Immune Response to the Recombinant Apa Protein from Mycobacterium tuberculosis Expressed in Streptomyces lividans After Intranasal Administration in Mice. Induction of Protective Response to Tubercle Bacillus Aerosols Exposure.

Identifying and evaluating potential vaccine candidates has become one of the main objectives to combat tuberculosis. Among them, mannosylated Apa antigen from Mycobacterium tuberculosis and the non-mannosylated protein expressed in Escherichia coli, have been studied. Although both proteins can induce a protective response in mice, it has been considered that native protein can be dispensed. In this work, we study the protective response induced by Apa expressed in E. coli and in Streptomyces lividans. The latter, like native is secreted as a double band of 45/47 kDa, however, only its 47 kDa band is mannosylated. Both antigens and BCG were intranasal administrated in mice, and animals were then challenged by aerosol with M. tuberculosis H37Rv. The results showed that both, Apa from S. lividans and E. coli conferred statistically significantly protection to animals compared to controls. The cytokine immune response was studied by an immunoassay after animals' immunization, revealing that Apa from S. lividans induced a statistically significant proliferation of T cell, as well as the expression of IFN-γ, IL-1ß, IL-17 and IL-10. In contrast, non-proliferation was obtained with non-mannosylated protein, but induction of IL-12 and IL-17 was observed. Together, these results demonstrate that both proteins were able to modulate a specific immune response against M. tuberculosis, that could be driven by different mechanisms possibly associated with the presence or not of mannosylation. Furthermore, stimulation of cells from BCG-vaccinated animals with the proteins could be an important tool, to help define the use of a given subunit-vaccine after BCG vaccination.

New Insights into the Methylation of Mycobacterium tuberculosis Heparin Binding Hemagglutinin Adhesin Expressed in Rhodococcus erythropolis.

In recent years, knowledge of the role that protein methylation is playing on the physiopathogenesis of bacteria has grown. In Mycobacterium tuberculosis, methylation of the heparin binding hemagglutinin adhesin modulates the immune response, making this protein a subunit vaccine candidate. Through its C-terminal lysine-rich domain, this surface antigen interacts with heparan sulfate proteoglycans present in non-phagocytic cells, leading to extrapulmonary dissemination of the pathogen. In this study, the adhesin was expressed as a recombinant methylated protein in Rhodococcus erythropolis L88 and it was found associated to lipid droplets when bacteria were grown under nitrogen limitation. In order to delve into the role methylation could have in host-pathogen interactions, a comparative analysis was carried out between methylated and unmethylated protein produced in Escherichia coli. We found that methylation had an impact on lowering protein isoelectric point, but no differences between the proteins were found in their capacity to interact with heparin and A549 epithelial cells. An important finding was that HbhA is a Fatty Acid Binding Protein and differences in the conformational stability of the protein in complex with the fatty acid were observed between methylated and unmethylated protein. Together, these results suggest that the described role for this mycobacteria protein in lipid bodies formation could be related to its capacity to transport fatty acids. Obtained results also provide new clues about the role HbhA methylation could have in tuberculosis and point out the importance of having heterologous expression systems to obtain modified proteins.

Mycobacterial di-O-acyl trehalose inhibits Th-1 cytokine gene expression in murine cells by down-modulation of MAPK signaling.

Protection against tuberculosis (TB) is based on cell-mediated immune responses. TB is often characterized by immunological dysfunction of peripheral blood mononuclear cells, especially at chronic stages. Lipids from the Mycobacterium tuberculosis cell wall have been shown to produce various suppressive effects on cell-mediated immunity. The cell-surface lipid di-O-acyl-trehalose (DAT) is able to inhibit T-cell proliferation and cytokine secretion in cells from naïve mice. In the present study, we addressed the mechanisms involved in the suppressive effect caused by DAT. We found that DAT decreased the proliferation of spleen cells induced with PMA-ionomycin, suggesting that the suppressive mechanisms target intracellular functions just after phospholipase C-gamma activation. Addressing this possibility, the effect of DAT was found to involve down-modulation of the di-acyl glycerol-dependent activation of the MAPK-ERK1/2 pathway, one of the crucial signaling pathways leading to adaptive cell immune response against TB. Moreover, the inhibitory effect of DAT on proliferation was reproduced in antigen-stimulated T cells from M. tuberculosis-infected mice, involving the lowering of Th1-type cytokine transcription levels. The present findings thus reveal a new kind of bioactivity for a long-known M. tuberculosis cell wall lipid, DAT.

Mycobacterial trehalose-containing glycolipid with immunomodulatory activity on human CD4+ and CD8+ T-cells.

Protection against Mycobacterium tuberculosis is based on cell-mediated immunity, most importantly involving CD4+ and CD8+ T-cell subsets. One of the key features of the tubercle bacillus is its cell envelope, characterized by extremely abundant and specific lipids. The cell-surface glycolipid 2,3-di-O-acyl-trehalose (DAT) has been consistently found in M. tuberculosis strains. In this study, analysis of proliferation, activation markers and cytokine release was performed in human peripheral blood mononuclear cells (PBMC) activated in the presence and absence of DAT. We present evidence that mycobacterial DAT is able to reduce antigen-induced proliferation of human CD4+ and CD8+ T-cell subsets. We show that the effect is associated with a decrease of cells expressing the T-cell surface activation markers CD25 and CD69, and down-modulation of IL-2, IL-12, TNF-alpha and IL-10 cytokines. Data indicating that fine acyl chain structural variations in the trehalose-containing lipid may be involved in the degree of immune modulation are also presented.

Peptide mimotopes of Mycobacterium tuberculosis carbohydrate immunodeterminants.

Cell-surface saccharides of Mycobacterium tuberculosis appear to be crucial factors in tuberculosis pathogenicity and could be useful antigens in tuberculosis immunodiagnosis. In the present study, we report the successful antigenic and immunogenic mimicry of mannose-containing cell-wall compounds of M. tuberculosis by dodecamer peptides identified by phage-display technology. Using a rabbit antiserum raised against M. tuberculosis cell-surface saccharides as a target for biopanning, peptides with three different consensus sequences were identified. Phage-displayed and chemically synthesized peptides bound to the anticarbohydrate antiserum. Rabbit antibodies elicited against the peptide QEPLMGTVPIRAGGGS recognize the mannosylated M. tuberculosis cell-wall antigens arabinomannan and lipoarabinomannan, and the glycosylated recombinant protein alanine/proline-rich antigen. Furthermore, antibodies were also able to react with mannan from Saccharomyces cerevisiae, but not with phosphatidylinositol dimannosides or arabinogalactan from mycobacteria. These results suggest that the immunogenic peptide mimics oligomannosidic epitopes. Interestingly, this report provides evidence that, in contrast with previously known carbohydrate mimotopes, no aromatic residues are necessary in a peptide sequence for mimicking unusual glycoconjugates synthesized by mycobacteria. The possible usefulness of the identified peptide mimotopes as surrogate reagents for immunodiagnosis and for the study of functional roles of the native non-peptide epitopes is discussed.

6,6'-Dimycoloyl trehalose from a rapidly growing Mycobacterium: an alternative antigen for tuberculosis serodiagnosis.

Mycobacterial O-acyltrehaloses have been described as highly specific and sensitive reagents for tuberculosis immunodiagnosis. An O-acyltrehalose-containing lipid fraction from the rapidly growing Mycobacterium fortuitum was found to include additional antigens, which presented high cross-reactivity with sera from tuberculosis-infected patients. Based on a combination of selective chemical degradations, thin-layer-chromatography analyses and (1)H-nuclear magnetic resonance spectroscopy, the antigenic by-product was identified as 6,6'-dimycoloyl trehalose, the so-called cord factor. The lipid was purified and tested in ELISA for pulmonary tuberculosis serodiagnosis. Sensitivity and specificity of the test were found to be 66.6-74.1% and 95.2-99.0%, respectively, showing a slightly higher efficiency as compared to the ELISA performed using 6,6'-dimycoloyl trehalose from Mycobacterium tuberculosis. No cross-reactivity was found with sera from Nocardia-infected individuals.

Structure and antigenicity of the major glycolipid from Taenia solium cysticerci.

Lipids were extracted from cysticerci of the human tapeworm Taenia solium isolated from various infected pigs and analysed by two-dimensional thin-layer chromatography. These consisted of both alkali-labile and alkali-stable glycolipids, and phosphorylated non-glycosylated lipids. Because abundant and immunogenic glycolipids of parasites have been implicated in host-parasite interactions, the major lipid, an alkali-stable glycolipid, was purified by chromatography and its structure and antigenicity were determined. The structure of the major glycolipid of T. solium, GSL-I, was elucidated through a combination of chemical degradative methods, gas chromatography/mass spectrometry analyses of the degradative products, matrix-assisted-laser desorption/ionisation time of flight mass spectrometry and nuclear magnetic resonance spectroscopy. This analytical strategy led to the identification of a family of beta-galactosylceramides composed mainly of phytosphinganine (2-hydroxylated sphinganine) N-acylated by C16-C24 fatty acids, with the predominance of 2-hydroxylated homologues. Enzyme-linked immunosorbent assay showed no correlation between the antibody titres directed against GSL-I in the human sera and the infective status; in contrast, a very high specific immunoreactivity and a sensitivity above 50% were observed when GSL-I was tested with cerebrospinal fluids from well characterised infected humans. Thus, although these results do not support the use of GSL-I alone as an antigen for the detection of neurocysticercosis, its use as part of an antigen cocktail for the diagnosis of the disease in cerebrospinal fluids merits further investigations.