IFNG-mediated immune responses enhance autophagy against Mycobacterium tuberculosis antigens in patients with active tuberculosis

Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen.     

IFNG-mediated immune responses enhance autophagy against Mycobacterium tuberculosis antigens in patients with active tuberculosis

Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen.     

Response to Mycobacterium tuberculosis antigens is enhanced by monoclonal antibodies.

T lymphocytes purified from peripheral blood of tuberculosis patients and from healthy skin test PPD-positive subjects show a remarkably increased proliferative response in culture to purified protein derivative (PPD) when three anti-mycobacterial monoclonal antibodies are added together with the antigen. This indicates an important role played by antibodies in the T cell response to the tuberculous infection.     

Multiple small RNAs identified in Mycobacterium bovis BCG are also expressed in Mycobacterium tuberculosis and Mycobacterium smegmatis

Tuberculosis (TB) is a major global health problem, infecting millions of people each year. The causative agent of TB, Mycobacterium tuberculosis, is one of the world’s most ancient and successful pathogens. However, until recently, no work on small regulatory RNAs had been performed in this organism. Regulatory RNAs are found in all three domains of life, and have already been shown to regulate virulence in well-known pathogens, such as Staphylococcus aureus and Vibrio cholera. Here we report the discovery of 34 novel small RNAs (sRNAs) in the TB-complex M. bovis BCG, using a combination of experimental and computational approaches. Putative homologues of many of these sRNAs were also identified in M. tuberculosis and/or M. smegmatis. Those sRNAs that are also expressed in the non-pathogenic M. smegmatis could be functioning to regulate conserved cellular functions. In contrast, those sRNAs identified specifically in M. tuberculosis could be functioning in mediation of virulence, thus rendering them potential targets for novel antimycobacterials. Various features and regulatory aspects of some of these sRNAs are discussed.     

Detection of in vitro and in vivo released antigens of diagnostic interest in Mycobacterium tuberculosis by immunoblotting

Identification of in vitro and in vivo released mycobacterial antigens are of considerable interest in diagnosis of Mycobacterium tuberculosis. Isolation of in vitro released antigen from M. tb excretory-secretory culture filtrate protein and in vivo released circulating tuberculous antigen from smear positive pulmonary tuberculosis sera by ammonium sulphate precipitation is reported. The antigens were resolved by SDS-PAGE and immunoblotting was performed using pooled serum of smear positive, smear negative pulmonary tuberculosis sera and normal sera to identify reactive tuberculous antigens. In vitro and in vivo released mycobacterial antigens showed reactivity at 100, 31, 43 and 20 kDa with smear positive and smear negative pulmonary tuberculosis patients. Further, the in vitro released antigen showed strong reactivity exclusively at 55 kDa antigen with smear positive and 24 kDa antigen with smear negative pulmonary tuberculosis sera. In vivo released antigen reacted exclusively at 170 and 16 kDa with smear positive and 19 kDa antigen with smear negative pulmonary tuberculosis patients. Antigens of 24 and 19 kDa which are reactive with sputum negative sera will be of diagnostic interest and need further study in patients with low bacillary load. The in vitro and in vivo released mycobacterial 100, 31,43 and 20 kDa antigens, reactive with patients sera are of diagnostic interest in tuberculosis.     

Recognition of novel glycolipid antigens from smooth variants of Mycobacterium tuberculosis

A major polar and three minor slightly less polar glycolipids were identified in extracts of two smooth (Canetti) strains of Mycobacterium tuberculosis. Immunostaining on thin-layer chromatograms and enzyme-linked immunosorbent assay (ELISA) of purified lipids demonstrated that the major and the two most polar of the minor glycolipids are potent antigens, reacting with homologous antisera and also with that raised against the type strain (H37Rv).     

Identification of the new T-cell-stimulating antigens from Mycobacterium tuberculosis culture filtrate

The proteins secreted by Mycobacterium tuberculosis are an important target for vaccine development. To identify the antigens from M. tuberculosis culture filtrate (CF) that strongly stimulate T-cells, the CF was fractionated by ion-exchange chromatography and then non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mini-whole gel elution. Each fraction was screened for its ability to induce interferon-gamma (IFN-gamma) production in peripheral blood mononuclear cells isolated from healthy tuberculin reactors. The protein bands that strongly induced IFN-gamma production were subjected to N-terminal sequencing. Two new proteins, a 17-kDa protein (Rv0164, MTSP17) and an 11-kDa (Rv3204, MTSP11) protein, were identified. The recombinant MTSP17 (rMTSP17) and rMTSP11 induced significant production of IFN-gamma and interleukin (IL)-12p40 in peripheral blood mononuclear cells from healthy tuberculin reactors. Interestingly, IL-12p40 production in response to rMTSP11 was significantly higher than that in response to rMTSP17 or the three components of the antigen 85 complex. These results suggest that MTSP11 antigen should be further evaluated as a component of a subunit vaccine.     

Exosomes released from infected macrophages contain Mycobacterium avium glycopeptidolipids and are proinflammatory

Mycobacterium avium is a major opportunistic pathogen in HIV-positive individuals and is responsible for increased morbidity and mortality in AIDS patients. M. avium express glycopeptidolipids (GPLs) as a major cell wall constituent, and recent studies suggest that GPLs play an important role in M. avium pathogenesis. In the present study we show that M. avium-infected macrophages release GPLs, which are trafficked from the phagosome through the endocytic network to multivesicular bodies. Prior studies have shown that multivesicular bodies can fuse with the plasma membrane releasing small 50 to 100 nm vesicles known as exosomes. We found that M. avium-infected macrophages release exosomes containing GPLs leading to the transfer of GPLs from infected to uninfected macrophages. Interestingly, exosomes isolated from M. avium-infected but not from uninfected macrophages can stimulate a proinflammatory response in resting macrophages. This proinflammatory response is dependent on Toll like receptor (TLR) 2, TLR4, and MyD88 suggesting that released exosomes contain M. avium-expressed TLR ligands. Our studies are the first to demonstrate that exosomes isolated from mycobacteria-infected macrophages can induce a proinflammatory response, and we hypothesize that exosomes play an important role in immune surveillance during intracellular bacteria infections.     

Pulmonary mononuclear cell responses to antigens of Mycobacterium tuberculosis in healthy household contacts of patients with active tuberculosis and healthy controls from the community

Protective immunity against Mycobacterium tuberculosis requires CD4+ lymphocyte-mediated immune responses and IFN-gamma activity. As the primary portal of entry of M. tuberculosis is the lung, pulmonary immune responses against multiple M. tuberculosis Ags were compared between both M. tuberculosis-exposed tuberculin skin test-positive healthy household contacts (HHC) of patients with active sputum smear and culture-positive tuberculosis and tuberculin skin test-positive healthy control individuals from the community (CC). Frequencies of M. tuberculosis Ag-specific IFN-gamma-producing cells, IFN-gamma concentrations in culture supernatants, and DNA synthesis in bronchoalveolar cells (BAC) and PBMC were studied in HHC (n = 10) and CC (n = 15). Using enzyme-linked immunospot assay we found higher frequencies of IFN-gamma-producing cells with specificity to M. tuberculosis-secreted Ag 85 (Ag 85) in BAC from HHC than in BAC from CC (p < 0.022) and relative to autologous PBMC, indicating compartmentalization of Ag 85-specific cells to the lungs. Further, IFN-gamma-producing cells with specificity to components A and B of Ag 85 were specifically compartmentalized to the lungs in HHC (p < 0. 05). IFN-gamma concentrations in culture supernatants of BAC and Ag-specific DNA synthesis were low and comparable in the two subject groups. Increased immune responses to Ag 85 at the site of repeated exposure to M. tuberculosis (the lung) may represent an important component of protective immunity against M. tuberculosis. Correlates of protective immunity against M. tuberculosis are required for assessment of the efficiency of anti-tuberculous vaccines.     

Whole blood assay to access T cell-immune responses to Mycobacterium tuberculosis antigens in healthy Brazilian individuals

The production of interferon gamma (IFNgamma) guarantees effective T cell-mediated immunity against Mycobacterium tuberculosis infection. In the present study, we simply compare the in vitro immune responses to Mycobacterium antigens in terms of IFNg production in a total of 10 healthy Brazilian volunteers. Whole blood and mononuclear cells were cultivated in parallel with PPD, Ag85B, and M. bovis hsp65, and five-days supernatants were harvested for cytokine detection by ELISA. The inter-assay result was that the overall profile of agreement in response to antigens was highly correlated (r(2) = 0.9266; p = 0.0102). Potential analysis is in current progress to dictate the usefulness of this method to access the immune responses also in tuberculosis patients and its contacts.     

Cytokine profiles in tuberculosis patients and healthy subjects in response to complex and single antigens of Mycobacterium tuberculosis

Peripheral blood mononuclear cells (PBMC) were obtained from tuberculosis (TB) patients and Mycobacterium bovis bacillus Calmette-Guerin vaccinated healthy subjects. PBMC were tested for secretion of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-5 (IL-5) and IL-10 in response to complex (whole cells, culture filtrate and cell walls), single secreted (Ag85B, ESAT6, MPT64, PstS and MPT70) and single cytosolic (DnaK, GroES and GroEL) antigens of Mycobacterium tuberculosis. In the absence of antigens, detectable concentrations of TNF-alpha, IFN-gamma and IL-10 were secreted by PBMC of both donor groups, but the concentrations of only IL-10 were significantly higher (P=0.015) in TB patients than in healthy subjects. In the presence of complex antigens, PBMC secreted IFN-gamma and TNF-alpha in response to all three preparations, whereas IL-10 was secreted in response to whole cells and cell walls only. In the presence of single antigens, IFN-gamma was secreted in response to Ag85B, ESAT6 and MPT64 in TB patients and ESAT6 in healthy donors. Except for GroEL and DnaK, single antigens did not induce TNF-alpha and IL-10 secretion from PBMC in either donor group. The secretion of IFN-gamma, but not IL-10, in the presence of Ag85B, ESAT6 and MPT64 supports their potential as subunit vaccine candidates against TB.     

Mycobacterium leprae RecA is structurally analogous but functionally distinct from Mycobacterium tuberculosis RecA protein

Mycobacterium leprae is closely related to Mycobacterium tuberculosis, yet causes a very different illness. Detailed genomic comparison between these two species of mycobacteria reveals that the decaying M. leprae genome contains less than half of the M. tuberculosis functional genes. The reduction of genome size and accumulation of pseudogenes in the M. leprae genome is thought to result from multiple recombination events between related repetitive sequences, which provided the impetus to investigate the recombination-like activities of RecA protein. In this study, we have cloned, over-expressed and purified M. leprae RecA and compared its activities with that of M. tuberculosis RecA. Both proteins, despite being 91% identical at the amino acid level, exhibit strikingly different binding profiles for single-stranded DNA with varying GC contents, in the ability to catalyze the formation of D-loops and to promote DNA strand exchange. The kinetics and the extent of single-stranded DNA-dependent ATPase and coprotease activities were nearly equivalent between these two recombinases. However, the degree of inhibition exerted by a range of ATP:ADP ratios was greater on strand exchange promoted by M. leprae RecA compared to its M. tuberculosis counterpart. Taken together, our results provide insights into the mechanistic aspects of homologous recombination and coprotease activity promoted by M. lepare RecA, and further suggests that it differs from the M. tuberculosis counterpart. These results are consistent with an emerging concept of DNA-sequence influenced structural differences in RecA nucleoprotein filaments and how these differences reflect on the multiple activities associated with RecA protein.     

Mycobacterium tuberculosis strains of the Beijing genotype are rarely observed in tuberculosis patients in South America

The frequency of the Beijing genotype of Mycobacterium tuberculosis as a cause of tuberculosis (TB) in South America was determined by analyzing genotypes of strains isolated from patients that had been diagnosed with the disease between 1997 and 2003 in seven countries of the subcontinent. In total, 19 of the 1,202 (1.6%) TB cases carried Beijing isolates, including 11 of the 185 patients from Peru (5.9%), five of the 512 patients from Argentina (1.0%), two of the 252 Brazilian cases (0.8%), one of the 166 patients from Paraguay (0.6%) and none of the samples obtained from Chile (35), Colombia (36) and Ecuador (16). Except for two patients that were East Asian immigrants, all cases with Beijing strains were native South Americans. No association was found between carrying a strain with the Beijing genotype and having drug or multi-drug resistant disease. Our data show that presently transmission of M. tuberculosis strains of the Beijing genotype is not frequent in Latin America. In addition, the lack of association of drug resistant TB and infection with M. tuberculosis of the Beijing genotype observed presently demands efforts to define better the contribution of the virulence and lack of response to treatment to the growing spread of Beijing strains observed in other parts of the world.     

Genetic diversity among Mycobacterium tuberculosis isolates from Mexican patients

Forty-six isolates of the Mycobacterium tuberculosis complex were typified by PCR of the IS6110 region and by Mycobacterium bovis specific primers JB21/JB22. Isolate MVG01 was typified as M. bovis, being the first record of a case of human tuberculosis caused by this species in Mexico. RAPD-PCR was used to describe the genetic diversity of the remaining 45 M. tuberculosis complex isolates. The corrected genotypic diversity value calculated for the analyzed population was 0.96, the estimated mean gene diversity was 0.235, and the corrected Shannon-Weiner index was 2.15. All allele-loci combinations generated showed significant linkage disequilibria. The distribution of genetic variation was analyzed both by the unweighted pair group method with arithmetic averages clustering and by principal coordinates analysis. Unweighted pair group method with arithmetic averages clustering resulted in a tree with four main clusters and one unclustered strain (MVG20), the principal coordinates analysis strain distribution pattern being consistent with this grouping. The obtained results suggest that the studied isolates belong to a clonal population having significant genetic diversity. Our genetic diversity results are comparable with those reported for other populations of M. tuberculosis, although only three RAPD primers were used.     

Mutations linked with antimicrobial resistance in Mycobacterium tuberculosis isolated from tuberculosis patients in the Samara region

A total of 234 M. tuberculosis isolates were used to demonstrate the leading role of mutations in, respectively, codon 531 of gene rpoB (90.0%) and codon 315 of gene katG (92.9%), in the development of resistance to rifampicin and isoniazid by the methods of reverse hybridization with oligonucleotide probes and the sequencing of gene stretches. The levels of primary resistance of M. tuberculosis to rifampicin, isoniazid and multiresistance, according to the molecular-genetic analysis, were 41.0%, 57.7% and 37.2% respectively. The coincidence of the results of the bacteriological and molecular-genetic analyses of the antimicrobial resistance of the isolates was 90.4% and 95.3% for isoniazid and rifampicin respectively. The prevalence of individual types of mutations, linked with antimicrobial resistance, in the presence of a considerable spread of strains of the family Beijing in the region may be indicative of the limited number of M. tuberculosis clones circulating in the region.