Reduced susceptibility of clinical strains of Mycobacterium tuberculosis to reactive nitrogen species promotes survival in activated macrophages.

BACKGROUND: Drugs such as isoniazid (INH) and pretomanid (PRT), used against Mycobacterium tuberculosis are active partly through generation of reactive nitrogen species (RNS). The aim of this study was to explore variability in intracellular susceptibility to nitric oxide (NO) in clinical strains of M. tuberculosis. METHOD: Luciferase-expressing clinical M. tuberculosis strains with or without INH resistance were exposed to RNS donors (DETA/NO and SIN-1) in broth cultures and bacterial survival was analysed by luminometry. NO-dependent intracellular killing in a selection of strains was assessed in interferon gamma/lipopolysaccharide-activated murine macrophages using the NO inhibitor L-NMMA. RESULTS: When M. tuberculosis H37Rv was compared to six clinical isolates and CDC1551, three isolates with inhA mediated INH resistance showed significantly reduced NO-susceptibility in broth culture. All strains showed a variable but dose-dependent susceptibility to RNS donors. Two clinical isolates with increased susceptibility to NO exposure in broth compared to H37Rv were significantly inhibited by activated macrophages whereas there was no effect on growth inhibition when activated macrophages were infected by clinical strains with higher survival to NO exposure in broth. Furthermore, the most NO-tolerant clinical isolate showed increased resistance to PRT both in broth culture and the macrophage model compared to H37Rv in the absence of mutational resistance in genes associated to reduced susceptibility against PRT or NO. CONCLUSION: In a limited number of clinical M. tuberculosis isolates we found a significant difference in susceptibility to NO between clinical isolates, both in broth cultures and in macrophages. Our results indicate that mycobacterial susceptibility to cellular host defence mechanisms such as NO need to be taken into consideration when designing new therapeutic strategies.

Comparative proteome profiling of MCF10A and 184A1 human breast epithelial cells emphasized involvement of CDK4 and cyclin D3 in cell proliferation.

Acquiring high proliferation rate is crucial for carcinogenic transformation of cells. We report here proteome profiling of human breast epithelial cells with low (184A1) and high (MCF10A) proliferation rates. We identified 183 proteins in 184A1 and 318 proteins in MCF10A cells. These datasets provide the most comprehensive proteome annotations of 184A1 and MCF10A cells. Proteins were taken for identification from 2-D gels in a systematic and unbiased way. Functional clustering of the identified proteins showed similarities in distribution of proteins to the same functional domains, indicating similarities in proteomes of 184A1 and MCF10A cells. Among observed differences in protein expression, we validated correlation of expression of endogenous cyclin-dependent kinase 4 (CDK4), cyclin D3, cdc25B, and p38γ with cell proliferation. Furthermore, down-regulation of CDK4 and cyclin D3 with specific siRNA inhibited cell proliferation, which emphasized the role of CDK4 and cyclin D3 in enhancement of cell proliferation rate of human breast epithelial cells.