Involvement of caspase-9 in the inhibition of necrosis of RAW 264 cells infected with Mycobacterium tuberculosis.

In order to know how caspases contribute to the intracellular fate of Mycobacterium tuberculosis and host cell death in the infected macrophages, we examined the effect of benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethane (z-VAD-fmk), a broad-spectrum caspase inhibitor, on the growth of M. tuberculosis H37Rv in RAW 264 cells. In the cells treated with z-VAD-fmk, activation of caspase-8, caspase-3/7, and caspase-9 was clearly suppressed, and DNA fragmentation of the infected cells was also reduced. Under this experimental condition, it was found that the treatment markedly inhibited bacterial growth inside macrophages. The infected cells appeared to undergo cell death of the necrosis type in the presence of z-VAD-fmk. We further found that z-VAD-fmk treatment resulted in the generation of intracellular reactive oxygen species (ROS) in the infected cells. By addition of a scavenger of ROS, the host cell necrosis was inhibited and the intracellular growth of H37Rv was significantly restored. Among inhibitors specific for each caspase, only the caspase-9-specific inhibitor enhanced the generation of ROS and induced necrosis of the infected cells. Furthermore, we found that severe necrosis was induced by infection with H37Rv but not H37Ra in the presence of z-VAD-fmk. Caspase-9 activation was also detected in H37Rv-infected cells, but H37Ra never induced such caspase-9 activation. These results indicated that caspase-9, which was activated by infection with virulent M. tuberculosis, contributed to the inhibition of necrosis of the infected host cells, presumably through suppression of intracellular ROS generation.

Streptomycin-dependent exhibition of cytokine-inducing activity in streptomycin-dependent Mycobacterium tuberculosis strain 18b.

Peritoneal exudate cells of mice were stimulated with a streptomycin-dependent Mycobacterium tuberculosis strain, 18b. Gamma interferon production by natural killer cells depending on interleukin-12 and interleukin-18 was induced only in the presence of a high dose of streptomycin. This study suggested the requirement of active bacterial metabolism for this host response.

[Comparison between relative analogy and homology of 16S rRNA partial sequences between Mycobacterium szulgai and Mycobacterium malmoense].

A lot of nucleotide sequences of some genes, especially 16S rRNA gene, are registered in the public data-base. In order to identify clinical mycobacterial strains, 16S rRNA gene partial nucleotide sequences from 15 strains of Mycobacterium malmoense and 24 strains of Mycobacterium szulgai which are stored in the Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association were determined. Then homology of the sequences to the data of type strains submitted to public database were determined. Relative analogy to type strains by delta DDH was also measured. In the area of nucleotide position 51-588 corresponding accession number X52930 which is Mycobacterium malmoense type strain 16S rRNA gene sequence data, the homology of some partial sequences with Mycobacterium malmoense strains to data of accession number X52930 were lower than that with Mycobacterium szulgai type strain data, accession number X52926. In the area of nucleotide position 31-568 or nucleotide position 31-588, the homology of all nucleotide sequence data to collect species data of type strains were higher than the homology to another species data. Nucleotide position 38, 40, 47 and 49 might be differential nucleotides conserved between Mycobacterium malmoense strains and Mycobacterium szulgai one. These results suggest that homology of about 500 bp's 16S rRNA gene nucleotide sequence data may not be enough for differential identification. Nevertheless, database of RIDOM, Ribosomal differentiation of Medical Microorganisms, identified partial nucleotide sequences between nucleotide position 51-588 corresponding accession number X52930 correctly, though some were lower than 97% homology (data not shown). Therefore quality-controlled 16S rRNA gene nucleotide sequence database could be used for differential identification.

[Microplate hybridization methods of reduced unspecific reassociation considering delta Tm--comparison between relative relatedness of DNA and key characters for Mycobacterium szulgai].

It has been recognized that colorimetric microdilution plate hybridization method (DDH) shows equivocal identification results for some strains of Mycobacterium gordonae, and that chemotaxonomic identification method reveals some intermediate pattern between Mycobacterium szulgai and M. gordonae. In the present study, the results obtained by chemotaxonomic identification method on 25 strains of M. szulgai were compared with those obtained by DDH method. In chemotaxonomic methods, 8 of 25 M. szulgai strains showed negative results on 14 days' tween80 hydrolysis, 14 strains revealed negative nitrate reduction by the Tsukamura's method but all were positive by the Virtanen's method. Smooth colonies were found in 3 strains including M. szulgai type strain, JATA3201 (ATCC35799). Relative relatedness (relative color index) of genomic DNA was measured by DDH instead of spectrophotometric genomic DNA-DNA relatedness. A relative relatedness of 25 M. szulgai strains tested showed higher levels than 80%, but the inter-species relatedness to the other mycobacteria also showed high levels of 50-75%, when hybridizing temperature was set at 40 degrees C. At 56 degrees C, intra-species relative relatedness in 4 strains were lower than 50%, indicating that this condition is not appropriate. When hybridization temperature raised to 56 degrees C after overnight at 40 degrees C, a relative relatedness among 25 strains were again high (> 80%), and those to the other Mycobacterial species were lower than 70%. When hybridized at 56 degrees C after overnight at 45 degrees C, an intra-species relative relatedness again showed higher levels than 70% in all 25 strains, and interspecies percentiles were lowered satisfactorily to < 25%. In conclusion, through avoiding reassociation of nonspecific DNA fragments during the hybridization process, 45 degrees C overnight followed by 56 degrees C hybridization (delta DDH method) was found to be the better condition for identification and classification of Mycobacterium szulgai.

[Relative relatedness of 11 mycobacteria species by microplate hybridization methods considering delta Tm].

Intra-species variance within Mycobacterium xenopi, Mycobacterium gordonae or Mycobacterium szulgai has been reported in identification employing chemotaxonomic characteristics, 16 S rRNA gene sequences or relative relatedness (relative color index) of genomic DNA-DNA Hybridization. Genomic DNA-DNA reassociation at the constant temperature was found to be unreliable for classification of mycobacterial species. However, nonspecific DNA reassociation could be avoided by hybridization at 56 degrees C after 45 degrees C overnight, and this technique was named delta DDH method in the preceding paper. The present report shows relative relatedness (relative color index) of genomic DNA in delta DDH method among mycobacterial species. Relative relatedness was below 70% among BCG, M. kansasii, M. simiae, M. asiaticum, M. szulgai, M. gordonae, M. xenopi and M. nonchromogenicum. The results satisfied the criteria for bacterial classification, which was proposed by the International Committee for Systematic Bacteriology in 1987. In regard to Mycobacterium avium complex, relative relatedness between M. avium and M. intracellulare were approximately 75%. It appeared that M. avium and M. intracellulare could be classified into one species. It has been recognized, moreover, that there are intermediate strains between M. avium and M. intracellulare. Previously, numerical classification raised a concept of Mycobacterium avium-intracellulare-scrofulaceum complex. The present study revealed that relative relatedness of M. avium and of M. intracellulare to M. scrofulaceum were around 75%, while the percentiles of M. scrofulaceum relative to M. avium and that to M. intracellulare were both less than 50%. The relative relatedness of M. ulcerans against M. marinum was nearly 65%, whereas the relative relatedness of M. marinum against M. ulcerans was approximately 90%. The data may be partly explained by the horizontal gene transfer mechanism.