MACROPHAGE GENOMICS · Genomic screens to identify and characterize mechanisms of mycobacterial killing by Macrophages
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2007-10-01 → 2009-09-30
- EU contribution
- €158,197
- Participants
- 1
- Scheme
- IIF
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Results in brief
Final Activity Report Summary - MACROPHAGE GENOMICS (Genomic Screens to Identify and Characterize Mechanisms of Mycobacterial Killing by Macrophages.)
Mycobacteria are opportunistic microbes that reside and multiply in host macrophages, mainly by inhibition of host pro-inflammatory response. NF-kB is a crucial pro-inflammatory transcription factor that is inhibited by pathogenic mycobacteria along with its down-stream cytokine TNFa. Microarray analysis of cells infected with non-pathogenic m. smegmatis revealed that a number of genes were up-regulated, most strikingly TNFa and Rab proteins involved in membrane fusion, in comparison to control un-infected cells. Quantitative-PCR analysis confirmed these micorarray results. TNFa was released rapidly from cells infected with the non-pathogen m. smegmatis as compared to infection with pathogenic m. avium or control un-infected cells, as observed by enzyme-linked immunosorbent assay (ELISA). Furthermore, the addition of recombinant TNFa led to mycobacteria killing whereas treatment with TNFa -protease inhibitor led to dramatic growth in mycobacterial survival. This growth was also achieved when the cells were incubated with TNF-RI blocking antibodies. However, blocking with TNF-RII had only marginal effect. By immunoflourescence microscopy, we showed that addition of TNFa led to more phago-lysosome fusion, thus to more killing of mycobacteria.
Data: CORDIS, © European Union
Project objective
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.
Original text from CORDIS.
Participants
- EUROPEAN MOLECULAR BIOLOGY LABORATORY, HEIDELBERG · HEIDELBERGCoordinatorCity levelGermany
Links
Data: CORDIS, © European Union