Neutrophil degranulation and extracellular ROS production are inactivated by Yersinia pseudotuberculosis YopE through a SKAP2 independent pathway

ABSTRACT 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 ( Y ersinia o uter p roteins), that possess distinct biochemical functions, into PMNs to inhibit PMN responses. We show that several Yops, YopE, YopH, and YopO, each partially interfered with CD63 plasma membrane localization, 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 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 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. Furthermore, the SKAP2-independent pathway inactivated by YopE is involved in primary granule release and ROS production. Overall, this work highlights multiple signaling pathways involved in mounting degranulation and ROS production and the diverse Yop-mediated mechanisms that WT- Yptb employs to effectively disarm them. Importantly, this work provides an avenue to untangle neutrophil signaling pathways targeted by pathogens using Cas9-ER-HoxB8 cells. IMPORTANCE When sensing invading bacteria, neutrophils become activated through multiple receptors that trigger signal-transduction cascades resulting in the generation of antimicrobial responses. The enteric pathogen, Yersinia pseudotuberculosis ( Yptb ), is equipped to effectively inhibit these responses using its collection of effector proteins (Yops). Here, we developed a system to overcome the limitations of performing genetic manipulations in neutrophils by implementing CRISPR/Cas9 technology in an engineered system of myeloid progenitor cells (Cas9-ER-HoxB8), which can be induced to differentiate into neutrophils. By infecting genetically modified neutrophils with Yptb strains expressing individual Yops, we identified distinct host signaling pathways that synergize to induce neutrophil antimicrobial responses. Our findings provide insight into several signaling events triggered by Yptb infection and show how YopE and YopH target distinct pathways to block vesicle trafficking and extracellular ROS production. This powerful genetic system can be applied to other pathogens to dissect the intricacies of neutrophil-pathogen interactions.