Structure of a novel glycopeptidolipid antigen containing a O-methylated serine isolated from Mycobacterium xenopi. Complete 1H-NMR and 13C-NMR assignment.

GPL X-1, a novel glycopeptidolipid (GPL) isolated from Mycobacterium xenopi (CIPT 140 35004), has recently been found to typify a new class of mycobacterial glycopeptidolipids devoid of C-mycoside core structure, the so-called serine-containing glycopeptidolipid [Rivière, M. & Puzo, G. (1991) J. Biol. Chem. 266, 9057-9063]. Here we report the purification and characterization of a novel serine-containing GPL termed GPL X-IIb, isolated from the M. xenopi strain NCTC 10042. On thin-layer chromatography, this GPL was found to be present in some other M. xenopi strains isolated from patients with pulmonary infections. The sugar and amino-acid compositions of this GPL were elucidated from the native form using a combination of two-dimensional homonuclear and heteronuclear scalar coupling NMR. The peptide and sugar sequences, as well as the methoxyl group locations on the C-3 of the 6-deoxy-alpha-L-talopyranoside (6dTalp) and on a Ser, were unambiguously determined by heteronuclear multiple-bond correlation experiments. GPL X-IIb was found to be composed of a lipotetrapeptide of the following structure C12-Ser-OMe-Ser-Phe-aThr-OMe (aThr = allothreonine). The sugar part is made up of 3OMe-alpha-L-6dTalp and the following disaccharide: alpha-L-Rhap-(1-->3)-2-O-Lau-alpha-L-Rhap (Rhap = rhanmopyranose). Unlike GPL X-I, the sugar attachment sites on the tetrapeptide were successfully determined from heteronuclear three-bond coupling correlation observed in the heteronuclear multiple bond correlation spectrum between the anomeric carbon resonances and the beta protons of aThr-OMe and Ser. It was established that the 3OMe-6dTalp glycosylates the Ser while the disaccharide is linked to the aThr-OMe. Thus both GPL X-I and GPL X-IIb share a common lipotetrapeptide core [with the exception of Ser(OMe)] but drastically differ in their oligosaccharide appendage. Thus, by analogy with the M. avium complex, the present report suggests that M. xenopi species can be divided in various serovars characterized by the unique structure of their C-mycoside GPL oligosaccharide appendage, enhancing the interest for this new type of serine-containing glycopeptidolipid.

[Use of the immunoblotting method in serodiagnosis of M. kansasii mycobacteriosis]. / Vyuzití imunoblotové metody v sérodiagnostice mykobakteriózy M. kansasii.

The indirect enzyme test ELISA with soluble complex antigen of M. kansasii, used for assessment of the titre of IgG serum antibodies in a group of patients suffering from mycobacteriosis M. kansasii and in a control group of patients suffering from tuberculosis did not reveal statistically significant differences between serological responses of these two groups. On the other hand, the immunoblot analysis revealed in sera selected at random from these two groups differences at the level of subprotein units against the complex antigen of M. kansasii. In a group of 15 sera of patients with M. kansasii the immunodominant area was 42-45 kD protein subunits, in the control group of 10 sera with M. tuberculosis the area was 35-39 kD. The western blot technique is more perspective for improvement of the serum diagnosis of M. kansasii, in particular where a specific antigen is not available.

Preparation and properties of monoclonal antibodies against Mycobacterium tuberculosis.

The above mentioned monoclonal antibodies of idiotype IgG 3/Kappa against TMC 120 and TMC 107 strains of M. tuberculosis were prepared by fusion of mouse myeloma line FO cells with splenocytes BALB/c of mice immunized with complex antigens. These were prepared from the bacterial mass previously inactivated with gamma-rays radiation, and applied without mycobacterial adjuvant agent. Species specificity of both monoclonal antibodies was determined by ELISA testing and dot blot test. The potential use of the mentioned preparations consists in species identifying and taxonomy of mycobacteria, mycobacterial antigen purification and prompt TBC diagnosis (serodiagnosis, identifying of specific antigens in pathological materials.

Screening of serum antibodies against Mycobacterium tuberculosis by enzyme-linked immunosorbent assay (ELISA) in healthy population.

Serum samples of venous blood of healthy individuals, chosen at random from two areas of the People's Democratic republic of Yemen, were examined for the presence of IgG antibodies against mycobacterial antigen by ELISA. From the district of Aden, the capital (A), there were 214 samples (72% vaccinated by BCG, 108 men and 106 women), other 235 ones originated from mountain area Laudar Muhairas (H) (66% vaccinated, 115 men and 120 women). The overall average of acquired titres was 1:81.4 in area A, and an average of 1:102.2 was recorded in area H. There was no substantial difference in the titres level in men of the two areas (1:86.9 and 1:90 resp.), however, a significant difference was observed in women: 1:75.7 in area A and 1:114.7 in area H. In area A the titres were ranging in all age groups from 1:61 to 1:83 and the differences between age groups and between vaccinated and unvaccinated individuals were not significant. In area H the titres were generally higher (1:72 to 1:164) in the unvaccinated and 1:79 to 1:204 in the vaccinated. The differences in the unvaccinated were not of a statistical importance: in the vaccinated, the titres of the 20-29 years old (1:204.5) substantially differed from the titres obtained in other groups. The differences in titres level between the two studied areas are explained by a different epidemiological situation, namely by a higher tuberculosis infestation of some age groups in area H.

Characterization of a monoclonal antibody to Mycobacterium kansasii.

The characterization of an IgG2/Kappa monoclonal antibody against a protein antigen obtained by gel chromatography of the complex sonicate of M. kansasii biomass is described. It recognized specifically epitope molecules in complex and fractionated M. kansasii antigens ranging from 14.4 to 20 kD. The 20 kD immunoreactive band is common to the complex antigen, its fraction A and to the fraction B which was employed as the immunogenic agent. The 14.4 and 18 kD bands are predominantly present in the fraction B. High dilution of this MoAb (1:200,000) employed successfully in immunoblot studies suggests an antibody of very high affinity which could be useful for affinity purification of mycobacterial antigens in serodiagnosis of mycobacterial infections and in demonstration of antigens in biological materials.

A monoclonal antibody to Mycobacterium kansasii: preparation and properties.

An IgG2/kappa monoclonal antibody specific for a subunit protein antigen (mol.w. 32 KD) obtained by means of gel chromatography of the cell-free sonicate of M. kansasii was generated by the fusion of spleenocytes from BALB/c mice immunized with this antigen and Ag-8 myeloma cells. Its specificity was demonstrated by ELISA and dot blot procedures. This MoAb has potential application in taxonomy and species identification of M. kansasii isolates, in studies relating to the molecular pattern of the bound antigen and in serodiagnostics of the mycobacterial disease due to M. kansasii.

An evaluation of the polyacrylamide gel electrophoresis fractionation method for the production of Mycobacterium tuberculosis skin test preparations. I. Production, physiochemical characterization and serological analyses.

As a part of a cooperative inter-laboratory WHO supported project raw tuberculins were produced and purified protein derivative (PPD, 18.7 g protein) was prepared. Employing a multistage preparative polyacrylamide gel electrophoresis (PAGE) method the PPD was separated into four fractions corresponding to 15, 7, 4.75 and 3.5% gel concentrations. The PAGE procedure resulted in three lots of material--each representing 11 electrophoretic runs. Immunodiffusion analyses showed that the largest number of precipitinogens was found in the 15% fractions and that some precipitinogens cross-reacted with preparations of Mycobacterium bovis BCG, M. intracellulare, M. kansasii, M. smegmatis and M. vaccae.

An evaluation of the polyacrylamide gel electrophoresis fractionation method for the production of Mycobacterium tuberculosis skin test preparations. II. Comparative skin testing on guinea-pigs.

As part of a cooperative inter-laboratory WHO supported project for the fractionation of Mycobacterium tuberculosis skin test preparations, four fractions (designated 15, 7, 4.75 and 3.5%) were evaluated by comparative skin tests on sensitized guinea-pigs. The 7% fraction was the most potent in both homologously and heterologously sensitized animals, and the 4.75% and 3.5% gel fractions showed the lowest activity. Significant levels of cross-reactivity in guinea-pigs immunized with M. bovis BCG, M. kansasii, M. avium and M. intracellulare were demonstrated for all fractions examined, thus reflecting the antigenic relationships among these mycobacteria. These four fractions may qualify as starting material for further studies aiming at a reduction of skin test cross-reactivity.

The patterns of complex and partially purified mycobacterial antigens in macrophage migration inhibition testing.

In rabbits immunized intratarsally by M. tuberculosis, M. kansasii and M. avium the responses to homologous and heterologous antigens were assessed by direct and indirect macrophage migration inhibition tests. Complex cytoplasmic antigens were obtained by disruption of bacterial mass and by ultracentrifugation of the supernatants. The partially purified antigens were prepared by gel chromatography of the complex antigens on a Sephadex G 150 column. The middle fraction (260/280 ratio approx. 1, molecular weight approx. 32 KD) was employed as partially purified antigen. In the direct tests the migration activity of immune spleen macrophages was significantly reduced by homologous complex and partially purified antigens (MI = 0.63 to 0.72) and it differed significantly from responses obtained with heterologous antigens (MI = 0.75 to 0.92); however, these were still lower than those in nonimmunized control animals where MI ranged from 0.89 to 1.01. In the indirect tests, the strongest responses were recorded again with homologous complex and partially purified antigens (MI = 0.43 to 0.53). The responses in heterologous systems differed even more markedly than in direct tests (MI = 0.65 to 0.81); and, these were again still significantly lower than in control animals (MI = 0.89 to 0.98). In both direct and indirect tests, the complex and partially purified antigens did not vary substantially in their immunogenic capacity. The presence of cross-reacting responses in heterologous systems can be explained by a close relatedness of mycobacteria used in the immunization schedule and by the presence of common epitopes in complex and purified testing antigens.

In vitro studies of cell-mediated immunity in rabbits sensitized with Mycobacterium kansasii and Mycobacterium tuberculosis.

Rabbits, sensitized with M. kansasii, responded by a profound inhibition of migration of macrophages elicited by both antigens: the migration index for homologous antigen was 0.51 in the direct test and 0.65 in the indirect test; for heterologous antigen the indexes were 0.53 and 0.67. However, significant differences in reactivity were found in rabbits sensitized with M. tuberculosis. In the homologous system, high reactivity was maintained and the migration index reached the value of 0.53 in the direct and 0.63 in the indirect test. On the other hand, the heterologous antigen M. kansasii influenced the migration in both direct and indirect assays significantly less, the migration indexes being 0.62 and 0.72. The differences were statistically significant at 1% and 5% levels.

Ultrastructural studies of mycobacterial phages isolated from the material of sarcoid patients.

Mycophages DNAIII, Legendre and Clark, isolated from sarcoid material by Mankiewicz, belongs with respect to their ultrastructural morphology, to the systematic group B according to Bradley or to group IV according to Tikhonenko. These phages contain 2-DNA, their head is a regular icosahedron, the tail consisting of a helix of protein subunits is attached to the head by a narrowed segment and is fixed in it by means of a disc-like structure. It is terminated by a ball-shoped or conical end structure. By their dimensions, these phages belong to the smallest ones in this systematic group. In comparison with other mycobacterial phages, the studied phages exhibit very low mechanical resistance. This characteristic is most pronounced in phage DNAIII. This group of mycophages is also very sensitive lipid solvents and increased temeprature. This phenomenon is also most pronounced in phage DNAIII. The explanation of these findings will be the subject of our further study.