No abstract

Perforin (PFN) delivery of granzymes (Gzm) into the target cell at the immunological synapse is the major pathway for inducing apoptosis of virus-infected cells and tumors. A validated model for how PFN delivers Gzm into the cytosol is still lacking. PFN was originally thought to work by forming pores in the target cell plasma membrane that allow Gzm entry. This model was questioned when it was shown that GzmB is endocytosed without PFN. Moreover, apoptosis could be triggered by adding PFN to washed cells that have previously endocytosed GzmB. In this study, we show that GzmB binds to the plasma membrane mostly via nonspecific charge interactions. Washing in saline does not remove bound Gzm. However, if externally bound GzmB is completely removed, subsequent addition of PFN does not release previously endocytosed GzmB and does not trigger apoptosis. Therefore, PFN must be coendocytosed with GzmB to deliver it into the cytosol.

SummaryBorrelia burgdorferi, the causative agent of Lyme disease, activates multiple signalling pathways leading to induction of pro-inflammatory mediators at sites of inflammation. Binding of B. burgdorferi to integrin a3b1 on human chondrocytes activates signalling leading to release of several pro-inflammatory mediators, but the B. burgdorferi protein that binds integrin a3b1 and elicits this response has remained unknown. A search of the B. burgdorferi genome for a canonical integrin binding motif, the RGD (Arg-GlyAsp) tripeptide, revealed several candidate ligands for integrins. In this study we show that one of these candidates, BBB07, binds to integrin a3b1 and inhibits attachment of intact B. burgdorferi to the same integrin. BBB07 is expressed during murine infection as demonstrated by recognition by infected mouse sera. Recombinant purified BBB07 induces proinflammatory mediators in primary human chondrocyte cells by interaction with integrin a3b1. This interaction is specific, as P66, another integrin ligand of B. burgdorferi, does not activate signalling through a3b1. In summary, we have identified a B. burgdorferi protein, BBB07, that interacts with integrin a3b1 and stimulates production of pro-inflammatory mediators in primary human chondrocyte cells.

Summary Identifying molecular targets of Yersinia virulence effectors, or Yops, during animal infection is challenging because few cells are targeted by Yops in an infected organ and isolating these sparse effector-containing cells is difficult. YopH, a tyrosine phosphatase, is essential for full virulence of Yersinia. Investigating the YopH-targeted signal-transduction pathway(s) in neutrophils during infection of a murine host, we find that several host proteins, including the essential signaling adapter SLP-76, are dephosphorylated in the presence of YopH in neutrophils isolated from infected tissues. YopH inactivated PRAM-1/SKAP-HOM and the SLP-76/Vav/PLCγ2 signal-transduction axes, leading to an inhibition of calcium response in isolated neutrophils. Consistent with a failure to mount a calcium response, IL-10 production was reduced in neutrophils containing YopH from infected tissues. Finally, a yopH mutant survived better in the absence of neutrophils, indicating that neutrophil inactivation by YopH by targeting PRAM-1/SKAP-HOM and SLP-76/Vav/PLCγ2 signaling hubs may be critical for Yersinia survival.

The C-type lectin receptor dectin-1 and the integrin Mac-1 have key roles in controlling fungal infection. Here, we demonstrate that dectin-1- and Mac-1-induced activation of protein kinase Cδ in neutrophils, independent of the Card9 adaptor, is required for reactive oxygen species production and for intracellular killing upon Candida albicans uptake. Protein kinase Cδ was also required for zymosan-induced cytokine generation in neutrophils. In macrophages, protein kinase Cδ deficiency prevented fungi-induced reactive oxygen species generation but had no effect on activation of TGF-β-activated kinase-1, an effector of Card9, or nuclear factor κB activation, nor did it affect phagolysosomal maturation, autophagy, or intracellular C. albicans killing. In vivo, protein kinase Cδ-deficient mice were highly susceptible to C. albicans and Aspergillus fumigatus infection, which was partially rescued with adoptively transferred wild-type neutrophils. Thus, protein kinase Cδ activation downstream of dectin-1 and Mac-1 has an important role in neutrophil, but not macrophage, functions required for host defense against fungal pathogens.