YopJ-Induced Caspase-1 Activation in Yersinia-Infected Macrophages: Independent of Apoptosis, Linked to Necrosis, Dispensable for Innate Host Defense

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJKIM) strains have high cytotoxic activity. In addition, YopJKIM-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJKIM-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJKIM-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJKIM. Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity

Intestinal Permeability of Cyclic Peptides: Common Key Backbone Motifs Identified

Insufficient oral bioavailability is considered as a key limitation for the widespread development of peptides as therapeutics. While the oral bioavailability of small organic compounds is often estimated from simple rules, similar rules do not apply to peptides, and even the high oral bioavailability that is described for a small number of peptides is not well understood. Here we present two highly Caco-2 permeable template structures based on a library of 54 cyclo­(-d-Ala-Ala₅-) peptides with different <i>N</i>-methylation patterns. The first (all-<i>trans</i>) template structure possesses two β-turns of type II along Ala⁶-d-Ala¹ and Ala³-Ala⁴ and is only found for one peptide with two <i>N</i>-methyl groups at d-Ala¹ and Ala⁶ [<b>(</b><i><b>N</b></i><b>Me­(1,6)</b>]. The second (single-<i>cis</i>) template possesses a characteristic <i>cis</i> peptide bond preceding Ala⁵, which results in type VI β-turn geometry along Ala⁴-Ala⁵. Although the second template structure is found in seven peptides carrying <i>N</i>-methyl groups on Ala⁵, high Caco-2 permeability is only found for a subgroup of two of them [<i><b>N</b></i><b>Me­(1,5)</b> and <i><b>N</b></i><b>Me­(1,2,4,5)</b>], suggesting that <i>N</i>-methylation of d-Ala¹ is a prerequisite for high permeability of the second template structure. The structural similarity of the second template structure with the orally bioavailable somatostatin analog cyclo­(-Pro-Phe-<i>N</i>Me-d-Trp-<i>N</i>Me-Lys-Thr-<i>N</i>Me-Phe-), and the striking resemblance with both β-turns of the orally bioavailable peptide cyclosporine A, suggests that the introduction of bioactive sequences on the highly Caco-2 permeable templates may result in potent orally bioavailable drug candidates