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Secretion Antigens of Mycobacterium tuberculosis:
Archives of Medical Research 30 (1999) 171–178
ORIGINAL ARTICLE
Secretion Antigens of Mycobacterium tuberculosis: A Comparison Between a Reference Strain and Seven Wild Isolates Oscar Rojas-Espinosa,* Javier Rangel-Moreno,* Angélica Amador-Jiménez,* Ruth Parra-Maldonado,** Patricia Arce-Paredes* and Javier Torres-López** *Departamento de Inmunología , Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), México, D.F., Mexico **Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, Centro Médico Nacional Siglo XXI, IMSS, México, D.F., Mexico Received for publication July 13, 1998; accepted March 24, 1999 (98/076).
Background. This study was carried out with the aim of detecting possible differences between proteins secreted by fresh wild isolates of Mycobacterium tuberculosis and from a reference strain of this microorganism, H37Rv TMCC 102. Materials and Methods. This reference strain of M. tuberculosis has been in our laboratory for over 10 years, where it has been maintained by serial subcultures in PBY and Lowenstein-Jensen media. Patterns of protein secretion and recognition by sera derived from both tuberculosis patients and normal individuals were analyzed by electrophoresis and Western blotting. Results. No major qualitative differences were observed among the several strains studied with respect to protein patterns or recognition of these proteins by test sera. Normal sera were found to react with almost all antigens recognized by tuberculosis sera, but with less intensity. However, a small protein of 14.5 kDa, secreted by both the wild and reference strains of M. tuberculosis, was recognized by 32 of the 40 tuberculous patient sera tested (80%), and was not recognized by any of the 40 serum samples derived from healthy individuals. Conclusions. This small protein seems to be a potentially important antigen for the serological diagnosis of tuberculosis and/or for use in the follow-up of patients who received treatment. © 1999 IMSS. Published by Elsevier Science Inc. Key Words: Mycobacteria tuberculosis, Diagnosis of tuberculosis, 14.5 kDa antigen.
Introduction A number of immunological tests (enzyme-linked immunosorbent assays) have been developed by several authors for the serodiagnosis of tuberculosis. These tests use antigens derived from reference strains of Mycobacterium tuberculoAddress reprint requests to: Dr. Oscar Rojas-Espinosa, Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, I.P.N., Carpio y Plan de Ayala, Col. Santo Tomás, 11340 México, D.F., México. Tel.: (1525) 729-6000, ext. 62370; FAX: (1525) 729-6000, ext. 62489. E-mail: [email protected] This work was partially supported by the Dirección de Estudios Profesionales y de Investigación (DEPI) del IPN (Project nos. DEPI 970488 and DEPI 980681). AAJ and JRM are fellows of Consejo Nacional de Ciencia y Tecnología (CONACYT), PAP is a fellow of COFAA (IPN) and ORE is a fellow of COFAA (IPN) and the Sistema Nacional de Investigadores (SNI), Mexico.
sis H37Rv, M. tuberculosis H37Ra, or even from M. bovis, BCG (1). In most laboratories, reference strains have been maintained for many years by periodic subculturing in selective media, such as Lowenstein-Jensen’s medium. Changes in structural and antigenic characteristics of the microorganism as a result of repeated subculturing is, therefore, a real possibility. For this reason, we compared patterns of antigen secretion by several recently isolated wild strains of M. tuberculosis with the patterns of antigen secretion displayed by a reference strain of M. tuberculosis H37Rv. This reference strain has been maintained in our laboratory for approximately 10 years. Several colleagues have noted changes, proportional to the time spent in storage, in the protein pattern of mycobacterial extracts prepared by sonication. These changes were observed even when samples were prepared as frozen-stored aliquots that were not sub-
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jected to repeated freezing and thawing. One explanation for the observed changes is that they were caused by the activity of proteolytic enzymes extracted from the microorganisms together with other structural proteins. This has prompted the addition of protease inhibitors during the preparation of the sonicates. The work presented here is from studies carried out using the supernatant (filtrate) of 7week-old cultures of M. tuberculosis. This approach was chosen for two reasons: first, to avoid working with the microorganisms themselves; and second, it was reasoned that the material collected from the 7-week culture filtrates should be maximally autohydrolyzed and, therefore, should remain stable thereafter. Comparisons made between the reference and the wild strains of M. tuberculosis were based on the following two parameters: detection of protein patterns observed in polyacrylamide gels stained with silver stain as described by Merril et al. (2), and recognition of antigens by sera derived from active pulmonary tuberculosis patients and from healthy (non-tuberculous) individuals.
Materials and Methods Reagents. Unless otherwise indicated, chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Buffer solutions included borate-buffer solution, BBS (0.3092 g of boric acid, and 0.4768 g of sodium tetraborate per liter of 0.15 M NaCl, pH 8.4); phosphate buffer solution, PBS (0.01 M phosphate in 0.15 M NaCl, pH 7.4), and phosphate-citrate buffer, PCB (0.1 M citric acid in 0.2 M disodium phosphate, pH 5.0).
Mycobacterium tuberculosis strains. Ten isolates were obtained from sputum or urine specimens from 10 patients with clinically diagnosed tuberculosis. Seven of the 10 isolates were confirmed to be authentic M. tuberculosis on the basis of morphological examination, staining, and biochemical criteria. Of these seven isolates, six were from pulmonary tuberculosis patients and one was from a renal tuberculosis patient. The M. tuberculosis reference strain was obtained approximately 10 years ago from the Trudeau Mycobacterial Collection (M. tuberculosis H37Rv TMC 102) and since then has been maintained in our laboratory by periodically subculturing in Lowenstein-Jensen’s medium.
Culture of M. tuberculosis. Both the reference and the wild strains of M. tuberculosis were grown in the protein-free Proskawer-Becker medium, as modified by Youmans (PBY medium). Cultures were maintained without shaking at 378C for 7 weeks; at this time, confluent superficial growth was always observed. The confluent bacillary mass was separated by decanting and filtration (through filter paper). Microorganisms were then removed from the filtrate by
0.22-mm Millipore filtration before being reduced to a minimum volume by dialysis against sucrose crystals as follows: 500–1000 mL of the filtrates were collected into cellulose dialysis sacs (12,000 Da cut-off). The sacs were then covered with sucrose crystals and in order to facilitate the draining of extracted fluid, the concentration system was placed in a tilted position. Fresh sucrose crystals were periodically added until no more liquid was extracted. This technique resulted in the reduction of the filtrate to about 1/10 of the original volume. The concentrated filtrate was then dialyzed against saline-borate solution (BBS, pH 8.4) to eliminate sucrose, metabolites, and other small sized components of the medium. Finally, the filtrate was recovered and its protein content measured by Lowry’s method and adjusted to 1.0 mg/mL. The sample was then Milliporesterilized and divided into 1.0-mL aliquots before being frozen for additional use. Proteins secreted by M. tuberculosis H37Rv. Analysis of the protein patterns in the filtrates of each wild and reference M. tuberculosis strain was carried out by polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing and reducing conditions using Laemmli buffer (3). Twenty to 30 mL of each filtrate (1.0-mg protein/mL) were fractionated in 12%-polyacrylamide-SDS gels. Ten microliters (1 mg protein/mL) of prestained molecular weight protein markers (SDS-7B, Sigma) were simultaneously run as reference proteins. The gels were run for 4 to 5 h at 20 mA per gel until the tracking dye had migrated 10 cm. Gels were either fixed for silver staining (2) or were transferred to Immobilon P membranes (polyvinylidene difluoride, PVDF, IPVH 000 10, Millipore Corporation, Bedford, MA, USA) for Western blot analysis. Sera. The reactivity of 40 serum samples from healthy (non-tuberculous) individuals and 40 serum samples taken from patients with pulmonary tuberculosis, against secreted protein antigens of M. tuberculosis H37Rv was measured by the ELISA test. Medical personnel at the Hospital General de México in Mexico City obtained the sera after receiving informed consent from the patients. A conventional ELISA was carried out as follows: (a) wells were coated with 2 mg of protein from culture filtrate in 100 mL of borate buffer solution, BBS, pH 8.4; (b) wells were washed three times with 150 mL phosphate buffer solution, pH 7.4 containing 0.5% Tween 20 (PBST); (c) blocking was carried out with 150 mL of 2% skim milk in BBS, for 30 min at 378C; (d) wells were incubated with 100 mL of sera diluted 1:100 (tuberculous and normal sera) and 1:20 (normal sera) in 2% skim milk in PBS for l h at 378C;
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(e) wells were again washed three times with 150 mL of PBST; (f) incubated for 1 h at 378C with 100 mL of horseradish peroxidase-labeled goat antibody to human immunoglobulins (1:2000 in 2% skim milk in PBS) (dilutions of serum and peroxidase labeled antibodies were chosen from a previous chessboard titration of the reagents); (g) again wells were washed three times with 150 mL of PBST and once with PBS; (h) 100 mL of a mixture containing 4.0 mg of o-phenylene diamine (Sigma P1526) and 10 mL of 30% hydrogen peroxide (Sigma H1009) in 10 mL of phosphate citrate buffer pH 5.0 was added to each well; (i) 20 mL of 4N sulfuric acid was added to arrest the reaction; and (j) absorbency readings were taken at 492 nm in an ELISA reader. Sera from healthy or tuberculous individuals were selected for their high readings and then used to detect mycobacterial antigens secreted by the several wild and reference strains by Western blot analysis. Western blot analysis. Proteins fractionated by SDS-PAGE (3) were transferred by electrophoresis onto Immobilon-P membranes using a semi-dry transfer unit (Hoefer TE 77, Hoefer Scientific Instruments, San Francisco, CA, USA) and the buffer system described by Towbin et al. (4). After transfer, the membranes were dried and cut into 0.5 cm wide vertical strips. The strips were then used to detect antigenic components using a conventional Western blot procedure that included treatment of the strips with absolute methanol (30 sec), rinsing with distilled water and then with borate buffer BBS, blocking with 2% skim milk in BBS (30 min), incubation with the sera (normal and tuberculous) diluted 1:100 in 2% skim milk in PBST (M-PBST) (2 h), washing with PBST (3 3 1 min), incubation with a peroxidase-labeled goat anti-human immunoglobulins serum (1:500 in M-PBST) (60 min), washing with PBST (4 3 1 min) and developing with a mixture containing 3.0 mg of 3-amino-9 ethyl carbazole (Sigma A5754) and 10 mL of 30 % hydrogen peroxide (Sigma H1009) in 10 mL of phosphate citrate buffer pH 5.0.
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several strains studied were observed. When differences were observed they were found to be quantitative rather than qualitative in nature (Figure 1). Antigens secreted by M. tuberculosis H37Rv recognized by sera from tuberculosis patients. As expected, tuberculous individuals were found to harbor antibodies that recognize a large number of secreted antigens. Taken together, the sera of all tuberculosis patients recognized over 60 different antigens in the Western blot system. The most frequently recognized antigens had molecular weights between 30 and 90 kDa. Approximately 80% of the 40 sera included in this study reacted with a component of 14.5 kDa. Figure 2 shows a representative result with 14 tuberculous sera (diluted 1:100) reactive to this antigen. The most reactive sera were randomly pooled into two groups, TbP1 and TbP2, in order to analyze the antigenic patterns of the seven wild isolates of M. tuberculosis. Antigens secreted by M. tuberculosis H37Rv recognized by sera from healthy individuals. All sera tested reacted with a wide variety of antigens secreted by the microorganism. However, the most frequently recognized antigens were those with molecular weights between 30 and 90 kDa. None of the normal sera diluted 1:100 reacted with the 14.5 kDa antigen recognized by the sera of tuberculosis patients, and this was so even when normal sera were tested diluted 1:20
Results Patterns of protein secretion by seven wild strains and the H37Rv reference strain of M. tuberculosis. Over 60 proteins were identified in the culture filtrates of each of the seven wild and the reference strains of M. tuberculosis, by silver staining (2). These proteins had molecular weights ranging from 10.0 to over 200.0 kDa. Small peptides with molecular weights lower than 10 kDa were also observed in the SDS-PAGE-electropherograms of some of the strains. On examining the gels, no obvious differences between the
Figure 1. Protein patterns of the seven wild strains (1–7) and the reference strain (R) of M. tuberculosis used in this study (PAGE-SDS, Merril’s silver stain). Quantitative but not qualitative differences are observed within the strains.
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(Figures 3 and 4, respectively). The most reactive sera, which were also found to be the most reactive in the ELISA system, were pooled into two groups, NSP1 and NSP2, in order to analyze the seven wild isolates of M. tuberculosis. Recognition of secreted antigens by pools of sera from both tuberculous and healthy individuals. The majority of proteins secreted by M. tuberculosis H37Rv TMC 102 were recognized by the pools of tuberculous sera, TbP1 and TbP2. The pools of normal sera, NSP1 and NSP2, recognized the same components as the tuberculous sera although with lower intensities. The obvious exception was again the 14.5 kDa protein that was only recognized by TbP1 and TbP2 but not by NSP1 and NSP2. Again, this was the case even when the normal sera pools were used at a higher concentration of 1:20 instead of at the concentration of 1:100, which was used for the Tb pools. In all cases the most strongly recognized antigens were those with molecular weights between 30 and 90 kDa (Figure 5).
Figure 2. Reactivity of 19 tuberculous sera to antigens secreted by M. tuberculosis H37Rv TMC 102. Note the wide range of reactivities of sera to secreted antigens and in particular, the reactivity to component 14.5 kDa. The most frequently recognized antigens have molecular weights of between 30 and 90 kDa. (Sera diluted 1:100).
Patterns of antigen recognition displayed by the M. tuberculosis H37Rv culture filtrates. Over 60 antigens were globally recognized in the culture filtrates of the reference strain of M. tuberculosis H37Rv, by the pools of tuberculous sera, TbP1 and TbP2, and by the pools of sera from healthy individuals, NSP1 and NSP2. The reactive components as detected in eight separate Western blot experiments were found to have molecular weights ranging from 12–135 kDa. However, the most frequently recognized were found to be antigens with molecular weights between 97 and 1312 kDa.
Figure 3. Reactivity of 10 normal sera to antigens secreted by M. tuberculosis H37Rv TMC 102. Ten normal sera (chosen from among the most reactive by ELISA) were tested diluted 1:100. Compared to Figure 2, this figure shows the lack of reactivity with antigen 14.5 kD. This antigen, however, is strongly recognized by the pool of tuberculous sera (Tb1).
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Figure 4. Reactivity of 20 normal sera to antigens secreted by M. tuberculosis H37Rv TMC 102. Note the wide range of reactivities of sera to the secreted antigens and the lack of reactivity to protein 14.5 kDa. The most frequently recognized antigens are those of molecular weights between 30 and 90 kDa (normal sera were diluted 1:20 to reinforce their reactivity). The components detected in the blots by the Indian ink (ii) stain are indicated to the left.
Patterns of antigen recognition displayed by the M. tuberculosis H37Rv culture filtrates. The antigens detected by the pools of sera TbP1 and TbP2 in the culture filtrates of the eight strains studied (including the reference strain) were those with molecular weights between 130 and 12 kDa (Table 1). These results were obtained from eight separate Western blot experiments; most antigens were detected in the seven wild strains tested, and corresponded to antigens also present in the H37Rv reference strain. Some antigens were present in some strains but were not clearly defined in others. Figure 5 shows the type of results obtained when the eight M. tuberculosis strains are tested against the pools of sera TbP1 and TbP2. Note that with all the strains tested the antigen most frequently recognized by TbP1 and TbP2 was that of 14.5 kDa. Recognition of antigens by sera from healthy, non-tuberculous individuals. Sera derived from healthy non-tuberculous individuals (as well as the pools of sera made up from them) recognized practically the same antigens secreted by M. tuberculosis, as those recognized by the pool of tuberculous sera. The notable exception was with the 14.5 kDa anti-
gen. This antigen was frequently recognized by the sera of tuberculous individuals and the pools of tuberculous sera, but was not recognized by sera of normal individuals tested (Figures 3 and 4). The secreted antigens globally recognized by the pools of normal sera NSP1 and NSP2 in the seven wild and the reference strains of M. tuberculosis were those with molecular weights between 111 kDa and 19 kDa (Table 1). As previously discussed, not all of the antigens in the culture filtrates of the eight strains tested were equally detected by the pools NSP1 and NSP2.
Discussion The discovery of antigenic proteins in the culture filtrate of M. tuberculosis has been well documented since the time of Koch, who in 1891 reported the production of the classical source of mycobacterial proteins, tuberculin (5,6). Since that time, due to the availability of a large series of monoclonal antibodies, the advent of RNA/DNA technology and the development of better methods for isolating molecules, the isolation and precise identification of dozens of compo-
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Figure 5. Reactivity of pooled tuberculous (TbP1 and TbP2) or normal (NSP1 and NSP2) sera to antigens secreted by M. tuberculosis H37Rv or by the wild isolates 1 and 2 of the microorganism. Note the strong reactivity of tuberculous sera with the 14.5 kDa secreted antigen and the lack of reactivity of NSP1 and NSP2 sera with this component. Similar results were obtained with the wild isolates 3–7.
nents secreted by M. tuberculosis has been made possible (7). Many molecules have been characterized, some of which have been found to be potent T-cell stimulants while others have been found to be involved in functions necessary for the survival of the mycobacteria. The following are a selection of molecules so far characterized (5): (a) the AG85 (30-32 kDa) complex, which is formed by three fibronectin-binding proteins and is possibly involved in the phagocytosis of mycobacteria by macrophages (8); (b) the phosphate binding protein (38 kDa), which is externally located and is probably implicated in the introduction of phosphate in the bacteria (9,10); (c) the Dna K (70 kDa) and the GroES (10 kDa) proteins, which are stress (heat shock) proteins (11) and are involved in the microorganism’s adaptation to adverse environmental conditions; (d) the L-alanine dehydrogenase (40 kDa), which is probably involved in cell wall synthesis (9); (e) the MPT64 (26 kDa) and MPT51 (27 kDa) proteins,
which have some structural homology to the complex AG85 proteins (12), but are of unknown function; (f) the proline-rich complex 45-47 kDa, which was initially identified in the culture filtrates of BCG (13,14);
Table 1. Antigens secreted by M. tuberculosis H37Rv (TMC 102) and by the seven wild isolates of M. tuberculosis most frequently recognized by pools TbP1, TbP2, NSP1 and NSP2a,b Reactivity with pools TbP1 and TbP2 130, 116, 114, 111, 103-102, 100-99, 97-95, 91, 89, 87-86, 83, 82-81, 80-79, 78, 77, 75, 74, 71, 70, 69, 67, 65-64, 62-59, 58, 57-55, 53, 52, 50, 49-48, 47, 46-44, 43, 42, 39 (40-38), 37-36, 35, 34, 33, 32-31, 30, 29, 27, (28-26), 25, 22, 21, 20, 19, 18, 17, 15, 14 (14.5), 12. Reactivity with pools NSP1 and NSP2: 111-109, 104, 95, 80, 78-77, 74, 72-71, 70, 69, 67, 63, 62, 60, 58-57, 5553, 52, 51, 50, 49-48, 46, 41-40, 39, 37, 36, 33, 31-30, 29, 27, 26-25, 2321, 20, 19. a
All figures are given in kDa. b Several other antigens were less frequently detected by the tuberculous (TbP 1 and 2) normal (NSP1, NSP2) serum pools.
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(g) the 23 kDa superoxide dismutase protein, which in its native state exists as a tetramer of 88 kDa (6,15); (h) a lipoprotein of 19 kDa, which is a molecule secreted and anchored on the external side of the cell wall (9); (i) the alpha crystalline protein (12 kDa), which is a lowmolecular-weight, heat-shock protein structurally related to the 18 kDa antigen of M. leprae (16), and (j) the ESTA-6 (6 kDa) antigen, a small protein which is highly reactive with immune T cells (17). In the present study, it appears that on the basis of their molecular weights, most of the mentioned molecules including several other antigens were recognized. This recognition was achieved either by silver staining or by Western blot analysis of the culture filtrates obtained from the M. tuberculosis H37Rv (TMC-102) reference strain and the culture filtrates of seven wild isolates of M. tuberculosis. The reference strain of M. tuberculosis H37Rv, TMC-102, was obtained more than 10 years ago from the Trudeau Institute and has been maintained since then by periodical subculturing on Lowenstein-Jensen’s medium. The seven strains of M. tuberculosis were freshly isolated from clinical cases of tuberculosis and showed patterns of secreted proteins similar to those displayed by the reference M. tuberculosis H37Rv strain. On the basis of the techniques used in this study, SDS-PAGE and the silver staining method of Merril, no major qualitative differences were observed among the eight strains studied, although several quantitative differences were noted. By means of Western blot analysis, the majority of secreted proteins were recognized by sera derived from tuberculosis patients as well as from the sera of normal non-tuberculous individuals. More than 60 secreted antigens were recognized by sera from tuberculous patients, although this number is probably below the actual number of potentially recognized antigens. Approximately the same number of secreted antigens were recognized by the sera from normal individuals, and these were found to be the same antigens with the exception of the 14.5 kDa antigen. This 14.5 kDa antigen was recognized by sera from the majority of tuberculosis patients tested (.80%), but was not recognized by sera from healthy individuals (0%). The high degree of recognition (of varying intensity) of M. tuberculosis secreted antigens by sera derived from normal non-tuberculous subjects suggests that healthy people harbor antibodies to most antigens of this microorganism; these antigens probably share epitopes with antigens produced by other mycobacteria or even by other microorganisms. The lack of reactivity of normal sera with 14.5 kDa protein might indicate that this is a component with low immunogenicity, or a component released in vivo in only minor amounts. For an individual to produce antibodies to this component, a large number of microorganisms leading to an extensive infection would be necessary. This occurs in patients with clinical tuberculosis but does not occur in healthy people who might, however, be infected (infection does not necessarily mean disease).
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In a study carried out to develop a diagnostic test for tuberculous meningitis, Chandramuki et al. (18) set up an enzyme-linked immunoassay, using lipoarabinomannan (LAM) and the 14 kDa, 19 kDa, and 38 kDa mycobacterial proteins as antigens. They found that the antibody responses to both the 14 kDa and the LAM antigens were more immunodominant than those directed against the other antigens used. IgG response in cerebrospinal fluid (CSF) to these antigens confirmed the diagnosis of tuberculous meningitis with a sensitivity of 53% and a specificity of 100%. In our study, we identified sera, derived from clinically diagnosed tuberculosis patients, which did not react with the 14.5 kDa antigen (20%). It is possible that these individuals represent cases of arrested tuberculosis that have been cured and are no longer active, or that the cases were misdiagnosed as being tuberculosis (an unlikely possibility). It is possible that the 14.5 kDa antigen described in the present paper be the same antigen referred to by Chandramuki et al. (18). This antigen does not seem to be an autolysis product, as boiling of the culture filtrate for 3–30 min or its treatment with 0.1N NaOH or 0.1N HCl did not modify the protein or the antigen patterns of the intact material (Rojas-Espinosa O, Rangel-Moreno J, Arce-Paredes P, Tinoco-Centeno M. manuscript in preparation). We consider the 14.5 kDa protein to be worthy of additional investigation due to its potential as an antigen with an important role in the serodiagnosis of tuberculosis. In addition, this antigen may be highly relevant in the follow-up of the disease. Such a study, with cases of treated and untreated tuberculosis, is currently underway in our laboratory, using the isolated and additionally characterized 14.5 kDa protein as the antigen.
References 1. Daniel TM, Debanne SM. The serodiagnosis of tuberculosis and other mycobacterial diseases by enzyme-linked immunosorbent assay. Am Rev Resp Dis 1987;135:1137. 2. Merril CR, Goldman D, Sedman SA, Ebert MM. Ultrasensitive stain for proteins in polyacrilamide gels shows regional variations in cerebrospinal fluid proteins. Science 1981;211:1437. 3. Laemmli UK. Cleavage of structural proteins during the assembly of the head on bacteriophage T4. Nature 1970;227:680. 4. Towbin H, Staehelin T. Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 1979;76:4350. 5. Andersen P. Host response and antigens involved in protective immunity to Mycobacterium tuberculosis. Scand J Immunol 1997;45:115. 6. Andersen P, Askgaard D, Ljungqvist L, Bennedsen J, Heron I. Proteins released from Mycobacterium tuberculosis during growth. Infect Immunol 1991;59:1905. 7. Orme IM, Andersen P, Boom WH. T cell responses to Mycobacterium tuberculosis. J Infect Dis 1993;167:1481. 8. Abou-Zeid C, Smith I, Grange JM, Ratliff TL, Steele J, Rook GA. The secreted antigens of Mycobacterium tuberculosis and their relationship to those recognized by the available antibodies. J Gen Microbiol 1988;134:531. 9. Young DB, Garbe TR. Lipoprotein antigens of Mycobacterium tuberculosis. Res Microbiol 1991;142:55.
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10. Andersen AB, Ljungqvist L, Olsen M. Evidence that protein antigen b of Mycobacterium tuberculosis is involved in phosphate metabolism. J Gen Microbiol 1990;136:477. 11. Young DB, Garbe TR. Heat shock proteins and antigens of Mycobacterium tuberculosis. Infect Immun 1991a;59:3086. 12. Wiker HG, Nagai S, Harboe M, Ljungqvist L. A family of cross-reacting proteins secreted by M. tuberculosis. Scand J Immunol 1992; 36:307. 13. Romain F, Laqueyrerie A, Militzer P, Pescher P, Chavarot P, Langranderie M, Auregan G, Gheorghiu M, Marchal G. Identification of a Mycobacterium bovis BCG 45/47-kilodalton antigen complex. An immunodominant target for antibody response after immunization with living bacteria. Infect Immun 1993;61:742. 14. Romain F, Augier J, Pescher P, Marchal G. Isolation of a proline-rich mycobacterial protein eliciting delayed-type hypersensitivity reactions
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only in guinea pigs immunized with living mycobacteria. Proc Natl Acad Sci USA 1993b;90:5322. Kusunose E, Ichihara K, Noda Y, Kusunose M. Superoxide dismutase from Mycobacterium tuberculosis. J Biochem 1976;80:1343. Verbon A, Hartskeerl RA, Schuitema A, Kolk AHJ, Young DB, Lathigra R. The 14,000 molecular weight antigen of Mycobacterium tuberculosis is related to the alpha-crystalline family of low molecular weight heat shock proteins. J Bacteriol 1992;174:1352. Andersen P, Andersen AB, Sorensen AL, Nagai S. Recall of longlived immunity to Mycobacterium tuberculosis infection in mice. J Immunol 1995;154:3359. Chandramuki A, Bothamley GH, Brennan PJ, Ivanyi J. Levels of antibody to defined antigens of Mycobacterium tuberculosis in tuberculous meningitis. J Clin Microbiol 1989;27:821.
ORIGINAL ARTICLE
Secretion Antigens of Mycobacterium tuberculosis: A Comparison Between a Reference Strain and Seven Wild Isolates Oscar Rojas-Espinosa,* Javier Rangel-Moreno,* Angélica Amador-Jiménez,* Ruth Parra-Maldonado,** Patricia Arce-Paredes* and Javier Torres-López** *Departamento de Inmunología , Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), México, D.F., Mexico **Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, Centro Médico Nacional Siglo XXI, IMSS, México, D.F., Mexico Received for publication July 13, 1998; accepted March 24, 1999 (98/076).
Background. This study was carried out with the aim of detecting possible differences between proteins secreted by fresh wild isolates of Mycobacterium tuberculosis and from a reference strain of this microorganism, H37Rv TMCC 102. Materials and Methods. This reference strain of M. tuberculosis has been in our laboratory for over 10 years, where it has been maintained by serial subcultures in PBY and Lowenstein-Jensen media. Patterns of protein secretion and recognition by sera derived from both tuberculosis patients and normal individuals were analyzed by electrophoresis and Western blotting. Results. No major qualitative differences were observed among the several strains studied with respect to protein patterns or recognition of these proteins by test sera. Normal sera were found to react with almost all antigens recognized by tuberculosis sera, but with less intensity. However, a small protein of 14.5 kDa, secreted by both the wild and reference strains of M. tuberculosis, was recognized by 32 of the 40 tuberculous patient sera tested (80%), and was not recognized by any of the 40 serum samples derived from healthy individuals. Conclusions. This small protein seems to be a potentially important antigen for the serological diagnosis of tuberculosis and/or for use in the follow-up of patients who received treatment. © 1999 IMSS. Published by Elsevier Science Inc. Key Words: Mycobacteria tuberculosis, Diagnosis of tuberculosis, 14.5 kDa antigen.
Introduction A number of immunological tests (enzyme-linked immunosorbent assays) have been developed by several authors for the serodiagnosis of tuberculosis. These tests use antigens derived from reference strains of Mycobacterium tuberculoAddress reprint requests to: Dr. Oscar Rojas-Espinosa, Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, I.P.N., Carpio y Plan de Ayala, Col. Santo Tomás, 11340 México, D.F., México. Tel.: (1525) 729-6000, ext. 62370; FAX: (1525) 729-6000, ext. 62489. E-mail: [email protected] This work was partially supported by the Dirección de Estudios Profesionales y de Investigación (DEPI) del IPN (Project nos. DEPI 970488 and DEPI 980681). AAJ and JRM are fellows of Consejo Nacional de Ciencia y Tecnología (CONACYT), PAP is a fellow of COFAA (IPN) and ORE is a fellow of COFAA (IPN) and the Sistema Nacional de Investigadores (SNI), Mexico.
sis H37Rv, M. tuberculosis H37Ra, or even from M. bovis, BCG (1). In most laboratories, reference strains have been maintained for many years by periodic subculturing in selective media, such as Lowenstein-Jensen’s medium. Changes in structural and antigenic characteristics of the microorganism as a result of repeated subculturing is, therefore, a real possibility. For this reason, we compared patterns of antigen secretion by several recently isolated wild strains of M. tuberculosis with the patterns of antigen secretion displayed by a reference strain of M. tuberculosis H37Rv. This reference strain has been maintained in our laboratory for approximately 10 years. Several colleagues have noted changes, proportional to the time spent in storage, in the protein pattern of mycobacterial extracts prepared by sonication. These changes were observed even when samples were prepared as frozen-stored aliquots that were not sub-
0188-0128/99 $–see front matter. Copyright © 1999 IMSS. Published by Elsevier Science Inc. PII S0188-0128(99)00 0 2 0 - 2
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jected to repeated freezing and thawing. One explanation for the observed changes is that they were caused by the activity of proteolytic enzymes extracted from the microorganisms together with other structural proteins. This has prompted the addition of protease inhibitors during the preparation of the sonicates. The work presented here is from studies carried out using the supernatant (filtrate) of 7week-old cultures of M. tuberculosis. This approach was chosen for two reasons: first, to avoid working with the microorganisms themselves; and second, it was reasoned that the material collected from the 7-week culture filtrates should be maximally autohydrolyzed and, therefore, should remain stable thereafter. Comparisons made between the reference and the wild strains of M. tuberculosis were based on the following two parameters: detection of protein patterns observed in polyacrylamide gels stained with silver stain as described by Merril et al. (2), and recognition of antigens by sera derived from active pulmonary tuberculosis patients and from healthy (non-tuberculous) individuals.
Materials and Methods Reagents. Unless otherwise indicated, chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Buffer solutions included borate-buffer solution, BBS (0.3092 g of boric acid, and 0.4768 g of sodium tetraborate per liter of 0.15 M NaCl, pH 8.4); phosphate buffer solution, PBS (0.01 M phosphate in 0.15 M NaCl, pH 7.4), and phosphate-citrate buffer, PCB (0.1 M citric acid in 0.2 M disodium phosphate, pH 5.0).
Mycobacterium tuberculosis strains. Ten isolates were obtained from sputum or urine specimens from 10 patients with clinically diagnosed tuberculosis. Seven of the 10 isolates were confirmed to be authentic M. tuberculosis on the basis of morphological examination, staining, and biochemical criteria. Of these seven isolates, six were from pulmonary tuberculosis patients and one was from a renal tuberculosis patient. The M. tuberculosis reference strain was obtained approximately 10 years ago from the Trudeau Mycobacterial Collection (M. tuberculosis H37Rv TMC 102) and since then has been maintained in our laboratory by periodically subculturing in Lowenstein-Jensen’s medium.
Culture of M. tuberculosis. Both the reference and the wild strains of M. tuberculosis were grown in the protein-free Proskawer-Becker medium, as modified by Youmans (PBY medium). Cultures were maintained without shaking at 378C for 7 weeks; at this time, confluent superficial growth was always observed. The confluent bacillary mass was separated by decanting and filtration (through filter paper). Microorganisms were then removed from the filtrate by
0.22-mm Millipore filtration before being reduced to a minimum volume by dialysis against sucrose crystals as follows: 500–1000 mL of the filtrates were collected into cellulose dialysis sacs (12,000 Da cut-off). The sacs were then covered with sucrose crystals and in order to facilitate the draining of extracted fluid, the concentration system was placed in a tilted position. Fresh sucrose crystals were periodically added until no more liquid was extracted. This technique resulted in the reduction of the filtrate to about 1/10 of the original volume. The concentrated filtrate was then dialyzed against saline-borate solution (BBS, pH 8.4) to eliminate sucrose, metabolites, and other small sized components of the medium. Finally, the filtrate was recovered and its protein content measured by Lowry’s method and adjusted to 1.0 mg/mL. The sample was then Milliporesterilized and divided into 1.0-mL aliquots before being frozen for additional use. Proteins secreted by M. tuberculosis H37Rv. Analysis of the protein patterns in the filtrates of each wild and reference M. tuberculosis strain was carried out by polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing and reducing conditions using Laemmli buffer (3). Twenty to 30 mL of each filtrate (1.0-mg protein/mL) were fractionated in 12%-polyacrylamide-SDS gels. Ten microliters (1 mg protein/mL) of prestained molecular weight protein markers (SDS-7B, Sigma) were simultaneously run as reference proteins. The gels were run for 4 to 5 h at 20 mA per gel until the tracking dye had migrated 10 cm. Gels were either fixed for silver staining (2) or were transferred to Immobilon P membranes (polyvinylidene difluoride, PVDF, IPVH 000 10, Millipore Corporation, Bedford, MA, USA) for Western blot analysis. Sera. The reactivity of 40 serum samples from healthy (non-tuberculous) individuals and 40 serum samples taken from patients with pulmonary tuberculosis, against secreted protein antigens of M. tuberculosis H37Rv was measured by the ELISA test. Medical personnel at the Hospital General de México in Mexico City obtained the sera after receiving informed consent from the patients. A conventional ELISA was carried out as follows: (a) wells were coated with 2 mg of protein from culture filtrate in 100 mL of borate buffer solution, BBS, pH 8.4; (b) wells were washed three times with 150 mL phosphate buffer solution, pH 7.4 containing 0.5% Tween 20 (PBST); (c) blocking was carried out with 150 mL of 2% skim milk in BBS, for 30 min at 378C; (d) wells were incubated with 100 mL of sera diluted 1:100 (tuberculous and normal sera) and 1:20 (normal sera) in 2% skim milk in PBS for l h at 378C;
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(e) wells were again washed three times with 150 mL of PBST; (f) incubated for 1 h at 378C with 100 mL of horseradish peroxidase-labeled goat antibody to human immunoglobulins (1:2000 in 2% skim milk in PBS) (dilutions of serum and peroxidase labeled antibodies were chosen from a previous chessboard titration of the reagents); (g) again wells were washed three times with 150 mL of PBST and once with PBS; (h) 100 mL of a mixture containing 4.0 mg of o-phenylene diamine (Sigma P1526) and 10 mL of 30% hydrogen peroxide (Sigma H1009) in 10 mL of phosphate citrate buffer pH 5.0 was added to each well; (i) 20 mL of 4N sulfuric acid was added to arrest the reaction; and (j) absorbency readings were taken at 492 nm in an ELISA reader. Sera from healthy or tuberculous individuals were selected for their high readings and then used to detect mycobacterial antigens secreted by the several wild and reference strains by Western blot analysis. Western blot analysis. Proteins fractionated by SDS-PAGE (3) were transferred by electrophoresis onto Immobilon-P membranes using a semi-dry transfer unit (Hoefer TE 77, Hoefer Scientific Instruments, San Francisco, CA, USA) and the buffer system described by Towbin et al. (4). After transfer, the membranes were dried and cut into 0.5 cm wide vertical strips. The strips were then used to detect antigenic components using a conventional Western blot procedure that included treatment of the strips with absolute methanol (30 sec), rinsing with distilled water and then with borate buffer BBS, blocking with 2% skim milk in BBS (30 min), incubation with the sera (normal and tuberculous) diluted 1:100 in 2% skim milk in PBST (M-PBST) (2 h), washing with PBST (3 3 1 min), incubation with a peroxidase-labeled goat anti-human immunoglobulins serum (1:500 in M-PBST) (60 min), washing with PBST (4 3 1 min) and developing with a mixture containing 3.0 mg of 3-amino-9 ethyl carbazole (Sigma A5754) and 10 mL of 30 % hydrogen peroxide (Sigma H1009) in 10 mL of phosphate citrate buffer pH 5.0.
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several strains studied were observed. When differences were observed they were found to be quantitative rather than qualitative in nature (Figure 1). Antigens secreted by M. tuberculosis H37Rv recognized by sera from tuberculosis patients. As expected, tuberculous individuals were found to harbor antibodies that recognize a large number of secreted antigens. Taken together, the sera of all tuberculosis patients recognized over 60 different antigens in the Western blot system. The most frequently recognized antigens had molecular weights between 30 and 90 kDa. Approximately 80% of the 40 sera included in this study reacted with a component of 14.5 kDa. Figure 2 shows a representative result with 14 tuberculous sera (diluted 1:100) reactive to this antigen. The most reactive sera were randomly pooled into two groups, TbP1 and TbP2, in order to analyze the antigenic patterns of the seven wild isolates of M. tuberculosis. Antigens secreted by M. tuberculosis H37Rv recognized by sera from healthy individuals. All sera tested reacted with a wide variety of antigens secreted by the microorganism. However, the most frequently recognized antigens were those with molecular weights between 30 and 90 kDa. None of the normal sera diluted 1:100 reacted with the 14.5 kDa antigen recognized by the sera of tuberculosis patients, and this was so even when normal sera were tested diluted 1:20
Results Patterns of protein secretion by seven wild strains and the H37Rv reference strain of M. tuberculosis. Over 60 proteins were identified in the culture filtrates of each of the seven wild and the reference strains of M. tuberculosis, by silver staining (2). These proteins had molecular weights ranging from 10.0 to over 200.0 kDa. Small peptides with molecular weights lower than 10 kDa were also observed in the SDS-PAGE-electropherograms of some of the strains. On examining the gels, no obvious differences between the
Figure 1. Protein patterns of the seven wild strains (1–7) and the reference strain (R) of M. tuberculosis used in this study (PAGE-SDS, Merril’s silver stain). Quantitative but not qualitative differences are observed within the strains.
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(Figures 3 and 4, respectively). The most reactive sera, which were also found to be the most reactive in the ELISA system, were pooled into two groups, NSP1 and NSP2, in order to analyze the seven wild isolates of M. tuberculosis. Recognition of secreted antigens by pools of sera from both tuberculous and healthy individuals. The majority of proteins secreted by M. tuberculosis H37Rv TMC 102 were recognized by the pools of tuberculous sera, TbP1 and TbP2. The pools of normal sera, NSP1 and NSP2, recognized the same components as the tuberculous sera although with lower intensities. The obvious exception was again the 14.5 kDa protein that was only recognized by TbP1 and TbP2 but not by NSP1 and NSP2. Again, this was the case even when the normal sera pools were used at a higher concentration of 1:20 instead of at the concentration of 1:100, which was used for the Tb pools. In all cases the most strongly recognized antigens were those with molecular weights between 30 and 90 kDa (Figure 5).
Figure 2. Reactivity of 19 tuberculous sera to antigens secreted by M. tuberculosis H37Rv TMC 102. Note the wide range of reactivities of sera to secreted antigens and in particular, the reactivity to component 14.5 kDa. The most frequently recognized antigens have molecular weights of between 30 and 90 kDa. (Sera diluted 1:100).
Patterns of antigen recognition displayed by the M. tuberculosis H37Rv culture filtrates. Over 60 antigens were globally recognized in the culture filtrates of the reference strain of M. tuberculosis H37Rv, by the pools of tuberculous sera, TbP1 and TbP2, and by the pools of sera from healthy individuals, NSP1 and NSP2. The reactive components as detected in eight separate Western blot experiments were found to have molecular weights ranging from 12–135 kDa. However, the most frequently recognized were found to be antigens with molecular weights between 97 and 1312 kDa.
Figure 3. Reactivity of 10 normal sera to antigens secreted by M. tuberculosis H37Rv TMC 102. Ten normal sera (chosen from among the most reactive by ELISA) were tested diluted 1:100. Compared to Figure 2, this figure shows the lack of reactivity with antigen 14.5 kD. This antigen, however, is strongly recognized by the pool of tuberculous sera (Tb1).
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Figure 4. Reactivity of 20 normal sera to antigens secreted by M. tuberculosis H37Rv TMC 102. Note the wide range of reactivities of sera to the secreted antigens and the lack of reactivity to protein 14.5 kDa. The most frequently recognized antigens are those of molecular weights between 30 and 90 kDa (normal sera were diluted 1:20 to reinforce their reactivity). The components detected in the blots by the Indian ink (ii) stain are indicated to the left.
Patterns of antigen recognition displayed by the M. tuberculosis H37Rv culture filtrates. The antigens detected by the pools of sera TbP1 and TbP2 in the culture filtrates of the eight strains studied (including the reference strain) were those with molecular weights between 130 and 12 kDa (Table 1). These results were obtained from eight separate Western blot experiments; most antigens were detected in the seven wild strains tested, and corresponded to antigens also present in the H37Rv reference strain. Some antigens were present in some strains but were not clearly defined in others. Figure 5 shows the type of results obtained when the eight M. tuberculosis strains are tested against the pools of sera TbP1 and TbP2. Note that with all the strains tested the antigen most frequently recognized by TbP1 and TbP2 was that of 14.5 kDa. Recognition of antigens by sera from healthy, non-tuberculous individuals. Sera derived from healthy non-tuberculous individuals (as well as the pools of sera made up from them) recognized practically the same antigens secreted by M. tuberculosis, as those recognized by the pool of tuberculous sera. The notable exception was with the 14.5 kDa anti-
gen. This antigen was frequently recognized by the sera of tuberculous individuals and the pools of tuberculous sera, but was not recognized by sera of normal individuals tested (Figures 3 and 4). The secreted antigens globally recognized by the pools of normal sera NSP1 and NSP2 in the seven wild and the reference strains of M. tuberculosis were those with molecular weights between 111 kDa and 19 kDa (Table 1). As previously discussed, not all of the antigens in the culture filtrates of the eight strains tested were equally detected by the pools NSP1 and NSP2.
Discussion The discovery of antigenic proteins in the culture filtrate of M. tuberculosis has been well documented since the time of Koch, who in 1891 reported the production of the classical source of mycobacterial proteins, tuberculin (5,6). Since that time, due to the availability of a large series of monoclonal antibodies, the advent of RNA/DNA technology and the development of better methods for isolating molecules, the isolation and precise identification of dozens of compo-
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Figure 5. Reactivity of pooled tuberculous (TbP1 and TbP2) or normal (NSP1 and NSP2) sera to antigens secreted by M. tuberculosis H37Rv or by the wild isolates 1 and 2 of the microorganism. Note the strong reactivity of tuberculous sera with the 14.5 kDa secreted antigen and the lack of reactivity of NSP1 and NSP2 sera with this component. Similar results were obtained with the wild isolates 3–7.
nents secreted by M. tuberculosis has been made possible (7). Many molecules have been characterized, some of which have been found to be potent T-cell stimulants while others have been found to be involved in functions necessary for the survival of the mycobacteria. The following are a selection of molecules so far characterized (5): (a) the AG85 (30-32 kDa) complex, which is formed by three fibronectin-binding proteins and is possibly involved in the phagocytosis of mycobacteria by macrophages (8); (b) the phosphate binding protein (38 kDa), which is externally located and is probably implicated in the introduction of phosphate in the bacteria (9,10); (c) the Dna K (70 kDa) and the GroES (10 kDa) proteins, which are stress (heat shock) proteins (11) and are involved in the microorganism’s adaptation to adverse environmental conditions; (d) the L-alanine dehydrogenase (40 kDa), which is probably involved in cell wall synthesis (9); (e) the MPT64 (26 kDa) and MPT51 (27 kDa) proteins,
which have some structural homology to the complex AG85 proteins (12), but are of unknown function; (f) the proline-rich complex 45-47 kDa, which was initially identified in the culture filtrates of BCG (13,14);
Table 1. Antigens secreted by M. tuberculosis H37Rv (TMC 102) and by the seven wild isolates of M. tuberculosis most frequently recognized by pools TbP1, TbP2, NSP1 and NSP2a,b Reactivity with pools TbP1 and TbP2 130, 116, 114, 111, 103-102, 100-99, 97-95, 91, 89, 87-86, 83, 82-81, 80-79, 78, 77, 75, 74, 71, 70, 69, 67, 65-64, 62-59, 58, 57-55, 53, 52, 50, 49-48, 47, 46-44, 43, 42, 39 (40-38), 37-36, 35, 34, 33, 32-31, 30, 29, 27, (28-26), 25, 22, 21, 20, 19, 18, 17, 15, 14 (14.5), 12. Reactivity with pools NSP1 and NSP2: 111-109, 104, 95, 80, 78-77, 74, 72-71, 70, 69, 67, 63, 62, 60, 58-57, 5553, 52, 51, 50, 49-48, 46, 41-40, 39, 37, 36, 33, 31-30, 29, 27, 26-25, 2321, 20, 19. a
All figures are given in kDa. b Several other antigens were less frequently detected by the tuberculous (TbP 1 and 2) normal (NSP1, NSP2) serum pools.
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(g) the 23 kDa superoxide dismutase protein, which in its native state exists as a tetramer of 88 kDa (6,15); (h) a lipoprotein of 19 kDa, which is a molecule secreted and anchored on the external side of the cell wall (9); (i) the alpha crystalline protein (12 kDa), which is a lowmolecular-weight, heat-shock protein structurally related to the 18 kDa antigen of M. leprae (16), and (j) the ESTA-6 (6 kDa) antigen, a small protein which is highly reactive with immune T cells (17). In the present study, it appears that on the basis of their molecular weights, most of the mentioned molecules including several other antigens were recognized. This recognition was achieved either by silver staining or by Western blot analysis of the culture filtrates obtained from the M. tuberculosis H37Rv (TMC-102) reference strain and the culture filtrates of seven wild isolates of M. tuberculosis. The reference strain of M. tuberculosis H37Rv, TMC-102, was obtained more than 10 years ago from the Trudeau Institute and has been maintained since then by periodical subculturing on Lowenstein-Jensen’s medium. The seven strains of M. tuberculosis were freshly isolated from clinical cases of tuberculosis and showed patterns of secreted proteins similar to those displayed by the reference M. tuberculosis H37Rv strain. On the basis of the techniques used in this study, SDS-PAGE and the silver staining method of Merril, no major qualitative differences were observed among the eight strains studied, although several quantitative differences were noted. By means of Western blot analysis, the majority of secreted proteins were recognized by sera derived from tuberculosis patients as well as from the sera of normal non-tuberculous individuals. More than 60 secreted antigens were recognized by sera from tuberculous patients, although this number is probably below the actual number of potentially recognized antigens. Approximately the same number of secreted antigens were recognized by the sera from normal individuals, and these were found to be the same antigens with the exception of the 14.5 kDa antigen. This 14.5 kDa antigen was recognized by sera from the majority of tuberculosis patients tested (.80%), but was not recognized by sera from healthy individuals (0%). The high degree of recognition (of varying intensity) of M. tuberculosis secreted antigens by sera derived from normal non-tuberculous subjects suggests that healthy people harbor antibodies to most antigens of this microorganism; these antigens probably share epitopes with antigens produced by other mycobacteria or even by other microorganisms. The lack of reactivity of normal sera with 14.5 kDa protein might indicate that this is a component with low immunogenicity, or a component released in vivo in only minor amounts. For an individual to produce antibodies to this component, a large number of microorganisms leading to an extensive infection would be necessary. This occurs in patients with clinical tuberculosis but does not occur in healthy people who might, however, be infected (infection does not necessarily mean disease).
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In a study carried out to develop a diagnostic test for tuberculous meningitis, Chandramuki et al. (18) set up an enzyme-linked immunoassay, using lipoarabinomannan (LAM) and the 14 kDa, 19 kDa, and 38 kDa mycobacterial proteins as antigens. They found that the antibody responses to both the 14 kDa and the LAM antigens were more immunodominant than those directed against the other antigens used. IgG response in cerebrospinal fluid (CSF) to these antigens confirmed the diagnosis of tuberculous meningitis with a sensitivity of 53% and a specificity of 100%. In our study, we identified sera, derived from clinically diagnosed tuberculosis patients, which did not react with the 14.5 kDa antigen (20%). It is possible that these individuals represent cases of arrested tuberculosis that have been cured and are no longer active, or that the cases were misdiagnosed as being tuberculosis (an unlikely possibility). It is possible that the 14.5 kDa antigen described in the present paper be the same antigen referred to by Chandramuki et al. (18). This antigen does not seem to be an autolysis product, as boiling of the culture filtrate for 3–30 min or its treatment with 0.1N NaOH or 0.1N HCl did not modify the protein or the antigen patterns of the intact material (Rojas-Espinosa O, Rangel-Moreno J, Arce-Paredes P, Tinoco-Centeno M. manuscript in preparation). We consider the 14.5 kDa protein to be worthy of additional investigation due to its potential as an antigen with an important role in the serodiagnosis of tuberculosis. In addition, this antigen may be highly relevant in the follow-up of the disease. Such a study, with cases of treated and untreated tuberculosis, is currently underway in our laboratory, using the isolated and additionally characterized 14.5 kDa protein as the antigen.
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