Fractionation and analysis of mycobacterial proteins.

The extraction and isolation of native bacterial proteins continue to be valuable technical pursuits in order to understand bacterial physiology, screen for virulence determinants, and describe antigens. In this chapter, methods for the manipulation of whole mycobacterial cells are described in detail. Specifically, the concentration of spent culture filtrate media is described in order to permit separation of soluble, secreted proteins; several discrete separation techniques, including precipitation of protein mixtures with ammonium sulfate and separation of proteins by hydrophobic chromatography are also provided. Similarly, the generation of whole cell lysate and facile separation of lysate into subcellular fractions to afford cell wall, cell membrane, and cytosol enriched proteins is described. Due to the hydrophobic nature of cell wall and cell membrane proteins, several extraction protocols to resolve protein subsets (such as extraction with urea and SDS) are also provided, as well as a separation technique (isoelectric focusing) that can be applied to separate hydrophobic proteins. Lastly, two commonly used analytical techniques, in-gel digestion of proteins for LC-MS and analysis of intact proteins by MALDI-ToF MS, are provided for rapid analysis of discrete proteins within subcellular or chromatographic fractions. While these methods were optimized for the manipulation of Mycobacterium tuberculosis cells, they have been successfully applied to extract and isolate Mycobacterium leprae, Mycobacterium ulcerans, and Mycobacterium avium proteins. In addition, a number of these methods may be applied to extract and analyze mycobacterial proteins from cell lines and host derived samples.

Deciphering the proteomic profile of Mycobacterium leprae cell envelope.

The complete sequence of the Mycobacterium leprae genome, an obligate intracellular pathogen, shows a dramatic reduction of functional genes, with a coding capacity of less than 50%. Despite this massive gene decay, the leprosy bacillus has managed to preserve a minimal gene set, most of it shared with Mycobacterium tuberculosis, allowing its survival in the host with ensuing pathological manifestations. Thus, the identification of proteins that are actually expressed in vivo by M. leprae is of high significance in understanding obligate, intracellular mycobacterial pathogenesis. In this study, a high-throughput proteomic approach was undertaken resulting in the identification of 218 new M. leprae proteins. Of these, 60 were in the soluble/cytosol fraction, 98 in the membrane and 104 in the cell wall. Although several proteins were identified in more than one subcellular fraction, the majority were unique to one. As expected, a high percentage of these included enzymes responsible for lipid biosynthesis and degradation, biosynthesis of the major components of the mycobacterial cell envelope, proteins involved in transportation across lipid barriers, and lipoproteins and transmembrane proteins with unknown functions. The data presented in this study contribute to our understanding of the in vivo composition and physiology of the mycobacterial cell envelope, a compartment known to play a major role in bacterial pathogenesis.

Granulosain I, a cysteine protease isolated from ripe fruits of Solanum granuloso-leprosum (Solanaceae).

A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.

Identification of proteins induced by polycyclic aromatic hydrocarbon in Mycobacterium vanbaalenii PYR-1 using two-dimensional polyacrylamide gel electrophoresis and de novo sequencing methods.

Protein profiles of Mycobacterium vanbaalenii PYR-1 grown in the presence of high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) were examined by two-dimensional gel electrophoresis (2-DE). Cultures of M. vanbaalenii PYR-1 were incubated with pyrene, pyrene-4,5-quinone (PQ), phenanthrene, anthracene, and fluoranthene. Soluble cellular protein fractions were analyzed and compared, using immobilized pH gradient (IPG) strips. More than 1000 gel-separated proteins were detected using a 2-DE analysis program within the window of isoelectric point (pI) 4-7 and a molecular mass range of 10-100 kDa. We observed variations in the protein composition showing the upregulation of multiple proteins for the five PAH treatments compared with the uninduced control sample. By N-terminal sequencing or mass spectrometry, we further analyzed the proteins separated by 2-DE. Due to the lack of genome sequence information for this species, protein identification provided an analytical challenge. Several PAH-induced proteins were identified including a catalase-peroxidase, a putative monooxygenase, a dioxygenase small subunit, a small subunit of naphthalene-inducible dioxygenase, and aldehyde dehydrogenase. We also identified proteins related to carbohydrate metabolism (enolase, 6-phosphogluconate dehydrogenase, indole-3-glycerol phosphate synthase, and fumarase), DNA translation (probable elongation factor Tsf), heat shock proteins, and energy production (ATP synthase). Many proteins from M. vanbaalenii PYR-1 showed similarity with protein sequences from M. tuberculosis and M. leprae. Some proteins were detected uniquely upon exposure to a specific PAH whereas others were common to more than one PAH, which indicates that induction triggers not only specific responses but a common response in this strain.

Truncated structural variants of lipoarabinomannan in Mycobacterium leprae and an ethambutol-resistant strain of Mycobacterium tuberculosis.

Current knowledge on the structure of lipoarabinomannan (LAM) has resulted primarily from detailed studies on a few selected laboratory strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium smegmatis. Our previous work was the first to report on the salient structural features of M. tuberculosis clinical isolates and demonstrated significant structural variations. A prime effort is to correlate a particular structural characteristic with observed differences in eliciting an immunobiological response, especially in the context of CD1-restricted presentation of LAM to T cells. T cell clones derived from the cutaneous lesions of leprosy patients have been shown to recognize specifically LAM from Mycobacterium leprae and not from M. tuberculosis Erdman or H37Rv. Herein we provide further fine structural data on LAM from M. leprae (LepLAM) and a tuberculosis clinical isolate, CSU20 (CSU20LAM), which was unexpectedly recognized by the supposedly LepLAM-specific CD1-restricted T cell clones. In comparison with the de facto laboratory LAM standard from M. tuberculosis H37Rv (RvLAM), LepLAM derived from in vivo grown M. leprae is apparently simpler in its arabinan architecture with a high degree of exposed, non-mannose-capped termini. On the other hand, CSU20, an ethambutol-resistant clinical isolate, makes a vastly heterogeneous population of LAM ranging from rather small and non-mannose-capped to full-length and fully capped variants. LepLAM and CSU20LAM contain a higher level of succinylation than RvLAM, which, in the context of truncated or less elaborated arabinan, may contribute to selective recognition by T cells. LAM from all species could be resolved into discrete forms by isoelectric focusing based apparently on their arabinan heterogeneity. In the light of our current and more recent findings, we reason that all immunobiological data should be cautiously interpreted and that the actual LAM variants that may be present in vivo during infection and pathogenesis need to be taken into consideration.

Connectivity patterns in tuberculosis and leprosy patients are indistinguishable from that of healthy donors.

Connectivity, the self-defined interactions between antigen-recognising molecules in a network system can in part be assessed by measuring the reactivity of a given serum against an ordered set of immunoglobulin (Ig)G F(ab')2 fractions, separated by means of isoelectric focusing so that, the serum reactivity against the whole set of fractions defines a characteristic pattern of connectivity. Deviations from the normal condition (healthy donors) have so far been documented for two autoimmune diseases: systemic lupus erythematosus (SLE) and pemphigus vulgaris, as well as for human immunodeficiency virus (HIV)-1 infection. We tested here if bacterial infections lead to alterations in connectivity. In addition, we wanted to test if two antigenically related bacteria would produce similar or otherwise distinctive connectivity patterns. Connectivity analysis was applied on the sera from tuberculosis and leprosy patients and the sera from healthy donors were used as control. No statistically significant differences between the three groups studied were found. These results have implications for theories that set the origin of autoimmune diseases in microbial infections. To the best of our knowledge, this is the first attempt to analyze the connectivity status in bacterial infections.

IgG humoral response against the antigen 85 complex homologues in leprosy.

Antigen 85 complex is the major protein component present in M. bovis BCG culture filtrate (CF). It consists of a family of three proteins: 85A, 85B and 85C. Combining isoelectric focusing and Western blot analysis, we have previously identified different antigenically related proteins present in the CF of other mycobacteria (M. tuberculosis, M. kansasii, M. avium, M. gordonae, M. fortuitum and M. phlei) using monoclonal antibodies (MoAbs) directed against the antigen 85 complex of M. bovis BCG. Humoral immune response directed against these cross-reactive homologues was analysed in sera from 20 patients with multibacillary leprosy (BL/LL), from 20 patients with paucibacillary leprosy (BT/TT) and from 15 healthy leprosy contacts. All the antigen 85 homologues identified in the seven CFs by MoAbs were also recognized by IgG present in sera from multibacillary leprosy patients, but not or very faintly in sera from paucibacillary leprosy patients or from healthy subjects. These results suggest that some of the M. leprae epitopes inducing a significant humoral response in multibacillary leprosy are common to the various 85 antigenically related proteins present in all mycobacterial species.

Characterization of the antigen 85 complex fibronectin-binding proteins derived from aged culture filtrates of Mycobacterium bovis BCG, Tice substrain.

The antigen 85 complex are major T-cell and B-cell antigens and fibronectin-binding proteins secreted by Mycobacterium tuberculosis, M. leprae and attenuated M. bovis (BCG vaccine). The Ag 85 complex was found to comprise a high proportion of the extracellular protein in filtrates of surface-pellicle cultures of Tice-substrain BCG vaccine, attaining a maximum of 25%. This proportion began to decrease prior to the end of the logarithmic growth phase, about 3 weeks after the start of the culture, mainly due to apparent degradation of the Ag 85 complex. Isolation of the main Ag 85 protein and determination of the first 36 residues of the NH2-terminus showed identity with the 85A protein isolated by others from various mycobacteria. Both the Ag 85A and B components were secreted in nearly constant proportions over a 6-week period. No Ag 85C protein was detected.

Effects of chemotherapy on antibody levels directed against PGL-I and 85A and 85B protein antigens in lepromatous patients.

IgG antibodies against antigens 85A and 85B from Mycobacterium bovis BCG, IgM antibodies against phenolic glycolipid-I (PGL-I) and circulating PGL-I antigen were measured in the serum of 11 patients with lepromatous leprosy receiving multidrug therapy (MDT). Before treatment, 6 patients were reactive to antigen 85A, 10 patients to antigen 85B, and 11 patients to PGL-I; circulating PGL-I was detected in the sera of all of them. After 2 years of MDT PGL-I antigen could no longer be detected in all of the patients, except for two who were not compliant with treatment. IgG antibodies directed against the 85A and 85B antigens and IgM antibodies against the PGL-I antigen also decreased significantly during treatment but more slowly. The determination of circulating PGL-I antigen remains the most appropriate tool for monitoring lepromatous leprosy under MDT.

T-cell responses of leprosy patients and healthy contacts toward separated protein antigens of Mycobacterium leprae.

Sonicated extracts of Mycobacterium leprae were separated by two-dimensional gel electrophoresis and electroeluted into 400 distinct soluble fractions. These fractions were probed with T lymphocytes from leprosy patients of different disease types, healthy contacts, and unexposed healthy individuals. Proliferative responses were visualized using three-dimensional stimulation profiles. T cells from many patients and contacts responded to a multitude of antigen fractions of different molecular masses and isoelectric points. T cells from unexposed individuals gave significant responses to lysates or whole organisms of M. leprae, but no or only marginal responses to separated antigen fractions. T cells of polar tuberculoid (TT) and the majority of polar lepromatous (LL) leprosy patients responded only to separated antigen fractions but not to lysates or whole organisms of M. leprae. The remaining LL patients were totally unresponsive and even failed to respond to separated M. leprae fractions. Thus, in some leprosy patients unresponsiveness to M. leprae seems to be caused by distinct components and can be broken by using separated antigen fractions; whereas in others, anergy remains. T cells of borderline tuberculoid (BT) patients, who were under chemotherapy, responded to separated antigen fractions as well as to lysates of M. leprae organisms. In contrast, BT patients who were untreated failed to react with any of the M. leprae preparations. Similarly, T cells of the majority of LL patients responding to separated fractions were under chemotherapy; whereas T cells from untreated LL patients gave no or only marginal responses to any of the M. leprae antigen preparations. These findings suggest some linkage between the degree of T-cell responsiveness and antileprosy drug treatment.

Further characterization and immunochemical studies on the carbohydrate specificity of jackfruit (Artocarpus integrifolia) lectin.

The lectin from jackfruit (Artocarpus integrifolia) seeds has been purified by Rivanol (6,9-diamino-2-ethoxyacridine lactate) treatment. The specific activity, molecular weights of parent lectin and its subunit, its glycoprotein nature, and hemagglutination-inhibition assays suggest that this preparation is identical to that obtained by affinity chromatography on melibiose-agarose adsorbent (Ahmed, H., and Chatterjee, B. P. (1986) in Lectins, Biology, Biochemistry, Clinical Biochemistry (Bøg-Hansen, T. C., and van Driessche, E., eds) Vol. 5, pp. 125-133, Walter de Gruyter, New York). The lectin strongly agglutinates human and several animal erythrocytes. The lectin contains five isolectins of pI values 7.1, 6.85, 5.5, 5.3, and 5.1. It is thermally stable and loses its activity above 75 degrees C. The hemagglutinating activity remains unchanged in the presence of bivalent cations viz., Ca2+, Mg2+, Mn2+, etc. It is a metalloprotein. The lectin retains its activity by dialysis with acetic acid followed by EDTA. It agglutinates Ehrlich ascites cells. Equilibrium dialysis of lectin with melibiose and quenching of fluorescence of 4-methylumbelliferyl-alpha-D-galactopyranoside by the lectin show that homotetrameric jackfruit lectin has two sugar-binding sites. The lectin precipitates well several galactomannans and glycoproteins having terminal D-Gal-alpha-(1----6)- or D-Gal-beta-(1----3)-D-GalNAc residues. It hardly or does not precipitate polysaccharides having terminal D-Gal-alpha-(1----3) residues. Quantitative precipitin-inhibition studies using various haptens suggest that the -OCH2- group at C-1 and -OH groups at C-4 and partially at C-6 in the alpha-glycoside of D-galactose configuration are important for lectin-sugar interaction.

Immunogenic "subunit" of the ICRC antileprosy vaccine.

The administration of a vaccine containing ICRC bacilli, which is currently undergoing clinical trials in India, induces persistent lepromin conversion in lepromatous leprosy (LL) patients and lepromin-negative healthy subjects, with "upgrading" of tissue response in the former. A sonicate of ICRC bacilli, when subjected to gel-filtration chromatography using high-pressure liquid chromatography (HPLC), yields a high molecular weight glycolipoprotein (PP-I) with an apparent molecular weight of 10(6) daltons. PP-I, which brings about lepromin conversion in lepromin-negative healthy subjects, is a major immunogen of the organism, and carries epitopes for both B and T cells. A similar high molecular weight glycolipoprotein (PP-I Mycobacterium leprae) has been isolated from the sonicate of M. leprae. The two PP-I fractions exhibit a close antigenic relatedness at both B- and T-cell levels. However, they differ in their chemical composition and carry different charges. PP-I of ICRC is not only a good immunogen. Its high lipid content provides the necessary built-in adjuvant that would make it a good candidate for a "subunit" antileprosy vaccine. Also, since it carries epitopes for both B and T cells, PP-I ICRC could be used for "molecular engineering" to obtain molecules which selectively stimulate T-cell immunity which is the dominant host defense against M. leprae.

Studies on the antigenic specificity of Mycobacterium leprae. I. Isoelectric and chromatofocusing separation.

Cell sonicates of Mycobacterium leprae and other mycobacteria were subjected to isoelectric focusing and chromatofocusing to evaluate their protein antigens and to determine if the patterns were significantly different. Isoelectric focusing showed that the proteins of all mycobacteria focused within the pH range of 3.5 to 5.5, except those of M. leprae which extended beyond 5.5 to 6.5. These studies have indicated for the first time that the protein antigens of mycobacteria are acidic in nature. Comparison between the proteins of untreated and autoclaved M. leprae showed distinct differences between the two preparations, in respect of loss of some antigens in the autoclaved M. leprae sonicate. This indicates that the bands that were not visible in the autoclaved M. leprae were those of heat-labile proteins. It is possible, however, that the absent bands could have been of a low order of intensity and hence were not discernible. On the other hand, the proteins could have coagulated due to the heat treatment, thus causing confirmational changes or ionic interactions with membrane components, due to their acidic nature. It is possible that the proteins in the autoclaved M. leprae are the ones that possess immunogenic properties since the protective ability of heat-killed M. leprae has already been established. Chromatofocusing studies have confirmed the isoelectric focusing data in respect of the number of antigens and their respective protein content, besides permitting the availability of the various fractions for further biological characterization.