Extended Data Fig. 8: Time-resolved HPLC–MS analysis of SopB phosphotransferase-phosphatase activities.
From: Kinase-independent synthesis of 3-phosphorylated phosphoinositides by a phosphotransferase
(a) Inorganic phosphate release assessed following treatment of liposomes with 2.5 µg recombinant SopBWT or SopBC460S. Liposome composition was (mol%) POPS:PtdIns(4,5)P2 (90:10) and 2.5 nmol PtdIns(4,5)P2 was provided per reaction. At the indicated time points, reactions were terminated by addition of 50 mM NEM. Data are mean ± s.e.m. of duplicate wells. Time 0 min corresponds to a no enzyme control. (b) Separation of PtdIns(3,4)P2 and PtdIns(4,5)P2 regio-isomers in a sample treated with SopBWT. An example HPLC–MS trace derived from LUVs treated with 0.5 µg (71.5 nM) SopBWT for 5 min. Note the appearance of PtdIns(3,4)P2 at ≈20.6 min. (c) Model of the PtdIns(3,4,5)P3 biosensor designed with tandem Bruton’s Tyrosine Kinase (BTK) PH domains (bPHx2). Each component was separated by flexible, serine- and glycine-rich linker sequences. NES, nuclear export signal. (d) Heterologous expression of wild-type SopB induces PM-translocation of bPHx2. Representative confocal micrographs of mCherry-tagged bPHx2 (inverted grey) co-transfected with EGFP-SopBC460S or EGFP-SopBWT. (e) Hypothesized PPIns conversions catalysed by SopB and potential modulation by host phosphatases. In the presence of PtdIns(4,5)P2 in vitro, SopB generates the species PtdIns(3,4,5)P3, PtdIns(3,4)P2, and PtdIns(3)P. The latter species are hypothesized to arise, at least in part, by sequential dephosphorylation of PtdIns(3,4,5)P3. In vivo, Salmonella infection favours the accumulation of PtdIns(3,4)P2 likely due to the high basal activity of host 5-phosphatases (INPP5B, SYNJ1/2, SHIP1/2, and others) that rapidly convert PtdIns(3,4,5)P3 to PtdIns(3,4)P2. It remains unclear if the rapid clearance of PtdIns(3,4)P2 following fission of the Salmonella-containing vacuole from the PM is due to the intrinsic activity of SopB or to the activity of host 4-phosphatases (INPP4A/B). Nonetheless, SopB is sufficient to give rise to PtdIns(3)P in vitro from PtdIns(4,5)P2, arguing for a second –likely minor– pathway to generate this inositide in addition to Vps34-mediated synthesis on the bacterial vacuole (Mallo et. al., 2008). Finally, phosphatidylinositol may arise by direct dephosphorylation of the 4- and 5-positions of PtdIns(4,5)P2 by SopB, or indirectly by dephosphorylation of products of the phosphotransferase reaction. Source numerical data are available in source data.
