5 A resolution.

Chaperonin-60 (cpn60) and chaperonin-10 (cpn10) are essential proteins involved in ATP-dependent folding of several intracellular proteins in the bacterial cell. Folding of the nascent substrate polypeptide takes place in the large central cavity formed by each ring of the tetradecameric cpn60. This large cavity is closed upon capping by the heptameric cpn10. Cpn10s interact with cpn60s primarily through a 17-residue mobile loop and regulate the release and binding of the substrate polypeptide from the cpn60 surface. Here, the structure of M. tuberculosis cpn10 is reported at 3.5 A resolution. The overall structure of the cpn10 monomer is formed of a four-stranded beta-barrel and two long stretches of highly flexible segments: the dome loop and the mobile loop. The seven subunits in the heptamer show very little conformational difference and exhibit nearly perfect sevenfold geometry. The binding sites for metal ions in the dome loop of cpn10 have been identified, suggesting the role of metal ions in the stabilization of the protein. Comparisons with the available cpn10 structures indicate several interesting features.

A postgenomic approach to identification of Mycobacterium leprae-specific peptides as T-cell reagents.

To identify Mycobacterium leprae-specific human T-cell epitopes, which could be used to distinguish exposure to M. leprae from exposure to Mycobacterium tuberculosis or to environmental mycobacteria or from immune responses following Mycobacterium bovis BCG vaccination, 15-mer synthetic peptides were synthesized based on data from the M. leprae genome, each peptide containing three or more predicted HLA-DR binding motifs. Eighty-one peptides from 33 genes were tested for their ability to induce T-cell responses, using peripheral blood mononuclear cells (PBMC) from tuberculoid leprosy patients (n = 59) and healthy leprosy contacts (n = 53) from Brazil, Ethiopia, Nepal, and Pakistan and 20 United Kingdom blood bank donors. Gamma interferon (IFN-gamma) secretion proved more sensitive for detection of PBMC responses to peptides than did lymphocyte proliferation. Many of the peptides giving the strongest responses in leprosy donors compared to subjects from the United Kingdom, where leprosy is not endemic, have identical, or almost identical, sequences in M. leprae and M. tuberculosis and would not be suitable as diagnostic tools. Most of the peptides recognized by United Kingdom donors showed promiscuous recognition by subjects expressing differing HLA-DR types. The majority of the novel T-cell epitopes identified came from proteins not previously recognized as immune targets, many of which are cytosolic enzymes. Fifteen of the tested peptides had > or =5 of 15 amino acid mismatches between the equivalent M. leprae and M. tuberculosis sequences; of these, eight gave specificities of > or =90% (percentage of United Kingdom donors who were nonresponders for IFN-gamma secretion), with sensitivities (percentage of responders) ranging from 19 to 47% for tuberculoid leprosy patients and 21 to 64% for healthy leprosy contacts. A pool of such peptides, formulated as a skin test reagent, could be used to monitor exposure to leprosy or as an aid to early diagnosis.

Hansen's disease in a patient with a history of sarcoidosis.

We report a rare case of concomitant Hansen's disease (HD) and sarcoidosis. Reticulin staining may be a helpful diagnostic tool in establishing the diagnosis of sarcoidosis in skin lesions. The diagnosis of HD can be established despite negative polymerase chain reaction results for the detection of Mycobacterium leprae DNA. Finally, a well-established diagnosis of sarcoidosis does not preclude the development of another granulomatous disorder. Hence, when new lesions developed in a patient with sarcoidosis despite appropriate therapy, other concurrent diagnoses should be pursued.

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

Factors responsible for the in vitro growth of Mycobacterium leprae in Dhople-Hanks (DH) medium, and also to improve the technique devised earlier, and the source of the M. leprae used as inoculum, were investigated. M. leprae were obtained from armadillos and nude mice, both inoculated earlier with human- or armadillo-derived M. leprae. The growth of M. leprae in DH medium was monitored using two biochemical indicators. Normal growth was obtained when inocula were from livers and spleens of M. leprae-infected armadillos. The M. leprae harvested from the footpads of nude mice failed to multiply in the same medium. Using inocula from livers and spleens of infected armadillos, a gradual decrease in inoculum size resulted in a proportionately slower multiplication of M. leprae. When the DH medium was supplemented with whole M. leprae, or cell-free extracts of M. leprae, from irradiated livers and spleens of infected armadillos, nude mouse-derived M. leprae exhibited growth in the DH medium in accord with that obtained using armadillo-derived M. leprae. Similar results were obtained with cell-free extracts of M. leprae harvested from non-irradiated livers and spleens of infected armadillos, but no growth was obtained when the medium was supplemented with extracts from livers or spleens of normal armadillos. These results indicate the possible existence of a growth factor in armadillo-derived M. leprae.

Secreted proteins of Mycobacterium leprae.

In mycobacteria, secreted proteins represent a distinct group, probably of particular importance for development of immune responses following infection. Quantification of individual proteins in culture fluid and corresponding disrupted bacilli permits determination of a localization index for identification of secreted proteins. This procedure cannot be applied to Mycobacterium leprae because secreted proteins are lost during isolation of bacilli from tissues. The DNA sequences of secreted proteins of Mycobacterium tuberculosis were compared with sequences of M. leprae. Genes for homologues of the 85a, 85b, 85c, mpt32 (apa), mpt51, erp, mtc28, Rv2376c, Rv3354 and Rv0526 genes were identified. All of these contain signal sequences typical for secretion in M. leprae. In several instances the local distance between marker genes and occurrence on the same or the complementary DNA strand was similar in these two species. The genomic organisation of genes for secreted proteins is thus very similar in M. leprae and M. tuberculosis, the homology being higher for the mature polypeptide chains than for the corresponding signal peptides.

Down-regulatory effect of Mycobacterium leprae cell wall lipids on phagocytosis, oxidative respiratory burst and tumour cell killing by mouse bone marrow derived macrophages.

The authors have previously demonstrated that lipids from Mycobacterium leprae cell walls inhibit macrophage functions and are endowed with anti-inflammatory properties in vivo. To investigate these observations further, the authors describe here the influence of dead M. leprae or of the lipids extracted from the cell wall of the mycobacterium, enclosed in liposomes, on the phagocytic, oxidative respiratory burst and tumouricidal ability of bone marrow derived macrophages in vitro. Dead M. leprae or its cell wall lipids abrogated the oxidative respiratory burst and phagocytic ability of mouse bone marrow derived macrophages. A dose-dependent inhibitory effect of the bacterial lipid extract on tumour cell killing by lipopolysaccharide (LPS)-activated bone marrow derived macrophages was demonstrated. However, when delipidated M. leprae was added to cultures of bone marrow derived macrophages, immune phagocytosis and superoxide production was up-regulated. Mycobacterium leprae or its lipids did not appear to be toxic to those cells assayed by the MTT (methyl thiazol tetrazolium) test. These data, added to our preceding observations, support the hypothesis that the down-regulatory activity of M. leprae wall lipids on macrophage function might be one of the evasive mechanisms of the bacterium to enable it to perpetuate itself in the host tissues.

Biometals in skin and sera of leprosy patients and their correlation to trace element contents of M. leprae and histological types of the disease; a comparative study with cutaneous tuberculosis.

The present study has provided information on the biometal contents of killed and dried Mycobacterium leprae as well as dermal granulomas induced by the invading mycobacteria in various histological types of leprosy patients. For comparison, the biometal contents of the contralateral leprosy-unaffected skin of the same patients also were measured. The study also reports changes of serum levels of the biometals in these patients which were compared with those in healthy control subjects and patients with skin tuberculosis. These data show that M. leprae is rich in zinc. During the course of the evolution of the disease there is gross alteration of the dynamics of the inflammatory cell population that infiltrates into leprosy granulomas, resulting in the alterations of trace element contents of the disease-affected skin lesions. Interestingly, the changes of the biometal contents in the granulomas of the patients with skin tuberculosis are similar to those in leprosy patients. It is postulated that the significant decrease of the contents of copper, zinc, iron, calcium and magnesium in the disease-affected skin in comparison to that of the contralateral healthy skin is a local effect, perhaps due to erosion or influx of biometal-deficient inflammatory cells into the affected skin with eventual loss of connective tissue of skin and mobilization of tissue-bound microelements into the vascular compartment. On the contrary, the changes in biometal levels in the sera of leprosy patients appear to be a general effect perhaps due to the release of interleukin-1, a product of inflammatory cells, causing hypercupremic, hypozincemic and hypoferremic responses in the hosts. Moreover, growth and multiplication of M. leprae, especially in polar lepromatous leprosy patients with a high bacillary load, demand essential biometals which may be mobilized into the bacterial bodies from the hosts. This perhaps results in the change in the homeostasis of the essential biometals in the hosts.

Inositol phosphate capping of the nonreducing termini of lipoarabinomannan from rapidly growing strains of Mycobacterium.

Previous studies have demonstrated that the nonreducing termini of the lipoarabinomannan (LAM) from Mycobacterium tuberculosis are extensively capped with mannose residues, whereas those from a fast growing Mycobacterium sp., once thought to be an attenuated strain of M. tuberculosis, are not. The noncapped LAM, termed AraLAM, is known to be more potent than the mannose-capped LAM (ManLAM) in inducing functions associated with macrophage activation. Using a combination of chemical and enzymatic approaches coupled with fast atom bombardment-mass spectrometry analysis, we demonstrated that LAMs from all M. tuberculosis strains examined (Erdman, H37Ra, and H37Rv), as well as the attenuated Mycobacterium bovis BCG strain, are mannose-capped with the extent of capping varying between 40 and 70%. The nonreducing termini of LAM from Mycobacterium leprae were also found to be capped with mannoses but at a significantly lower level. A novel inositol phosphate capping motif was identified on a minor portion of the otherwise uncapped arabinan termini of LAMs from the fast growing Mycobacterium sp. and Mycobacterium smegmatis ATCC 14468 and mc(2)155. In addition, an inositol phosphate tetra-arabinoside was isolated from among endoarabinase digestion products of AraLAM and was shown to induce tumor necrosis factor-alpha production. Accordingly, we concluded that AraLAM is characteristic of some rapidly growing Mycobacterium spp. It is distinct from ManLAMs of M. tuberculosis, M. bovis BCG, and Mycobacterium leprae not only in the absence of mannose-capping but also in containing some terminal inositol phosphate substituents which may account for its particular potency in inducing macrophage activation.