Role of sphingosine kinase and sphingolipids in phagosome biology relevant for pathogenic mycobacteria infection in macrophages

• Applicant: Professor Gareth Griffiths, Ph.D.
• Subject Area: Anatomy and Physiology
• Term: from 2007 to 2009
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 37702489

Sphingosine kinase converts sphingosine (Sp) to sphingosine-1-phosphate (S1P). Sp kinase acts as a switch since substrate and product have opposite effects on cells; Sp for example promotes apoptosis, while S1 P favours cell survival. We showed that Sp kinase activity is present in membranes of latex bead phagosomes (LBP) isolated from 1 macrophages; there, high Sp inhibits membrane-catalysed actin assembly, which S1P stimulates. In macrophages, high S1P favours killing of mycobacteria enclosed in phagosomes, a pro-inflammatory response linked to actin assembly, while Sp behaves as an anti-inflammatory factor favouring mycobacterial growth. The ability of pathogenic mycobacteria such as M.tuberculosis to block the fusion of its phagosomes with lysosomes was recently shown to be dependent on their ability to inhibit Sp kinase. The goal here is to characterize Sp kinase on isolated phagosomes and phagosomes in macrophages in more detail. These analyses will be facilitated by using GFP-Sp kinase constructs and by RNAi experiments to selectively remove Sp kinase and its inter-linked molecules. LBP actin assembly is dependent on ezrin and P14,5P2 and is regulated by PLD. From the literature Sp Kinase binds directly to PLD, while PLD itself binds both Gactin, which inhibits its activity, and F-actin, which stimulates PLD. Sp kinase also indirectly binds PKA while cAMP/PKA inhibit phagosome actin assembly. These interactions have not all been analysed in one cell system. The isolated LBP and phagosomes inside macrophages are attractive systems to decipher the role of sphingolipids in a defined membrane process, actin assembly- that appears to be part of the pro-inflammatory response. This is relevant for killing pathogenic mycobacteria and modulation of sphingosine kinase activity could be considered as a therapeutic approach against these pathogens.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1267: Sphingolipids - Signals and Disease