Signaling roles of Dictyostelium discoideum TirA in response to gram-negative bacteria

Innate immune Toll-like receptors (TLRs) signal via toll/interleukin-1 receptor (TIR) domains, resulting in initiation of various signaling pathways, including MAPK pathways. The model organism Dictyostelium discoideum , a social amoeba that phagocytizes bacteria for nutrients, lacks full-length TLRs but encodes for several TIR domain-containing proteins. One such protein, TirA, is required for the growth of D. discoideum in the presence of Gram-negative bacteria. Furthermore, addition of LPS increases D. discoideum ’s bactericidal activities in a TirA- and ERK- dependent manner. Here we are investigating TirA’s role in the activation of ERK-MAPK pathways in response to bacteria and folic acid. ERK phosphorylation was found to be reduced in TirA-deficient cells as compared to WT cells in response to Gram-negative bacteria, but not in response to Gram-positive bacteria or to folic acid. Furthermore expression of uduB , a MAPK-regulated transcript, has previously been found to be upregulated in TirA-deficient cells during steady-state growth on Gram-negative bacteria. Here we show that upregulation of uduB can be detected within 4 h of exposure of TirA-deficient cells to Gram-negative bacteria. In addition to these procedures we are also working to solve the structure of D. discoideum TIR domains through the use of X-ray crystallography. By obtaining this structural information new insights into the conserved mechanisms of TIR domain signaling in may be revealed.