The mycobacterial DNA-binding protein 1 (MDP1) from Mycobacterium bovis BCG influences various growth characteristics.

BACKGROUND: Pathogenic mycobacteria such as M. tuberculosis, M. bovis or M. leprae are characterised by their extremely slow growth rate which plays an important role in mycobacterial virulence and eradication of the bacteria. Various limiting factors influence the generation time of mycobacteria, and the mycobacterial DNA-binding protein 1 (MDP1) has also been implicated in growth regulation. Our strategy to investigate the role of MDP1 in mycobacterial growth consisted in the generation and characterisation of a M. bovis BCG derivative expressing a MDP1-antisense gene. RESULTS: The expression rate of the MDP1 protein in the recombinant M. bovis BCG containing the MDP1-antisense plasmid was reduced by about 50% compared to the reference strain M. bovis BCG containing the empty vector. In comparison to this reference strain, the recombinant M. bovis BCG grew faster in broth culture and reached higher cell masses in stationary phase. Likewise its intracellular growth in mouse and human macrophages was ameliorated. Bacterial clumping in broth culture was reduced by the antisense plasmid. The antisense plasmid increased the susceptibility of the bacteria towards Ampicillin. 2-D protein gels of bacteria maintained under oxygen-poor conditions demonstrated a reduction in the number and the intensity of many protein spots in the antisense strain compared to the reference strain. CONCLUSION: The MDP1 protein has a major impact on various growth characteristics of M. bovis BCG. It plays an important role in virulence-related traits such as aggregate formation and intracellular multiplication. Its impact on the protein expression in a low-oxygen atmosphere indicates a role in the adaptation to the hypoxic conditions present in the granuloma.

Gelatinase activity in Mycobacterium bovis protein extract.

Proteases are well-recognized as virulence factors in different pathologies, resulting in tissue damage potential. Despite efforts over the past few years to identify mycobacterial protein antigens, there is little information regarding the role of mycobacterial proteinase activities. In this study, by zymography techniques, we have detected and partially studied some biochemical properties of Mycobacterium bovis proteases, such as pH dependency of activity and susceptibility to classical proteinase inhibitors. We observed optimal proteolytic activity at pH 8. Some proteinases were inhibited by classic inhibitors of serine proteases, such as PMSF, AEBSF, and 3-4 DCI. In some AEBSF pre-treated preparations we observed residual gelatinase activity in Rf 0.32. This gelatinase was stimulated by Zn2+ and inhibited by OPA (1 mM). This last effect was reversed by exposure to equimolar quantitative OPA/Zn+2 (1 mM/1 mM). These results suggest the existence of serine proteinase and metalloproteinase types in protein extracts of Mycobacterium bovis.

Evaluation of methods for isolation of DNA from slowly and rapidly growing mycobacteria.

Mycobacteria generally have thick cell walls and contain large amounts of lipid, making them resistant to DNA extraction. Five methods, namely, extensive enzymic digestion method (M1), 2-min mechanical glass-bead disruption method (M2), thermal shock method (M3), modified conventional enzymic digestion method (M4), and manual disruption with modified conventional enzymic digestion method (M5), were used to compare their effectiveness and simplicity in extracting DNA from slowly growing mycobacteria (Mycobacterium leprae, M. lepraemurium and M. bovis BCG), and a rapidly growing mycobacterium (M. phlei). The highest DNA yield was obtained by M2 from M. lepraemurium which produced 2.82 micrograms DNA/mg wet weight of cells, representing a theoretical yield of 78%. M3 gave the lowest DNA yield; 0.01 microgram DNA/mg wet weight of cells of M. lepraemurium was obtained. M4, in which proteinase K was used, is more effective than M1, in which subtilisin and pronase were used. M5 yielded a higher amount of DNA, but it required more manipulations to extract DNA as compared to M4. Extraction of DNA of M. leprae from nude mice is more difficult than that of M. leprae from armadillos by all of the methods used. These results suggest that the biosynthetic capabilities of these two forms of M. leprae may vary, depending on their cultural conditions and/or strain differences. Our results have shown that both M2 and M4 are the simplest, most effective and time-saving methods which are suitable for every routine laboratory to extract DNA from slowly and rapidly growing mycobacteria.

Characterization of the antigen 85 complex fibronectin-binding proteins derived from aged culture filtrates of Mycobacterium bovis BCG, Tice substrain.

The antigen 85 complex are major T-cell and B-cell antigens and fibronectin-binding proteins secreted by Mycobacterium tuberculosis, M. leprae and attenuated M. bovis (BCG vaccine). The Ag 85 complex was found to comprise a high proportion of the extracellular protein in filtrates of surface-pellicle cultures of Tice-substrain BCG vaccine, attaining a maximum of 25%. This proportion began to decrease prior to the end of the logarithmic growth phase, about 3 weeks after the start of the culture, mainly due to apparent degradation of the Ag 85 complex. Isolation of the main Ag 85 protein and determination of the first 36 residues of the NH2-terminus showed identity with the 85A protein isolated by others from various mycobacteria. Both the Ag 85A and B components were secreted in nearly constant proportions over a 6-week period. No Ag 85C protein was detected.

[Functional characteristics of the antioxidative system in mycobacteria grown on perfluorodecalin-modified media]. / Osobennosti funktsionirovaniia antiokislitel'noi sistemy mikobakterii, vyrashchennykh na sredakh, modifitsirovannykh perftordekalinom.

The growth of mycobacteria on perfluorodecalin-modified media was shown to be accompanied by distinct alterations in the activity of the antioxidant enzyme system in M. bovis BCG and M. lufu. In M. bovis BCG the levels of glutathione transferase and glutathione peroxidase-hydrogen peroxidase activity are decreased by 45.47% and 100.88%, respectively. In M. lufu, on the contrary, the level of superoxide dismutase is increased by 42.23%, with no changes observed in the levels of glutathione transferase and glutathione peroxidases. The data obtained suggest physiological heterogeneity of mycobacteria and, thus, open prospects for the differential approaches to the problem of increasing the efficacy of in vitro cultivation of various mycobacterial species, including M. leprae.

Macrophage activity in Mycobacterium leprae infection.

The outcome of an M. leprae infection is likely to depend upon the balance between the invading organism and the host's immune response. Macrophages are known to play a major role in this response and because M. leprae is an intracellular parasite, being found commonly in the macrophages of infected hosts, we have attempted to examine the macrophage/M. leprae relationship. Our model has been the athymic nude mouse which has been shown to be susceptible to lepromatous infection but whose macrophages when cultured in vitro actually kill phagocytosed M. leprae. We have shown that in vitro this killing effect is probably mediated, at least to some extent, by macrophage-generated hydrogen peroxide. Further, we have examined macrophages from nude and normal mice at various stages of M. leprae infection in time of their ability to produce hydrogen peroxide and superoxide. It would appear from our results that activation of macrophages to produce these two bactericidal metabolites increases with increasing bacterial load. However, it would seem that T-cell mediated mechanisms are also required for effective control of infection as the hyperactive macrophages seen in the nude mouse are unable to control M. leprae growth in contrast to the limited infection seen in normal mice.

Effect of T-cell depletion on the growth of BCG in the mouse footpad.

The growth of Mycobacterium bovis (BCG Montreal) and M. tuberculosis Erdman was determined in normal and T-cell depleted (THXB) mice when injected subcutaneously into a hind footpad. The bacilli multiplied only to a limited extent within the footpad itself but the infection quickly spread to the draining popliteal lymph node to eventually reach the liver, spleen and lung. The amount of systemic growth seen in the THXB mice was 10-100 times greater than in the normal controls, all of which developed a tuberculin hypersensitivity and an immune response in 14-18 days. T-cell depletion completely inhibited the expression of tuberculin sensitivity by the infected host as well as ablating the antituberculous response against both the vaccinating BCG population and a superinfecting Erdman challenge inoculum. Incorporation studies in the THXB mice indicated a striking reduction in cell division within the draining lymph node but there was an unexpected elevation in the level of incorporation by the lung cells as the BCG infection progressed. The significance of these findings is discussed in relation to the possible use of the BCG footpad model for studies of leprosy immunity.