Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and

Vaccines have had broad medical impact, but existing vaccine technologies and production methods are limited in their ability to respond rapidly to evolving and emerging pathogens, or sudden outbreaks. Here, we develop a rapid-response, fully synthetic, singledose, adjuvant-free dendrimer nanoparticle vaccine platform wherein antigens are encoded by encapsulated mRNA replicons. To our knowledge, this system is the first capable of generating protective immunity against a broad spectrum of lethal pathogen challenges, including H1N1 influenza, Toxoplasma gondii, and Ebola virus. The vaccine can be formed with multiple antigenexpressing replicons, and is capable of eliciting both CD8⁺ T-cell and antibody responses. The ability to generate viable, contaminant-free vaccines within days, to single or multiple antigens, may have broad utility for a range of diseases

Using a genetically encoded sensor to identify inhibitors of Toxoplasma gondii Ca2+ Signalling

This work was supported in part by National Institutes of Health Grants AI-110027 and AI-096836 (to S. N. J. M.) and 1DP5OD017892 (to S. L.).The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca2+. Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca2+ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca2+ 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 Ca2+. We define the pool of Ca2+ 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 Ca2+ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca2+. The enhancers identified are capable of releasing intracellular Ca2+ 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 Ca2+-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca2+ 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 Ca2+, underscoring the importance of these pathways and the therapeutic potential of their inhibition.Publisher PDFPeer reviewe

Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca 2+ Signaling

The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca2+. Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca2+ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca2+ 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 Ca2+. We define the pool of Ca2+ 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 Ca2+ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca2+. The enhancers identified are capable of releasing intracellular Ca2+ 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 Ca2+-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca2+ 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 Ca2+, underscoring the importance of these pathways and the therapeutic potential of their inhibition

Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9

This work was supported in part by NIH grant 1DP5OD017892 to S.LToxoplasma 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.Publisher PDFPeer reviewe

Identifying the target of an antiparasitic compound in Toxoplasma using thermal proteome profiling

Copyright © 2020 American Chemical Society. Apicomplexan parasites include the causative agents of malaria and toxoplasmosis. Cell-based screens in Toxoplasma previously identified a chemical modulator of calcium signaling (ENH1) that blocked parasite egress from host cells and exhibited potent antiparasitic activity. To identify the targets of ENH1, we adapted thermal proteome profiling to Toxoplasma, which revealed calcium-dependent protein kinase 1 (CDPK1) as a target. We confirmed the inhibition of CDPK1 by ENH1 in vitro and in parasites by comparing alleles sensitive or resistant to ENH1. CDPK1 inhibition explained the block in egress; however, the effects of ENH1 on calcium homeostasis and parasite viability were CDPK1-independent, implicating additional targets. Thermal proteome profiling of lysates from parasites expressing the resistant allele of CDPK1 identified additional candidates associated with the mitochondria and the parasite pellicle-compartments that potentially function in calcium release and homeostasis. Our findings illustrate the promise of thermal profiling to identify druggable targets that modulate calcium signaling in apicomplexan parasites

A decrease in SUMO-conjugated proteins accompanies <i>Shigella</i> infection.

<p>A comparison of SUMO-conjugated proteins from HeLa cells infected with wild type <i>Shigella</i> (M90T), a non-invasive strain (BS176), or uninfected cells for 1, 2, and 3 hours, both strains contained a plasmid expressing the <i>afaE</i> adhesin. Whole cell lysates were prepared from infected cells, separated on 7% polyacrylamide gels and immunoblotted using antibodies specific for SUMO1 (A) or SUMO2/3 (B). In (C) whole cell lysates from HeLa cells infected with wild type <i>Shigella</i> (M90T), a non-invasive mutant defective in the T3SS (<i>mxiD</i>), or an invasive mutant that does not produce a number of effectors (<i>mxiE</i>), was analyzed by immunoblotting using SUMO1-specific antibodies. Immunoblotting using GAPDH served as a loading control.</p

<i>Shigella</i> infection increases PML-NB number.

<p>HeLa cells were not infected (UI), infected with a non-invasive strain (BS176), or infected with an invasive wild-type strain (M90T) as indicated, both strains contained a plasmid expressing the <i>afaE</i> adhesin (A). Cells were incubated for 3 hours, fixed and immunostained for PML (red). M90T-infected cells with normal and high levels of PML nuclear bodies are shown as indicated. DNA was visualized with DAPI (blue) and white arrow heads indicate PML NB “doublets”. Scale bars = 10μm. The average number of PML bodies per cell for each treatment condition was quantified (B) and the <i>p</i>-value of Student’s t-test was calculated (n.s. = not significant). The percentage of cells containing either 0, 1–5, 6–10 or 11+ PML bodies was also determined (C) and the <i>p</i>-value of Student’s t-test was calculated. Values in B and C represent the mean +/- SE, n = 3. Total PML protein levels for each treatment condition was determined by Western blot analysis (D).</p

<i>Shigella</i> targets SUMO conjugating enzyme Ubc9.

<p>In (A-C) whole cell lysates prepared from HeLa cells infected for 1, 2 or 3 hours with the indicated strains of <i>Shigella</i> bearing a plasmid expressing the <i>afaE</i> adhesin were analyzed by immunoblotting using antibodies specific for SAE1 (A), SAE2 (B), or Ubc9 (C). In (D) HeLa cells were treated with the proteasome inhibitor MG132 prior to infection, or not, as indicated prior to infection and preparation of lysates. Lysates were analyzed for Ubc9 by immunoblotting. In (D) HeLa cells were infected with the indicated strains and lysates were analyzed by immunoblotting for UbcH5.</p

<i>Shigella</i> Infection Interferes with SUMOylation and Increases PML-NB Number

<div><p>Shigellosis is a severe diarrheal disease that affects hundreds of thousands of individuals resulting in significant morbidity and mortality worldwide. Shigellosis is caused by <i>Shigella</i> spp., a gram-negative bacterium that uses a Type 3 Secretion System (T3SS) to deliver effector proteins into the cytosol of infected human cells. <i>Shigella</i> infection triggers multiple signaling programs that result in a robust host transcriptional response that includes the induction of multiple proinflammatory cytokines. PML nuclear bodies (PML-NBs) are dynamic subnuclear structures that coordinate immune signaling programs and have a demonstrated role in controlling viral infection. We show that PML-NB number increases upon <i>Shigella</i> infection. We examined the effects of <i>Shigella</i> infection on SUMOylation and found that upon <i>Shigella</i> infection the localization of SUMOylated proteins is altered and the level of SUMOylated proteins decreases. Although <i>Shigella</i> infection does not alter the abundance of SUMO activating enzymes SAE1 or SAE2, it dramatically decreases the level of the SUMO conjugating enzyme Ubc9. All <i>Shigella</i>-induced alterations to the SUMOylation system are dependent upon a T3SS. Thus, we demonstrate that <i>Shigella</i> uses one or more T3SS effectors to influence both PML-NB number and the SUMOylation machinery in human cells.</p></div