Fig. 4: YspE1/YspE2 homologs in Y. pseudotuberculosis cannot degrades hGBPs.
From: Subversion of GBP-mediated host defense by E3 ligases acquired during Yersinia pestis evolution
a Heatmap of intra- and inter species sequence identity for yspE1 homologs among Y. pestis or Y. pseudotuberculosis strains with complete genomes available in the NCBI database. The identity values are shown in a color scale ranging from orange to blue, with purple and red in between. b Phylogenic tree (adapted from reference 2) labeled with different colors according to the presence of gene homologs of yspE1/yspE2. Y. pestis strains within green ellipse encode homologous proteins nearly identical to YspE1 and strains within blue ellipse completely lost the homolog of yspE1/yspE2 gene locus. The biovar Microtus Y. pestis 91001 was labeled with an arrow head. GBPs degradation activity of YspE1 (c) and YspE2 (d) homologs in Y. pseudotuberculosis IP2666pIB1, YPIII and PB1 + . Plasmids expressing FLAG-tagged YspE1/YspE2 homologs in three representative Y. pseudotuberculosis were individually transfected into IFN-γ primed HeLa cells stably expressing GFP-tagged hGBP1 or hGBP2 as indicated and the transfected cells were lysed and immunoblotted with anti-hGBP antibodies. Domain-swapping experiments between YspE1/YspE2 and their homologs in IP2666pIB1 (e) and YPIII (f). g Domain-swapping experiments between YspE1 and it homolog in PB1 + . Plasmids expressing hybrid proteins by swapping the LRR domain and NEL domain between the homologous protein pairs as indicated were transfected into HeLa cells expressing GFP-tagged hGBP1 and the level of hGBP1 was measured by immunoblotting. h, i represented the quantitative and statistical analysis results of c and d, respectively, from three independent experiments. j–l Represented the quantitative and statistical analysis results of e, f and g, respectively, from three independent experiments. Image J was used to obtain the quantitative results and bars represent mean ± S.D. Two-way ANOVA was performed to analyze the significance of difference in protein levels (***p < 0.001; ****p < 0.0001; ns, not significant).
