Regulation of Toxoplasma Motility by Calcium-Dependent Protein Kinases

Apicomplexans are protozoan parasites of animals, which must penetrate host cells to find a niche in which to replicate. To humans, they represent the most ubiquitous and deadliest eukaryotic pathogens, including Toxoplasma gondii and Plasmodium spp., the etiological agents of toxoplasmosis and malaria, respectively. A common mechanism enables these parasites to penetrate biological barriers and invade host cells actively, through a process termed gliding motility. This type of motility, unique to apicomplexans, relies on the directional translocation of adhesins via an actomyosin motor complex anchored in a vesicular network underlying the plasma membrane of the parasite. A variety of stimuli can trigger calcium increases in the parasite cytoplasm initiating motion, in part by regulating the secretion of adhesins from specialized organelles, called micronemes. Our studies investigate how these calcium signals are transduced to regulate T. gondii motility. Apicomplexans, like other chromalveolates and plants, possess calcium-dependent protein kinases: CDPKs) that are directly activated by calcium binding and have been proposed to participate in the transduction of calcium signals. A number of CDPKs are conserved among apicomplexans, and showed distinct subcellular localizations upon tagged expression in T. gondii, consistent with roles in different calcium-activated cellular pathways. Using a combination of chemical and genetic approaches we demonstrated that two of the conserved CDPKs are required for motility at different stages in the T. gondii life cycle. Generating a conditional knockout, we showed that TgCDPK1 is required for microneme secretion during egress and invasion. The essential role of this kinase was further supported through the use of bulky pyrazolo [3,4-d] pyrimidine: PP) analogues that inhibit TgCDPK1, and mirrored the effects of the conditional knockout. The specificity of these compounds is conferred by the expansion of the ATP-binding pocket in TgCDPK1 caused by the presence of a glycine at a key position called the `gatekeeper\u27, a feature unique among active parasite kinases. Mutating this residue to a methionine made TgCDPK1 resistant to the inhibitors, and enabled us to mutate the gatekeeper residue of a related kinase, TgCDPK3, rendering it sensitive to inhibition by PP analogues. This chemical-genetic strategy allowed us to implicate TgCDPK3 in the initiation of motility during egress, but demonstrate that its function is dispensable during invasion. Together, these observations provide the first evidence that related CDPKs regulate distinct signaling pathways, which distinguish the signaling events governing motility during egress and invasion by T. gondii. We also attempted to further understand the role of TgCDPK1 by examining its cellular targets. This process was also facilitated by the atypical ATP-binding pocket of TgCDPK1, which was able to bind bulky ATP analogues that allowed us to track its direct targets. This approach has allowed us to identify a number of putative TgCDPK1 targets. One such target, a dynamin-related protein, is phosphorylated in vivo in a CDPK1-dependent manner, consistent with a role in the regulation of motility. Together these observations provide a foundation for further characterization of CDPK signaling and the regulation of parasite motility. In particular, the chemical-genetic approaches adapted to parasites in these studies, represent a systematic means to dissecting these essential pathways in apicomplexans

Identification of PNG kinase substrates uncovers interactions with the translational repressor TRAL in the oocyte-to-embryo transition

The Drosophila Pan Gu (PNG) kinase complex regulates hundreds of maternal mRNAs that become translationally repressed or activated as the oocyte transitions to an embryo. In a previous paper (Hara et al., 2017), we demonstrated PNG activity is under tight developmental control and restricted to this transition. Here, examination of PNG specificity showed it to be a Thrkinase yet lacking a clear phosphorylation site consensus sequence. An unbiased biochemical screen for PNG substrates identified the conserved translational repressor Trailer Hitch (TRAL). Phosphomimetic mutation of the PNG phospho-sites in TRAL reduced its ability to inhibit translation in vitro. In vivo, mutation of tral dominantly suppressed png mutants and restored Cyclin B protein levels. The repressor Pumilio (PUM) has the same relationship with PNG, and we also show that PUM is a PNG substrate. Furthermore, PNG can phosphorylate BICC and ME31B, repressors that bind TRAL in cytoplasmic RNPs. Therefore, PNG likely promotes translation at the oocyte-to-embryo transition by phosphorylating and inactivating translational repressors.National Institutes of Health (U.S.) (Grant GM39341)National Institutes of Health (U.S.) (Grant GM118090

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

Ca2+ monitoring in Plasmodium falciparum using the yellow cameleon-Nano biosensor

Calcium (Ca2+)-mediated signaling is a conserved mechanism in eukaryotes, including the human malaria parasite, Plasmodium falciparum. Due to its small size (300?nM). We determined that the mammalian SERCA inhibitor thapsigargin and antimalarial dihydroartemisinin did not perturb SERCA activity. The change of the cytosolic Ca2+ level in P. falciparum was additionally detectable by flow cytometry. Thus, we propose that the developed YC-Nano-based system is useful to study Ca2+ signaling in P. falciparum and is applicable for drug screening.We are grateful to Japanese Red Cross Blood Society for providing human RBC and plasma. We also thank Tanaka R, Ogoshi (Sakura) M and Matsumoto N for technical assistance and Templeton TJ for critical reading. This study was conducted at the Joint Usage / Research Center on Tropical Disease, Institute of Tropical Medicine, Nagasaki University, Japan. KP was a Tokyo Biochemical Research Foundation (TBRF, http://www.tokyobrf.or.jp) post-doctoral fellow and PEF was a Japanese Society of Promotion Sciences (JSPS) post-doctoral fellow. This work was supported in part by the TBRF (K.P.), JSPS (P.E.F.), Takeda Science Foundation (K.Y.), Grants-in-Aids for Scientific Research 24590509 (K.Y.), 22390079 (O.K.), and for Scientific Research on Innovative Areas 23117008 (O.K.), MEXT, Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Effect of surgical experience and spine subspecialty on the reliability of the {AO} Spine Upper Cervical Injury Classification System

OBJECTIVE The objective of this paper was to determine the interobserver reliability and intraobserver reproducibility of the AO Spine Upper Cervical Injury Classification System based on surgeon experience (< 5 years, 5–10 years, 10–20 years, and > 20 years) and surgical subspecialty (orthopedic spine surgery, neurosurgery, and "other" surgery). METHODS A total of 11,601 assessments of upper cervical spine injuries were evaluated based on the AO Spine Upper Cervical Injury Classification System. Reliability and reproducibility scores were obtained twice, with a 3-week time interval. Descriptive statistics were utilized to examine the percentage of accurately classified injuries, and Pearson’s chi-square or Fisher’s exact test was used to screen for potentially relevant differences between study participants. Kappa coefficients (κ) determined the interobserver reliability and intraobserver reproducibility. RESULTS The intraobserver reproducibility was substantial for surgeon experience level (< 5 years: 0.74 vs 5–10 years: 0.69 vs 10–20 years: 0.69 vs > 20 years: 0.70) and surgical subspecialty (orthopedic spine: 0.71 vs neurosurgery: 0.69 vs other: 0.68). Furthermore, the interobserver reliability was substantial for all surgical experience groups on assessment 1 (< 5 years: 0.67 vs 5–10 years: 0.62 vs 10–20 years: 0.61 vs > 20 years: 0.62), and only surgeons with > 20 years of experience did not have substantial reliability on assessment 2 (< 5 years: 0.62 vs 5–10 years: 0.61 vs 10–20 years: 0.61 vs > 20 years: 0.59). Orthopedic spine surgeons and neurosurgeons had substantial intraobserver reproducibility on both assessment 1 (0.64 vs 0.63) and assessment 2 (0.62 vs 0.63), while other surgeons had moderate reliability on assessment 1 (0.43) and fair reliability on assessment 2 (0.36). CONCLUSIONS The international reliability and reproducibility scores for the AO Spine Upper Cervical Injury Classification System demonstrated substantial intraobserver reproducibility and interobserver reliability regardless of surgical experience and spine subspecialty. These results support the global application of this classification system