CDPK2A and CDPK1 form a signaling module upstream of Toxoplasma motility.

IMPORTANCE: This work uncovers interactions between various signaling pathways that govern Toxoplasma gondii egress. Specifically, we compare the function of three canonical calcium-dependent protein kinases (CDPKs) using chemical-genetic and conditional-depletion approaches. We describe the function of a previously uncharacterized CDPK, CDPK2A, in the Toxoplasma lytic cycle, demonstrating that it contributes to parasite fitness through regulation of microneme discharge, gliding motility, and egress from infected host cells. Comparison of analog-sensitive kinase alleles and conditionally depleted alleles uncovered epistasis between CDPK2A and CDPK1, implying a partial functional redundancy. Understanding the topology of signaling pathways underlying key events in the parasite life cycle can aid in efforts targeting kinases for anti-parasitic therapies.

Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling.

The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca(2+) Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca(2+) indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca(2+) signaling in the model apicomplexan Toxoplasma gondii In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca(2+) We define the pool of Ca(2+) regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca(2+) signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca(2+) The enhancers identified are capable of releasing intracellular Ca(2+) stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum Inhibition of Ca(2+)-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca(2+) stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca(2+), underscoring the importance of these pathways and the therapeutic potential of their inhibition.

Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9.

Toxoplasma gondii is a parasite of humans and animals, and a model for other apicomplexans including Plasmodium spp., the causative agents of malaria. Despite many advances, manipulating the T. gondii genome remains labor intensive, and is often restricted to lab-adapted strains or lines carrying mutations that enable selection. Here, we use the RNA-guided Cas9 nuclease to efficiently generate knockouts without selection, and to introduce point mutations and epitope tags into the T. gondii genome. These methods will streamline the functional analysis of parasite genes and enable high-throughput engineering of their genomes.

Opposing roles for membrane bound and soluble Fas ligand in glaucoma-associated retinal ganglion cell death.

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.