Search

There is a substantial need for biomarkers to distinguish latent stage from active Mycobacterium tuberculosis infections, for predicting disease progression. To induce the reactivation of tuberculosis, we present a new experimental animal model modified based on the previous model established by our group. In the new model, the reactivation of tuberculosis is induced without administration of immunosuppressive agents, which might disturb immune responses. To identify the immunological status of the persistent and chronic stages, we analyzed immunological genes in lung tissues from mice infected with M. tuberculosis. Gene expression was screened using cDNA microarray analysis and confirmed by quantitative RT-PCR. Based on the cDNA microarray results, 11 candidate cytokines genes, which were obviously up-regulated during the chronic stage compared with those during the persistent stage, were selected and clustered into three groups: (1) chemokine genes, except those of monocyte chemoattractant proteins (MCPs; CXCL9, CXCL10, CXCL11, CCL5, CCL19); (2) MCP genes (CCL2, CCL7, CCL8, CCL12); and (3) TNF and IFN-γ genes. Results from the cDNA microarray and quantitative RT-PCR analyses revealed that the mRNA expression of the selected cytokine genes was significantly higher in lung tissues of the chronic stage than of the persistent stage. Three chemokines (CCL5, CCL19, and CXCL9) and three MCPs (CCL7, CCL2, and CCL12) were noticeably increased in the chronic stage compared with the persistent stage by cDNA microarray (p < 0.01, except CCL12) or RT-PCR (p < 0.01). Therefore, these six significantly increased cytokines in lung tissue from the mouse tuberculosis model might be candidates for biomarkers to distinguish the two disease stages. This information can be combined with already reported potential biomarkers to construct a network of more efficient tuberculosis markers.

Abstract: l-Alanine dehydrogenase from Mycobacterium tuberculosis catalyzes the NADH-dependent reversible conversion of pyruvate and ammonia to l-alanine. Expression of the gene coding for this enzyme is up-regulated in the persistent phase of the organism, and alanine dehydrogenase is therefore a potential target for pathogen control by antibacterial compounds. We have determined the crystal structures of the apo- and holo-forms of the enzyme to 2.3 and 2.0 Å resolution, respectively. The enzyme forms a hexamer of identical subunits, with the NAD-binding domains building up the core of the molecule and the substrate-binding domains located at the apical positions of the hexamer. Coenzyme binding stabilizes a closed conformation where the substrate-binding domains are rotated by about 16° toward the dinucleotide-binding domains, compared to the open structure of the apo-enzyme. In the structure of the abortive ternary complex with NAD+ and pyruvate, the substrates are suitably positioned for hydride transfer between the nicotinamide ring and the C2 carbon atom of the substrate. The approach of the nucleophiles water and ammonia to pyruvate or the reaction intermediate iminopyruvate, respectively, is, however, only possible through conformational changes that make the substrate binding site more accessible. The crystal structures identified the conserved active-site residues His96 and Asp270 as potential acid/base catalysts in the reaction. Amino acid replacements of these residues by site-directed mutagenesis led to inactive mutants, further emphasizing their essential roles in the enzymatic reaction mechanism. [Copyright &y& Elsevier]

There is a substantial need for biomarkers to distinguish latent stage from active Mycobacterium tuberculosis infections, for predicting disease progression. To induce the reactivation of tuberculosis, we present a new experimental animal model modified based on the previous model established by our group. In the new model, the reactivation of tuberculosis is induced without administration of immunosuppressive agents, which might disturb immune responses. To identify the immunological status of the persistent and chronic stages, we analyzed immunological genes in lung tissues from mice infected with M. tuberculosis . Gene expression was screened using cDNA microarray analysis and confirmed by quantitative RT-PCR. Based on the cDNA microarray results, 11 candidate cytokines genes, which were obviously up-regulated during the chronic stage compared with those during the persistent stage, were selected and clustered into three groups: (1) chemokine genes, except those of monocyte chemoattractant proteins (MCPs; CXCL9, CXCL10, CXCL11, CCL5, CCL19); (2) MCP genes (CCL2, CCL7, CCL8, CCL12); and (3) TNF and IFN-γ genes. Results from the cDNA microarray and quantitative RT-PCR analyses revealed that the mRNA expression of the selected cytokine genes was significantly higher in lung tissues of the chronic stage than of the persistent stage. Three chemokines (CCL5, CCL19, and CXCL9) and three MCPs (CCL7, CCL2, and CCL12) were noticeably increased in the chronic stage compared with the persistent stage by cDNA microarray ( p < 0.01, except CCL12) or RT-PCR ( p < 0.01). Therefore, these six significantly increased cytokines in lung tissue from the mouse tuberculosis model might be candidates for biomarkers to distinguish the two disease stages. This information can be combined with already reported potential biomarkers to construct a network of more efficient tuberculosis markers.