Akt activation by the Salmonella effector SopB

Salmonella Typhimurium is a major cause of food poisoning worldwide and infections can be lethal in young children or immunocompromised hosts. These bacteria have the ability to invade host cells and grow intracellulary in specialized compartments, called Salmonella-containing vacuoles (SCVs). To accomplish this, S. Typhimurium translocates effector proteins directly into host cells. One such effector, SopB, has been shown to play an important role in disease caused by S. Typhimurium. SopB can modulate signal transduction pathways in host cells during the infection, including phosphoinositide (PI) metabolism, which leads to activation of the prosurvival kinase Akt, protecting infected epithelial cells from apoptosis. Akt is an important serinehreonine protein kinase that regulates cell survival, cell cycle and metabolism and is normally activated at the plasma membrane. Preliminary data indicate that SopB-mediated PI(3,4)P2 /PI(3,4,5)P 3 production at invasion ruffles is necessary for Akt activation and that class I and class II PI3-kinases are not involved in their formation. Therefore the mechanism of SopB- induced Akt activation is different from canonical Akt activation by growth factors and is currently unknown. To investigate the mechanism by which S. Typhimurium activates Akt during intracellular colonization, the specific aims of my research proposal are: 1. Determine the role of PI-kinases in SopB-mediated PI(3,4)P2 /PI(3,4,5)P 3 formation: Preliminary work showed that SopB-induced PI(3,4)P2 /PI(3,4,5)P 3 formation at invasion ruffles is necessary for Akt activation. By using PI3-kinase inhibitors and siRNA I could show that no class I and class II PI3-kinase is involved in SopB-mediated PI(3,4)P2 /PI(3,4,5)P 3 generation. I could detect recruitment of the PH-Akt domain to the membrane after SopB transformation in yeast, suggesting PI(3,4)P2 /PI(3,4,5)P 3 formation. Yeast cells have a simplified PI metabolism and lack PI(3,4,5)P3 under normal conditions. I will test the role of PI-kinases in SopB-mediated PI(3,4)P2 /PI(3,4,5)P 3 generation with mutant yeast strains lacking individual PI-kinases and in HeLa cells with specific siRNA against all known PI4- and PIP-kinases. 2. Determine if SopB-mediated PI(3,4)P2 /PI(3,4,5)P 3 formation occurs via a phosphotransfer reaction: I will test in vitro if SopB alone is sufficient to produce PI(3,4,5)P3 from PI(4,5)P2 . I will address the possibility that SopB acts as a kinase, potentially transferring phosphate from the 4- or 5-position of PI(4,5)P2 to the 3-position of PI(4,5)P2 , generating PI(3,4,5)P3 . Taken together, these studies will provide new insights into how S. Typhimurium colonizes host cells to establish a replicative niche during infection. In addition, it will help to gain more insight into PI metabolism and downstream signaling that regulates Akt activation.