Upon sensing Yersinia pseudotuberculosis (Yptb), receptor-mediated pathways are stimulated to trigger polymorphonuclear (PMN) antimicrobial responses. Yptb injects multiple Type 3 secreted effector proteins, Yops (Yersinia outer proteins), that possess distinct biochemical functions, into PMNs to inhibit PMN responses. Here, we show that several Yops, YopE, YopH, and YopO, each partially interfered with CD63 mobilization to the plasma membrane, a marker for primary degranulation. The host pathways involved in CD63 mobilization are complex and it is not completely understood how Yops collaborate to inactivate this process. Here, CRISPR/Cas9 technology was used in an immortalized system of myeloid progenitor cells (Cas9-ER-HoxB8) to generate a panel of knockout PMN cell lines. To probe the impact of different Yops on the neutrophil pathways activated upon encountering Yptb, we interrogated the panel of genetically modified neutrophils with genetically modified bacteria. This approach of targeted gene deletion to inactivate specific pathways/proteins uncovered host pathways that synergize to induce CD63 mobilization that are distinctly targeted by YopE and YopH. YopE specifically inhibited CD63 mobilization in the absence of SKAP2, a YopH target, whereas YopH inhibited CD63 mobilization in the absence of RhoG, a YopE target, indicating that these Yops inactivate distinct signaling pathways contributing to CD63 mobilization. Furthermore, the SKAP2-independent pathway inactivated by YopE is involved in primary granule release and ROS production. Overall, this work highlights the diverse Yop-mediated mechanisms that WT-Yptb employs to effectively disarm PMN responses and provides an avenue to untangle neutrophil signaling pathways targeted by pathogens using Cas9-ER-HoxB8 cells.
Leus, P. A., Manan Mejias, C. M., Ren, A., de la Rosa, M., Lofgren, E., Bunnell, S. C., Sykes, D. B., Mecsas, J.
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