Caenorhabditis elegans glp-4 encodes a valyl aminoacyl tRNA synthetase

Germline stem cell proliferation is necessary to populate the germline with sufficient numbers of cells for gametogenesis and for signaling the soma to control organismal properties such as aging. The Caenorhabditis elegans gene glp-4 was identified by the temperature-sensitive allele bn2 where mutants raised at the restrictive temperature produce adults that are essentially germ cell deficient, containing only a small number of stem cells arrested in the mitotic cycle but otherwise have a morphologically normal soma. We determined that glp-4 encodes a valyl aminoacyl transfer RNA synthetase (VARS-2) and that the probable null phenotype is early larval lethality. Phenotypic analysis indicates glp-4(bn2ts) is partial loss of function in the soma. Structural modeling suggests that bn2 Gly296Asp results in partial loss of function by a novel mechanism: aspartate 296 in the editing pocket induces inappropriate deacylation of correctly charged Val-tRNA(val). Intragenic suppressor mutations are predicted to displace aspartate 296 so that it is less able to catalyze inappropriate deacylation. Thus glp-4(bn2ts) likely causes reduced protein translation due to decreased levels of Val-tRNA(val). The germline, as a reproductive preservation mechanism during unfavorable conditions, signals the soma for organismal aging, stress and pathogen resistance. glp-4(bn2ts) mutants are widely used to generate germline deficient mutants for organismal studies, under the assumption that the soma is unaffected. As reduced translation has also been demonstrated to alter organismal properties, it is unclear whether changes in aging, stress resistance, etc. observed in glp-4(bn2ts) mutants are the result of germline deficiency or reduced translation

Effect of Intensive Versus Standard Blood Pressure Control on Stroke Subtypes

In the SPRINT (Systolic Blood Pressure Intervention Trial), the number of strokes did not differ significantly by treatment group. However, stroke subtypes have heterogeneous causes that could respond differently to intensive blood pressure control. SPRINT participants (N=9361) were randomized to target systolic blood pressures of \u3c120 mm Hg (intensive treatment) compared with \u3c140 mm Hg (standard treatment). We compared incident hemorrhage, cardiac embolism, large- and small-vessel infarctions across treatment arms. Participants randomized to the intensive arm had mean systolic blood pressures of 121.4 mm Hg in the intensive arm (N=4678) and 136.2 mm Hg in the standard arm (N=4683) at one year. Sixty-nine strokes occurred in the intensive arm and 78 in the standard arm when SPRINT was stopped. The breakdown of stroke subtypes across treatment arms included hemorrhagic (intensive treatment, n=6, standard treatment, n=7) and ischemic stroke subtypes (large artery atherosclerosis: intensive treatment n=11, standard treatment, n=13; cardiac embolism: intensive treatment n=11, standard treatment n=15; small artery occlusion: intensive treatment n=8, standard treatment n=8; other ischemic stroke: intensive treatment n=3, standard treatment n=1). Fewer strokes occurred among participants without prior cardiovascular disease in the intensive (n=43) than the standard arm (n=61), but the difference did not reach predefined statistical significance level of 0.05 (P=0.09). The interaction between baseline cardiovascular risk factor status and treatment arm on stroke risk did not reach significance (P=0.05). Similar numbers of stroke subtypes occurred in the intensive BP control and standard control arms of SPRINT

glp-4 Encodes the Valyl Amino-acyl tRNA Synthetase VARS-2

From the Washington University Senior Honors Thesis Abstracts (WUSHTA), Volume 4, Spring 2012. Published by the Office of Undergraduate Research. Joy Zalis Kiefer, Director of Undergraduate Research / Assistant Dean in the College of Arts & Sciences; E. Holly Tasker, Editor; Kristin Sobotka, Undergraduate Research Coordinator. Mentor: Tim Sched

glp-4 Encodes the Valyl Amino-acyl tRNA Synthetase VARS-2

Mentor: Tim Schedl From the Washington University Undergraduate Research Digest: WUURD, Volume 7, Issue 2, Spring 2012. Published by the Office of Undergraduate Research, Joy Zalis Kiefer Director of Undergraduate Research and Assistant Dean in the College of Arts & Sciences; Kristin Sobotka, Editor

Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii

This work performs transcriptomic analysis of U-I cells, captures the earliest stage of a host cell’s interaction with Toxoplasma gondii, and dissects the effects of individual classes of parasite effectors on host cell biology.The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single-cell transcriptomic analysis at 1 to 3 h postinfection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild-type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host’s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent proinflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggest that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes

Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of <i>Toxoplasma gondii</i>

<div><p><i>Toxoplasma gondii</i> is an obligate intracellular parasite that can infect virtually all nucleated cells in warm-blooded animals. The ability of <i>Toxoplasma</i> tachyzoites to infect and successfully manipulate its host is dependent on its ability to transport “GRA” proteins that originate in unique secretory organelles called dense granules into the host cell in which they reside. GRAs have diverse roles in <i>Toxoplasma</i>’s intracellular lifecycle, including co-opting crucial host cell functions and proteins, such as the cell cycle, c-Myc and p38 MAP kinase. Some of these GRA proteins, such as GRA16 and GRA24, are secreted into the parasitophorous vacuole (PV) within which <i>Toxoplasma</i> replicates and are transported across the PV membrane (PVM) into the host cell, but the translocation process and its machinery are not well understood. We previously showed that TgMYR1, which is cleaved by TgASP5 into two fragments, localizes to the PVM and is essential for GRA transport into the host cell. To identify additional proteins necessary for effector transport, we screened <i>Toxoplasma</i> mutants defective in c-Myc up-regulation for their ability to export GRA16 and GRA24 to the host cell nucleus. Here we report that novel proteins MYR2 and MYR3 play a crucial role in translocation of a subset of GRAs into the host cell. MYR2 and MYR3 are secreted into the PV space and co-localize with PV membranes and MYR1. Consistent with their predicted transmembrane domains, all three proteins are membrane-associated, and MYR3, but not MYR2, stably associates with MYR1, whose N- and C-terminal fragments are disulfide-linked. We further show that fusing intrinsically disordered effectors to a structured DHFR domain blocks the transport of other effectors, consistent with a translocon-based model of effector transport. Overall, these results reveal a novel complex at the PVM that is essential for effector translocation into the host cell.</p></div

MYR2 (<i>TGGT1_270700</i>) is essential for effector translocation.

<p><b>(A)</b> Schematic of <i>TGGT1_270700</i> (<i>MYR2</i>) disruption. To disrupt the <i>MYR2</i> locus, a CRISPR-Cas9 plasmid encoding a sgRNA against <i>TGGT1_270700</i> was co-transfected with linearized pGRA1-HA-HPT plasmid into RH<i>Δhpt</i> parasites. Arrows indicate location of primers used to confirm integration of vector in the gene locus (and consequent disruption of the gene ORF) by PCR. (<b>B)</b> Disruptive integration of the HXGPRT vector in an exon of <i>MYR2</i> was confirmed by PCR and sequencing. (<b>C)</b> Effect of <i>MYR2</i> disruption and complementation on effector translocation. Wild-type (WT) and RH<i>Δmyr2</i> tachyzoites were transiently transfected with a plasmid expressing Myc-tagged GRA24. For complementation, the <i>Δmyr2</i> strain was co-transfected with GRA24-Myc and pGRA1:MYR2-3xHA plasmids. Transfected tachyzoites were allowed to infect HFFs for 16–24 hours. The infected monolayers were then fixed and GRA24 and MYR2 localization was assessed with anti-myc tag and anti-HA antibodies, respectively. Scale bar indicates 5 μm. White arrow indicates host nucleus lacking detectable GRA24 in the cell infected with RH<i>Δmyr2</i> tachyzoites. <b>(D)</b> Quantitation of percentage of infected cells showing GRA24 localization in the host nucleus based on three independent experiments, each with analysis of 10 fields on at least three coverslips. Statistics were performed with one-way ANOVA and Tukey’s multiple comparison’s test. **** indicates P < 0.0001.</p

MYR1, MYR2, and MYR3 are necessary for fully efficient growth <i>in vitro</i>.

<p>(A) HFFs were infected with tachyzoites of the indicated strains for 20 hours, fixed, and then stained with antibody to SAG1 to assess the number of parasites per vacuole. The results indicate the percentage of vacuoles containing 2, 4, 8, or 16 parasites. The averages are based on 3 independent replicates, and error bars reflect SEM. (B, C) HFFs were infected with tachyzoites of the indicated strains for 6 days, fixed with methanol, and then stained with crystal violet. Plaque size was measured using ImageJ. The averages are based on results from at least 3 independent biological replicates, each with 2–3 technical replicates, and error bars indicate SEM. *p < 0.005 using one-way ANOVA and Tukey’s multiple comparison’s test.</p

Fusion of GRA16 to DHFR domain blocks effector transport.

<p>(A) DHFR domain blocks GRA16 transport to host nucleus. HFFs were infected with RH tachyzoites ectopically expressing GRA16-HA or GRA16-DHFR and fixed at 20 hpi. GRA16-HA was stained with anti-HA antibodies and GRA16-DHFR was stained with anti-DHFR antibodies. (B) GRA16-DHFR expression reduces GRA24 transport to the nucleus. Parental RH and RH::<i>GRA16-DHFR</i> tachyzoites were transiently transfected with GRA24-Myc plasmid and fixed at 20–24 hpi. GRA24 was stained with anti-Myc tag antibodies and its localization to the host nucleus was quantified. The averages are based on normalized results from three independent experiments. Error bars indicate standard error of the mean, and statistics were performed with Student’s t-test. *** indicates P < 0.001. (C) GRA24 localization to host nucleus does not depend on GRA16 translocation. HFFs were infected with RH wild type, RH<i>Δgra16</i>, and RH<i>Δgra16</i>::<i>GRA16-HA</i> tachyzoites transiently expressing Myc-tagged GRA24 and fixed at 20–24 hpi. Staining of the host nucleus with antibodies to the Myc-tag is expressed as a percentage of all cells infected with parasites expressing the GRA24 construct. Results are shown for one representative experiment of 3 performed. Scale bar indicates 5 μm.</p

MYR3 (<i>TGGT1_237230</i>) is essential for effector translocation.

<p><b>(A)</b> Schematic of <i>TGGT1_237230</i> (<i>MYR3</i>) disruption. MYR3 gene disruption was performed as described in (2A), but using a sgRNA and primers specific to <i>MYR3</i>. (<b>B)</b> Disruptive integration of the HXGPRT vector in an exon of <i>MYR3</i> was confirmed by PCR and sequencing. (<b>C)</b> Effect of <i>MYR3</i> disruption and complementation on effector translocation. GRA24-Myc was transfected into WT, <i>Δmyr3</i>, and <i>Δmyr3</i>::<i>MYR3</i> tachyzoites and its localization was assessed by IFA. Scale bar indicates 5 μm. White arrow indicates host nucleus lacking detectable GRA24 in the cell infected with RH<i>Δmyr3</i> tachyzoites. (<b>D)</b> Quantitation of percentage of infected cells showing GRA24 localization in the host nucleus based on examination of at least 10 fields per each of three coverslips. Error bars reflect standard deviation for three to four replicates. Results are representative of 5 experiments.</p