Yersinia pseudotuberculosis YopH targets SKAP2-dependent and independent signaling pathways to block neutrophil antimicrobial mechanisms during infection

Abstract

Yersinia suppress neutrophil responses by using a type 3 secretion system (T3SS) to inject 6-7 Yersinia effector proteins (Yops) effectors into their cytoplasm. YopH is a tyrosine phosphatase that causes dephosphorylation of the adaptor protein SKAP2, among other targets in neutrophils. SKAP2 functions in reactive oxygen species (ROS) production, phagocytosis, and integrin-mediated migration by neutrophils. Here we identify essential neutrophil functions targeted by YopH, and investigate how the interaction between YopH and SKAP2 influence Yersinia pseudotuberculosis (Yptb) survival in tissues. The growth defect of a ΔyopH mutant was restored in mice defective in the NADPH oxidase complex, demonstrating that YopH is critical for protecting Yptb from ROS during infection. The growth of a ΔyopH mutant was partially restored in Skap2-deficient (Skap2KO) mice compared to wildtype (WT) mice, while induction of neutropenia further enhanced the growth of the ΔyopH mutant in both WT and Skap2KO mice. YopH inhibited both ROS production and degranulation triggered via integrin receptor, G-protein coupled receptor (GPCR), and Fcγ receptor (FcγR) stimulation. SKAP2 was required for integrin receptor and GPCR-mediated ROS production, but dispensable for degranulation under all conditions tested. YopH blocked SKAP2-independent FcγR-stimulated phosphorylation of the proximal signaling proteins Syk, SLP-76, and PLCγ2, and the more distal signaling protein ERK1/2, while only ERK1/2 phosphorylation was dependent on SKAP2 following integrin receptor activation. These findings reveal that YopH prevents activation of both SKAP2-dependent and-independent neutrophilic defenses, uncouple integrin-and GPCR-dependent ROS production from FcγR responses based on their SKAP2 dependency, and show that SKAP2 is not required for degranulation.

RIPK1 activates distinct gasdermins in macrophages and neutrophils upon pathogen blockade of innate immune signalling

“…Neutrophils play an important role in host defence against Yersinia infection (25,26) and are the most abundant cell type that are injected with effector proteins during Yersinia infection (27). Therefore, we next examined the contribution of GSDMD and GSDME to neutrophil cell lysis and cytokine secretion following Yptb infection.…”

Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin andCandidastimulation

“…Neutrophil ROS production is driven by signal transduction pathways downstream of several receptors, including integrin, Dectin-1, Fcγ, and G-protein-coupled receptors (GPCRs), that activate the components of the NADPH oxidase complex (Nguyen et al, 2017). Recently, we and others reported that S rc K inase A ssociated P hosphoprotein-2 (SKAP2) was essential for ROS production in response to stimulation by integrin and GPCRs, as well as gram-negative bacteria K. pneumoniae and Yersinia pseudotuberculosis (Nguyen et al, 2020, Shaban et al, 2020, Boras et al, 2017). In addition, SKAP2 is critical for integrin-stimulated cytoskeletal changes in macrophages and neutrophils (Alenghat et al, 2012, Tanaka et al, 2016, Boras et al, 2017), and for K. pneumoniae -mediated Syk phosphorylation in neutrophils (Nguyen et al, 2020).…”

“…In addition, SKAP2 is critical for integrin-stimulated cytoskeletal changes in macrophages and neutrophils (Alenghat et al, 2012, Tanaka et al, 2016, Boras et al, 2017), and for K. pneumoniae -mediated Syk phosphorylation in neutrophils (Nguyen et al, 2020). Further highlighting the role of SKAP2 in the innate immune response, Skap2-/- mice are more susceptible to K. pneumoniae , exhibiting a higher K. pneumoniae bacterial burden than wild-type controls (Nguyen et al, 2020) and SKAP2 is an essential target of the virulence factor, YopH, in neutrophils during Y. pseudotuberculosis infection (Shaban et al, 2020).…”

“… Abstract Signaling cascades that convert the recognition of pathogens to efficient inflammatory responses by immune cells, specifically neutrophils, are critical for host survival. SKAP2, an adaptor protein, is required for reactive oxygen species (ROS) generation following stimulation by integrins, formyl peptide receptors and gram-negative bacteria Klebsiella pneumoniae and Yersinia pseudotuberculosis in vitro (Nguyen et al, 2020, Shaban et al, 2020, Boras et al, 2017). SKAP2 is also required for the host defense against K. pneumoniae and ΔyopH Y. pseudotuberculosis infection in vivo in mouse models (Shaban et al, 2020, Nguyen et al, 2020).…”

“…SKAP2, an adaptor protein, is required for reactive oxygen species (ROS) generation following stimulation by integrins, formyl peptide receptors and gram-negative bacteria Klebsiella pneumoniae and Yersinia pseudotuberculosis in vitro (Nguyen et al, 2020, Shaban et al, 2020, Boras et al, 2017). SKAP2 is also required for the host defense against K. pneumoniae and ΔyopH Y. pseudotuberculosis infection in vivo in mouse models (Shaban et al, 2020, Nguyen et al, 2020). Another class of pattern recognition receptors (PRR) is the C-type lectin receptors (CLR), such as Dectin-1, Dectin-2 and Mincle, that are critical to trigger innate immune responses.…”