Mechanistic insights into the first Lygus-active ß-pore forming protein.

The cotton pests Lygus hesperus and Lygus lineolaris can be controlled by expressing Cry51Aa2.834_16 in cotton. Insecticidal activity of pore-forming proteins is generally associated with damage to the midgut epithelium due to pores, and their biological specificity results from a set of key determinants including proteolytic activation and receptor binding. We conducted mechanistic studies to gain insight into how the first Lygus-active ß-pore forming protein variant functions. Biophysical characterization revealed that the full-length Cry51Aa2.834_16 was a stable dimer in solution, and when exposed to Lygus saliva or to trypsin, the protein underwent proteolytic cleavage at the C-terminus of each of the subunits, resulting in dissociation of the dimer to two separate monomers. The monomer showed tight binding to a specific protein in Lygus brush border membranes, and also formed a membrane-associated oligomeric complex both in vitro and in vivo. Chemically cross-linking the ß-hairpin to the Cry51Aa2.834_16 body rendered the protein inactive, but still competent to compete for binding sites with the native protein in vivo. Our study suggests that disassociation of the Cry51Aa2.834_16 dimer into monomeric units with unoccupied head-region and sterically unhindered ß-hairpin is required for brush border membrane binding, oligomerization, and the subsequent steps leading to insect mortality.

IL-18R-mediated HSC quiescence and MLKL-dependent cell death limit hematopoiesis during infection-induced shock.

Severe infection can dramatically alter blood production, but the mechanisms driving hematopoietic stem and progenitor cell (HSC/HSPC) loss have not been clearly defined. Using Ixodes ovatus Ehrlichia (IOE), a tick-borne pathogen that causes severe shock-like illness and bone marrow (BM) aplasia, type I and II interferons (IFNs) promoted loss of HSPCs via increased cell death and enforced quiescence. IFN-αß were required for increased interleukin 18 (IL-18) expression during infection, correlating with ST-HSC loss. IL-18 deficiency prevented BM aplasia and increased HSC/HSPCs. IL-18R signaling was intrinsically required for ST-HSC quiescence, but not for HSPC cell death. To elucidate cell death mechanisms, MLKL- or gasdermin D-deficient mice were infected; whereas Mlkl-/- mice exhibited protected HSC/HSPCs, no such protection was observed in Gsdmd-/- mice during infection. MLKL deficiency intrinsically protected HSCs during infection and improved hematopoietic output upon recovery. These studies define MLKL and IL-18R signaling in HSC loss and suppressed hematopoietic function in shock-like infection.