Download Physique?S4, TIF file, 6.5 MB. Copyright ? 2016 Kim et al. Predicted amino acid sequence of ROP54 (yellow, signal peptide; white, arginine-rich domains). (B) Helical wheel prediction of underlined regions (amino acids 83 to 120 and 123 to 155) of the ROP54 amino acid sequence (http://rzlab.ucr.edu/scripts/). Download Physique?S3, TIF file, 8.2 MB. Copyright ? 2016 Kim et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. Physique?S4. Exogenously expressed ROP54 does not localize to the PVM upon contamination. (A) Gene model Tafamidis meglumine showing ROP54 lacking its predicted signal peptide (amino acids [aa] 1 to 26). This construct was transiently transfected into HT1080 cells. (B) IFA results demonstrating cytoplasmic localization of ROP54 (aa 27 to 479) in HT1080 cells upon contamination with parasites. (C) Two N-terminally truncated ROP54 constructs were also used for ectopic expression in HT1080 cells. (D) Western blot results with host cell lysate with ectopically expressed truncated constructs of ROP54 from experiments shown in panels A and C. Download Physique?S4, TIF file, 6.5 MB. Copyright ? 2016 Kim et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. Physique?S5. competition of ROP54HAII and showed comparable ratios of parasites in the peritoneum versus spleen. Day 7 spleens from the competition assay were harvested to evaluate relative amounts ROP54HAII and parasites. No significant difference was observed when comparing the relative amounts of ROP54HAII and parasites in the peritoneal lavage fluid and spleen at day 7. Conditions were evaluated using a one-way ANOVA ( 0.05). Download Figure?S5, TIF file, 2.2 MB. Copyright ? 2016 Kim Tafamidis meglumine et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. Figure?S6. ROP54 does not modulate loading of IRGb6 onto PVM. (A) Colocalization of ROP54 and IRGb6 (1?h and 12 h postinfection) was demonstrated in macrophages activated with IFN- and LPS. (B) IRGb6-positive vacuoles were Rabbit polyclonal to NGFRp75 enumerated and compared between ROP54HAII and parasites. No difference in loading was observed. Download Figure?S6, TIF file, 6.9 MB. Copyright ? 2016 Kim et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International Tafamidis meglumine license. Table?S1. Primers used in this study (all primers are listed in the 5-to-3 direction; please refer to Materials and Methods in the main text for descriptions of the primers) Download Table?S1, PDF file, 0.1 MB. Copyright ? 2016 Kim et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. Table?S2. Proteins identified via mass spectrometry of ROP54 immunoprecipitation (IP-Western blotting data demonstrated that ROP54 does not form complexes with ROP5; therefore, proteins that had a higher score than ROP5 that had either a predicted hypothetical protein, signal peptide, or cell expression profile similar to an ROP are listed) Download Table?S2, PDF file, 0.04 MB. Copyright ? 2016 Kim et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. ABSTRACT uses unique secretory organelles called rhoptries to inject an array of effector proteins into the host cytoplasm that hijack host cell functions. We have discovered a novel rhoptry pseudokinase effector, ROP54, which is injected into the host cell upon invasion and traffics to the cytoplasmic face of the parasitophorous vacuole membrane (PVM). Disruption of in a type II strain of does not affect growth but results in a 100-fold decrease in virulence mutant parasites. While no difference in IRGb6 loading was seen, we observed a substantial increase in GBP2 loading Tafamidis meglumine on the parasitophorous vacuole (PV) of infections by modulating GBP2 Tafamidis meglumine loading onto parasite-containing vacuoles. IMPORTANCE The interactions between intracellular microbes and their host cells can lead to the discovery of novel drug targets. During infections, host cells express an array of immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs) that load onto the parasite-containing vacuole to clear the parasite. To counter this mechanism, the parasite secretes effector proteins that traffic to the vacuole to disarm the immunity-related.