ABSTRACT Neutrophils (PMNs, polymorphonuclear cells) are pivotal innate immune cells that directly mediate pathogen clearance through release of reactive oxygen species (ROS), degranulation, neutrophilic extracellular traps (NETs) and phagocytosis. The bacterial pathogen, Yersinia pseudotuberculosis (Yptb) antagonizes these antimicrobial actions by injecting critical virulence proteins, called Yops, into PMNs. We seek to understand mechanisms that mediate clearance of bacterial pathogens in tissue infection by understanding the processes that are disrupted by YopH and YopO. We have shown that SKAP2 is one essential target of YopH in PMNs in tissue infection; however, SKAP2 is not the only essential target of YopH in tissue infection. Missense mutations in SKAP2 are associated with the development of autoimmune disorders and cancers. This suggests that PMN dysfunction downstream of SKAP2 signaling has wide ranging implications beyond infection control. We have further shown that SKAP2-dependent mechanisms in neutrophils are critical for limiting growth of another Gram-negative pathogen, Klebsiella pneumoniae (Kp), in lungs. Using PMNs derived from myeloid progenitor (MP) hematopoietic stem cells, we found that SKAP2 is essential for extracellular (ROS) production but is not required for degranulation after Kp infection. Surprisingly, SKAP2 is required for Syk phosphorylation after infection by Kp, but not for Syk phosphorylation after stimulation of CLRs and integrin receptors, indicating that neutrophil recognition of Kp is via a distinct receptor, possibly a G protein coupled receptor (GPCR). The objective of this application is to use our expertise in murine infection models, MP-PMN genetic manipulation, bacterial genetics and live-cell visualization of protein complexes, to understand pathways in PMNs that are targeted by YopH and YopO. We will further investigate whether these targets play crucial roles in the control Kp. To this end, our specific aims are to (1) Dissect the modular functions of SKAP2 required for generating anti-microbial responses in neutrophils after infection with Kp. (2) Understand the effects of YopO on GPCR signaling in PMNs during murine infection. (3) Identify the SKAP2-independent pathways that are targeted by YopH to inactivate neutrophil degranulation. After completion of these studies, we will understand how SKAP2 and other Yop-targeted proteins work in PMNs to control infection by Kp and Yptb. A thorough understanding of molecular mechanisms that signal for the release of the tissue damaging bactericidal factors, ROS and granules, by PMNs during infection should lead to novel and targeted approaches to manipulate these pathways to enhance these activities during infection with multidrug resistant bacteria and to stop excessive damage due to uncontrolled PMN responses in a variety of auto-immune diseases.