Non-pathogenic mycobacteria are efficiently phagocytosed by macrophages and killed within phagosomes by acid hydrolases, reactive nitrogen intermediates, and likely other still unidentified factors that are part of the macrophage pro-inflammatory response. In contrast, pathogenic mycobacteria such as M. tuberculosis survive and grow within phagosomes by blocking macrophage pro-inflammatory responses, including the NF-kB transcription system, phago-lysosome fusion, acidification and nitric oxide release. Activation of NF-kB has recently been shown, by the host group, to be essential for macrophages to kill the non-pathogen M. smegmatis.
Here two genomic screening assays are proposed: the first (A) will use RNA micrroarray chips to identify macrophage mRNA¿s t hat are up-regulated in response to M.smegmatis or M.bovis BCG, that behaves like a pathogen. Emphasis will be given to identify macrophage proteins that are potentially involved in killing M.smegmatis; some of these proteins are predicted to be under the control of the NF-kB system. It is also expected that the potential killing proteins may not be up-regulated in BCG-infected cells, that therefore serve as a control.
The second screen (B) involves a recently developed high throughput RNAi approach that will be used to identify proteins that, when knocked down block the ability of GFP-M.smegmatis to fuse with lysosomes labelled with a red marker (rhodamine gold or lysotracker red). These proteins should be positive regulators of phagosome maturation and their identification should facilitate future efforts to understand how the pathogenic mycobacteria block phagosome maturation.
In parallel, RNAi knockdown of putative macrophage killing factors identified in screen A will allow us to test the hypothesis that these proteins indeed facilitate killing of M.smegmatis. The final proof of killing will be sought by testing purified factors for their effects on M.smegmatis growth and survival in vitro.
Field of science
- /natural sciences/biological sciences/biochemistry/biomolecules/proteins
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