Significance: Complement activation occurs when bacteria invade the circulating blood, leading not only to removal of the pathogen but also to inflammation, organ damage, and poor prognosis for septic patients. We used a baboon model of Escherichia coli bacteremia to determine the effects of a C5 inhibitor on bacteriolysis, bacteria clearance, and sepsis progression. We observed that complement-mediated bacteriolysis has a detrimental effect by inducing release of LPS and fulminant inflammation. Inhibition of C5 cleavage and subsequent formation of the lytic terminal complex C5b-9 diminished LPS release, blocked sepsis-induced inflammation, decreased the associated consumptive coagulopathy, and protected organ function. Overall, treatment with C5 inhibitor significantly improved the survival of septic baboons, suggesting a potentially important strategy to treat bacteremic sepsis.

Our recent studies revealed that the inositol phosphatase Src homology 2 (SH2) domain-containing inositol phosphatase (SHIP) is phosphorylated and associated with Shc exclusively under negative signaling conditions in B cells, which is due to recruitment of the SHIP SH2 domain to the FcγRIIb. In addition, we reported that SHIP-Shc interaction involves both SHIP SH2 and Shc phosphotyrosine binding domains. These findings reveal a paradox in which the single SH2 domain of SHIP is simultaneously engaged to two different proteins: Shc and FcγRIIb. To resolve this paradox, we examined the protein interactions of SHIP. Our results demonstrated that isolated FcγRIIb contains SHIP but not Shc; likewise, Shc isolates contain SHIP but not FcγRIIb. In contrast, SHIP isolates contain both proteins, revealing two separate pools of SHIP: one bound to FcγRIIb and one bound to Shc. Kinetic studies reveal rapid SHIP association with FcγRIIb but slower and more transient association with Shc. Affinity measurements using a recombinant SHIP SH2 domain and phosphopeptides derived from FcγRIIb (corresponding to Y273) and Shc (corresponding to Y317) revealed an approximately equal rate of binding but a 10-fold faster dissociation rate for FcγRIIb compared with Shc phosphopeptide and yielding in an affinity of 2.1 μM for FcγRIIb and 0.26 μM for Shc. These findings are consistent with a model in which SHIP transiently associates with FcγRIIb to promote SHIP phosphorylation, whereupon SHIP binds to Shc and dissociates from FcγRIIb.

During advanced stages of inhalation anthrax, Bacillus anthracis accumulates at high levels in the bloodstream of the infected host. This bacteremia leads to sepsis during late-stage anthrax; however, the mechanisms through which B. anthracis -derived factors contribute to the pathology of infected hosts are poorly defined. Peptidoglycan, a major component of the cell wall of Gram-positive bacteria, can provoke symptoms of sepsis in animal models. We have previously shown that peptidoglycan of B. anthracis can induce the production of proinflammatory cytokines by cells in human blood. Here, we show that biologically active peptidoglycan is shed from an active culture of encapsulated B. anthracis strain Ames in blood. Peptidoglycan is able to bind to surfaces of responding cells, and internalization of peptidoglycan is required for the production of inflammatory cytokines. We also show that the peptidoglycan traffics to lysosomes, and lysosomal function is required for cytokine production. We conclude that peptidoglycan of B. anthracis is initially bound by an unknown extracellular receptor, is phagocytosed, and traffics to lysosomes, where it is degraded to a product recognized by an intracellular receptor. Binding of the peptidoglycan product to the intracellular receptor causes a proinflammatory response. These findings provide new insight into the mechanism by which B. anthracis triggers sepsis during a critical stage of anthrax disease.

Key Points: PGN forms immune complexes with preexisting human anti-PGN antibodies to activate the classical complement pathway. Human platelets are activated by PGN–anti-PGN immune complexes through platelet FcγRIIa and through platelet binding C5b.