The Mycobacterium avium complex includes two closely related species, Mycobacterium avium and Mycobacterium intracellulare. They are opportunistic pathogens in humans and responsible for severe disease in a wide variety of animals. Yet, little is known about factors involved in their pathogenicity. Here, we identified, purified and characterized adhesins belonging to the heparin-binding hemagglutinin (HBHA) and laminin-binding protein (LBP) family from M. intracellulare ATCC13950 and examined clinical isolates from patients with different pathologies associated with M. intracellulare infection for the presence and conservation of HBHA and LBP. Using a recombinant derivative strain of M. intracellulare ATCC13950 producing green fluorescent protein and luciferase, we found that the addition of heparin inhibited mycobacterial adherence to A549 cells, whereas the addition of laminin enhanced adherence. Both HBHA and LBP were purified by heparin-Sepharose chromatography and their methylation profiles were determined by mass spectrometry. Patients with M. intracellulare infection mounted strong antibody responses to both proteins. By using PCR and immunoblot analyses, we found that both proteins were highly conserved among all 17 examined clinical M. intracellulare isolates from patients with diverse disease manifestations, suggesting a conserved role of these adhesins in M. intracellulare virulence in humans and their potential use as a diagnostic tool.
Despite its potential for early diagnosis of Mycobacterium avium subsp. paratuberculosis (MAP) infection, the IFN-γ release assay is not used routinely, because of low specificity of the established crude antigen preparation Johnin (PPDj). Limited data are available assessing the potential of MAP-derived protein and lipopeptide antigens to replace PPDj in assays for goats, while cattle and sheep have been studied more extensively. Furthermore, MAP infection is claimed to interfere with the diagnosis of bovine tuberculosis when other crude antigen preparations (PPDb, PPDa) are applied. In this study, the diagnostic potential of MAP-derived recombinant protein antigens, synthetic MAP lipopentapeptides and of Mycobacterium bovis- specific peptide cocktails was assessed compared to crude mycobacterial antigen preparations in experimentally infected goats. Goats were inoculated with MAP, or Mycobacterium avium subsp. hominissuis (MAH) as surrogate for environmental mycobacteria, non-exposed animals served as controls. Mycobacterium avium Complex-specific antibody and PPDj-induced IFN-γ responses were monitored in vivo . Infection status was assessed by pathomorphological findings and bacteriological tissue culture at necropsy 1 year after inoculation. The IFN-γ response to 13 recombinant protein antigens of MAP, two synthetic MAP lipopentapeptides and three recombinant peptide cocktails of Mycobacterium bovis was investigated at three defined time points after infection. At necropsy, MAP or MAH infection was confirmed in all inoculated goats, no signs of infection were found in the controls. Antibody formation was first detected 3–6 weeks post infection (wpi) in MAH-inoculated and 11–14 wpi in the MAP-inoculated goats. Maximum PPDj-induced IFN-γ levels in MAH and MAP exposed animals were recorded 3–6 and 23–26 wpi, respectively. Positive responses continued with large individual variation. Antigens Map 0210c, Map 1693c, Map 2020, Map 3651cT(it), and Map 3651c stimulated increased whole blood IFN-γ levels in several MAP-inoculated goats compared to MAH inoculated and control animals. These IFN-γ levels correlated with the intensity of the PPDj-induced responses. The two synthetic lipopentapeptides and the other MAP-derived protein antigens had no discriminatory potential. Stimulation with Mycobacterium bovis peptide cocktails ESAT6-CFP10, Rv3020c, and Rv3615c did not elicit IFN-γ production. Further work is required to investigate if test sensitivity will increase when mixtures of the MAP-derived protein antigens are applied.
Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a live vaccine that has been used in routine vaccination against tuberculosis for nearly 80 years. However, its efficacy is controversial. The failure of BCG vaccination may be at least partially explained by the induction of poor or inappropriate host responses. Dendritic cells (DCs) are likely to play a key role in the induction of immune response to mycobacteria by polarizing the reactivity of T lymphocytes toward a Th1 profile, contributing to the generation of protective cellular immunity against mycobacteria. In this study we aimed to investigate the production of Th1 and Th2 cytokines by naive CD4+ T cells to mycobacterial antigen-pulsed DCs in the group of young, healthy BCG vaccinated volunteers. The response of naive helper T cells was compared with the response of total blood lymphocytes. Our present results clearly showed that circulating naive CD45RA+CD4+ lymphocytes from BCG-vaccinated subjects can become effector helper cells producing IFN-gamma and IL-5 under the stimulation by autologous dendritic cells presenting mycobacterial protein antigen-PPD or infected with live M. bovis BCG bacilli.
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