In-vitro study of mouse peritoneal macrophage response to virulent and avirulent strains of mycobacteria.

An immunological study of pathogenesis of tuberculosis was carried out in BALB-c mice in-vitro. Peritoneal macrophages obtained from BALB-c mice were challenged with virulent (H37Rv) and avirulent (H37Ra, BCG, M. phlei) strains of mycobacteria. Activated peritoneal macrophages showed enlargement, presence of intracellular bacteria and vacuolation. These significant changes in macrophage morphology were clearly evidenced in cells infected with virulent strains of Mycobacterium tuberculosis i.e. H37Rv while being absent in cells infected with avirulent H37Ra, BCG and M. phlei. Virulent mycobacteria (H37Rv) survive the phagocytic action of macrophages by residing inside the vacuoles. The capacity of virulent and avirulent strain to stimulate TNF-alpha production from peritoneal macrophage of BALB-c mice was also examined at different time interval i.e. 1,2,4,6 and 8th day by measuring cytolytic activity of culture supernatant against murine fibroblast cell line. The pattern of highest TNF release was in case of H37Rv and least with M. phlei as measured in culture supernatant after 1,2,4,6 and 8th day.

Fusion of azurophil granules with phagosomes and activation of the tyrosine kinase Hck are specifically inhibited during phagocytosis of mycobacteria by human neutrophils.

Pathogenic mycobacteria parasitize macrophages and reside within phagosomes, which do not fuse with lysosomal granules. Mycobacteria are also internalized by neutrophils, which possess at least two types of granules, specific and azurophil granules, the latter being specialized lysosomes. Here, we investigated the ability of mycobacteria to inhibit the fusion of these granules with their phagosomes in human neutrophils. It was found that when pathogenic (Mycobacterium kansasii and Mycobacterium avium) or nonpathogenic (Mycobacterium smegmatis and Mycobacterium phlei) mycobacteria were internalized by neutrophils, they induced the inhibition of azurophil granule fusion with phagosomes even when they were serum opsonized. In contrast, secretion of specific granule content and production of O2-, both of which contribute to the neutrophil bactericidal response, were triggered. Hck is a Src family tyrosine kinase associated with azurophil granules. During internalization of zymosan, azurophil granules fused with phagosomes and Hck was activated and translocated to the phagosomal membrane, whereas in neutrophils engulfing mycobacteria, Hck did not translocate and remained unactivated. The activation of the tyrosine kinase Fgr was not affected. These results indicate that 1) pathogenic and nonpathogenic mycobacteria trigger similar bactericidal responses in neutrophils, 2) phagocytosis and fusion of azurophil granules can be uncoupled by mycobacteria, and 3) Hck could be one of the key elements of the azurophil secretory pathway that are altered during phagocytosis of mycobacteria.

Strategies used by pathogenic and nonpathogenic mycobacteria to synthesize rRNA.

One rRNA operon of all mycobacteria studied so far is located downstream from a gene thought to code for the enzyme UDP-N-acetylglucosamine carboxyvinyl transferase (UNAcGCT), which is important to cell wall synthesis. This operon has been designated rrnAf for fast-growing mycobacteria and rrnAs for slow growers. We have investigated the upstream sequences and promoter activities of rrnA operons of typical fast growers which also possess a second rrn (rrnBf) operon and of the rrnA operons of the fast growers Mycobacterium abscessus and Mycobacterium chelonae, which each have a single rrn operon per genome. These fast growers have a common strategy for increasing the efficiency of transcription of their rrnA operons, thereby increasing the cells' potential for ribosome synthesis. This strategy involves the use of multiple (three to five) promoters which may have arisen through successive duplication events. Thus we have identified a hypervariable multiple promoter region (HMPR) located between the UNAcGCT gene and the 16S rRNA coding region. Two promoters, P1 and PCL1, appear to play pivotal roles in mycobacterial rRNA synthesis; they are present in all of the species examined and are the only promoters used for rRNA synthesis by the pathogenic slow growers. P1 is located within the coding region of the UNAcGCT gene, and PCL1 has a characteristic sequence that is related to but distinct from that of the additional promoters. In fast-growing species, P1 and PCL1 produce less than 10% of rRNA transcripts, so the additional promoters found in the HMPR are important in increasing the potential for rRNA synthesis during rapid growth. In contrast, rrnB operons appear to be regulated by a single promoter; because less divergence has taken place, rrnB appears to be younger than rrnA.

[A new method of determining the virulence of nontubercular mycobacteria and weakly virulent Mycobacterium tuberculosis]. / Novyi metod opredeleniia virulentnosti netuberkuleznykh i slabovirulentnykh tuberkuleznykh mikobakterii.

A new method for enhancing the sensitivity of white mice to infection with faintly virulent mycobacteria is proposed. The method consists in the sensitization of animals with pertussis monovaccine introduced in a single-intraperitoneal injection 7 days before the infection of animals with the culture under study. The method permitted the detection of residual virulence in all opportunistic mycobacteria under study.