Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It

IFN-γ is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs) are strongly induced by IFN-γ. We studied the behavior of murine GBP1 (mGBP1) upon infection with T. gondii in vitro and confirmed that IFN-γ-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV) correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-γ-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii.National Institutes of Health (U.S.)Massachusetts Life Sciences Center (New Investigator Award)National Institute of General Medical Sciences (U.S.) (Pre-Doctoral Grant in the Biological Sciences (5-T32-GM007287-33))Studienstiftung des deutschen VolkesCancer Research Institute (New York, N.Y.)Cleo and Paul Schimmel FoundationBayer HealthcareHuman Frontier Science Program (Strasbourg, France

Preferential Brain Homing following Intranasal Administration of Trypanosoma cruzi▿

The Chagas’ disease parasite Trypanosoma cruzi commonly infects humans through skin abrasions or mucosa from reduviid bug excreta. Yet most studies on animal models start with subcutaneous or intraperitoneal injections, a distant approximation of the skin abrasion route. We show here that atraumatic placement of T. cruzi in the mouse nasal cavity produced low parasitemia, high survival rates, and preferential brain invasion compared to the case with subcutaneously injected parasites. Brain invasion was particularly prominent in the basal ganglia, peaked at a time when parasitemia was no longer detectable, and elicited a relatively large number of inflammatory foci. Yet, based on motor behavioral parameters and staining with Fluoro-Jade C, a dye that specifically recognizes apoptotic and necrotic neurons, brain invasion did not cause neurodegenerative events, in contrast to the neurodegeneration in the enteric nervous system. The results indicate that placement of T. cruzi on the mucosa in the mouse nasal cavity establishes a systemic infection with a robust yet harmless infection of the brain, seemingly analogous to disease progression in humans. The model may facilitate studies designed to understand mechanisms underlying T. cruzi infection of the central nervous system

Microtubule plus‐end binding proteins facilitate intracellular trypanosome infection

Mammalian cell invasion by the protozoan parasite Trypanosoma cruzi involves host cell microtubule dynamics. Microtubules support kinesin-dependent anterograde trafficking of host lysosomes to the cell periphery where targeted lysosome exocytosis elicits remodelling of the plasma membrane and parasite invasion. Here, a novel role for microtubule plus-end tracking proteins (+TIPs) in the co-ordination of T. cruzi trypomastigote internalization and post-entry events is reported. Acute silencing of CLASP1, a +TIP that participates in microtubule stabilization at the cell periphery, impairs trypomastigote internalization without diminishing the capacity for calcium-regulated lysosome exocytosis. Subsequent fusion of the T. cruzi vacuole with host lysosomes and its juxtanuclear positioning are also delayed in CLASP1-depleted cells. These post-entry phenotypes correlate with a generalized impairment of minus-end directed transport of lysosomes in CLASP1 knock-down cells and mimic the effects of dynactin disruption. Consistent with GSK3β acting as a negative regulator of CLASP function, inhibition of GSK3β activity enhances T. cruzi entry in a CLASP1-dependent manner and expression of constitutively active GSK3β dampens infection. This study provides novel molecular insights into the T. cruzi infection process, emphasizing functional links between parasite-elicited signalling, host microtubule plus-end tracking proteins and dynein-based retrograde transport. Highlighted in this work is a previously unrecognized role for CLASPs in dynamic lysosome positioning, an important aspect of the nutrient sensing response in mammalian cells

Determinants of GBP recruitment to Toxoplasma gondii vacuoles and the parasitic factors that control it.

IFN-γ is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs) are strongly induced by IFN-γ. We studied the behavior of murine GBP1 (mGBP1) upon infection with T. gondii in vitro and confirmed that IFN-γ-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV) correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-γ-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors

Objective: Islets from the same pancreas show remarkable variability in glucose sensitivity. While mitochondrial respiration is essential for glucose-stimulated insulin secretion, little is known regarding heterogeneity in mitochondrial function at the individual islet level. This is due in part to a lack of high-throughput and non-invasive methods for detecting single islet function. Methods: We have developed a novel non-invasive, high-throughput methodology capable of assessing mitochondrial respiration in large-sized individual islets using the XF96 analyzer (Agilent Technologies). Results: By increasing measurement sensitivity, we have reduced the minimal size of mouse and human islets needed to assess mitochondrial respiration to single large islets of >35,000 μm2 area (∼210 μm diameter). In addition, we have measured heterogeneous glucose-stimulated mitochondrial respiration among individual human and mouse islets from the same pancreas, allowing population analyses of islet mitochondrial function for the first time. Conclusions: We have developed a novel methodology capable of analyzing mitochondrial function in large-sized individual islets. By highlighting islet functional heterogeneity, we hope this methodology can significantly advance islet research. Keywords: Islets, Mitochondria, Respirometry, Glucos

Transcriptome Remodeling in Trypanosoma cruzi and Human Cells during Intracellular Infection

Intracellular colonization and persistent infection by the kinetoplastid protozoan parasite, Trypanosoma cruzi, underlie the pathogenesis of human Chagas disease. To obtain global insights into the T. cruzi infective process, transcriptome dynamics were simultaneously captured in the parasite and host cells in an infection time course of human fibroblasts. Extensive remodeling of the T. cruzi transcriptome was observed during the early establishment of intracellular infection, coincident with a major developmental transition in the parasite. Contrasting this early response, few additional changes in steady state mRNA levels were detected once mature T. cruzi amastigotes were formed. Our findings suggest that transcriptome remodeling is required to establish a modified template to guide developmental transitions in the parasite, whereas homeostatic functions are regulated independently of transcriptomic changes, similar to that reported in related trypanosomatids. Despite complex mechanisms for regulation of phenotypic expression in T. cruzi, transcriptomic signatures derived from distinct developmental stages mirror known or projected characteristics of T. cruzi biology. Focusing on energy metabolism, we were able to validate predictions forecast in the mRNA expression profiles. We demonstrate measurable differences in the bioenergetic properties of the different mammalian-infective stages of T. cruzi and present additional findings that underscore the importance of mitochondrial electron transport in T. cruzi amastigote growth and survival. Consequences of T. cruzi colonization for the host include dynamic expression of immune response genes and cell cycle regulators with upregulation of host cholesterol and lipid synthesis pathways, which may serve to fuel intracellular T. cruzi growth. Thus, in addition to the biological inferences gained from gene ontology and functional enrichment analysis of differentially expressed genes in parasite and host, our comprehensive, high resolution transcriptomic dataset provides a substantially more detailed interpretation of T. cruzi infection biology and offers a basis for future drug and vaccine discovery efforts

Dynamic host response signatures in <i>T</i>. <i>cruzi</i>-infected human fibroblasts.

<p>Expression patterns for selected genes in the most strongly modulated pathways in <i>T</i>. <i>cruzi</i> infected HFF. Genes in the following categories are highlighted. <b>(C) Mitotic Cell cycle:</b> <i>AURKA</i> (ENSG00000087586); <i>CDC6</i> (ENSG00000094804); <i>CDC20</i> (ENSG00000117399); <i>CDC25A</i> (ENSG00000164045); <i>CDK1</i> (ENSG00000170312); <i>CCNA2</i> (ENSG00000145386); <i>NEK2</i> (ENSG00000117650); <i>ORC1L</i> (ENSG00000085840); <i>PLK1</i> ENSG00000137807); KIF23 (ENSG00000137807). <b>(B) Cytokines/Chemokines:</b> <i>IL-8</i> (ENSG00000169429); <i>IL-6</i> (ENSG00000136244); <i>CCL8</i> (ENSG00000108700); <i>CCL2</i> (ENSG00000108691); <i>CXCL10</i> (ENSG00000169245). <b>(C) Type I Interferon:</b> <i>OAS</i> (ENSG00000089127); <i>IFNB</i> (ENSG00000171855); <i>ISG44</i> (ENSG00000137959); <i>GBP1</i> (ENSG00000117228); <i>ISG15</i> (ENSG00000187608). <b>(D) Mevalonate/Sterol biosynthesis:</b> <i>HMGCR</i> (ENSG00000113161); <i>HMGCS1</i> (ENSG00000112972); <i>DHCR7</i> (ENSG00000172893); <i>FDPS</i> (ENSG00000160752); <i>FDFT1</i> (ENSG00000079459); <i>HSD17B7</i> (ENSG00000132196); <i>LSS</i> (ENSG00000160285); <i>MVD</i> (ENSG00000167508); <i>SQLE</i> (ENSG00000104549); <i>MSMO1</i> (ENSG00000052802); <i>SC5D</i> (ENSG00000109929). All values are reported as log2 fold-change of the difference in expression of infected and matched uninfected controls at each time point as listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005511#ppat.1005511.s017" target="_blank">S7 Table</a>.</p

Simultaneous interrogation of parasite and host transcriptomes.

<p><b>(A) Intracellular <i>T</i>. <i>cruzi</i> life cycle and sample collection scheme.</b> Extracellular <i>T</i>. <i>cruzi</i> trypomastigotes actively penetrate mammalian cells where they receive cues to differentiate into amastigote forms that replicate in the host cell cytoplasm for 3–5 days, beginning at ~22 hpi with a doubling time of ~12 hr. Amastigote division ceases on day 4 or 5 post-infection and parasites differentiate back into trypomastigotes that rupture the host cell to initiate a new cellular infection cycle. For RNA-Seq analysis, total RNA was isolated from axenic <i>T</i>. <i>cruzi</i> epimastigotes (insect vector stage), extracellular trypomastigotes and from amastigote-containing human fibroblast monolayers at 6 time points spanning 4–72 hpi. Pie charts indicate the proportion of mapped sequence reads assigned to the parasite (pink) or human (grey). (<b>B) Distribution of global gene expression levels in a representative subset of <i>T</i>. <i>cruzi</i> and human samples.</b> Box plots showing comparisons of the distribution of per-gene counts (log2 counts per million with an offset of 1) normalized for sequencing library size. The ends of the whiskers represent the lowest datum still within 1.5 interquartile range (IQR) of the lower quartile, and the highest datum still within 1.5 IQR of the upper quartile. Genes with extremely high or low expression levels are shown as open circles above and below the whiskers, respectively. <b>(C) Principal component analysis plots of global transcriptome profiles.</b> Principal component analysis (PCA) plot for RNA-Seq data with <i>T</i>. <i>cruzi</i> and human samples plotted separately. The two first principal components (PC1 and PC2) are plotted with the proportion of variance explained by each component next to the axes labels. Each sample is represented by a dot and the color label corresponding to the sample group, such as the number of hours (hr) post-infection.</p

Validation of predicted metabolic features of <i>T</i>. <i>cruzi</i> developmental stages.

<p><b>(A)</b> Heatmap of expression values of annotated <i>T</i>. <i>cruzi</i> genes predicted to function in intermediary metabolism with a focus on glycolysis, TCA cycle and Ox-PHOS. <b>(B)</b> Calculated basal respiratory capacity <b>(C)</b> ATP-linked respiration and <b>(D)</b> spare-respiratory capacity of extracellular trypomastigotes and isolated intracellular amastigotes (60 hpi) as pmol of oxygen consumed per min (oxygen consumption rate; OCR) normalized to <i>T</i>. <i>cruzi</i> DNA (ng) per well. <b>(E)</b> ATP content measured in isolated trypomastigotes and amastigotes in KHB buffer without a consumable carbon source at time points indicated. <b>(F)</b> OCR response to glutamine (10 mM) and ELQ271 (10 μM) in isolated <i>T</i>. <i>cruzi</i> trypomastigotes and amastigotes. <b>(G)</b> Dose-dependent inhibition of <i>T</i>. <i>cruzi</i> amastigote growth in HFF following addition of ELQ271 at 18 hpi and relative infection measured at 72 hpi. Host cell viability and growth (inset) is unaffected by the compound over the course of the assay. Graphs shown are representative of 3 independent experiments.</p

Temporal expression of metabolic pathway genes in mammalian-infective stages of <i>T</i>. <i>cruzi</i>.

<p>Relative mRNA expression of selected genes in intracellular <i>T</i>. <i>cruzi</i> amastigote stages (4–72 hpi) compared to extracellular trypomastigotes (<b>T</b>). Genes in the following metabolic pathways are highlighted: (<b>A) Mevalonate pathway</b>: mevalonate kinase (TcCLB.436521.9), mevalonate diphosphate decarboxylase (TcCLB.507993.330), squalene monooxygenase (TcCLB.509589.20), farnesyl pyrophosphate synthase (TcCLB.508323.9), 3-hydroxy-3-methylglutaryl-CoA reductase (TcCLB.509167.20). <b>(B) Fatty Acid Synthesis:</b> fatty acid elongase 1 (ELO1) (TcCLB.506661.30), fatty acid elongase 2 (ELO2) (TcCLB.506661.20), fatty acid elongase 3 (ELO3) (TcCLB.506661.10). <b>(C) Glycolysis:</b> glyceraldehyde-3-phosphate dehydrogenase (TcCLB.506943.60); hexokinase (TcCLB.508951.20), triosephosphate isomerase (TcCLB.508647.200), phosphoglycerate kinase (TcCLB.511419.40), aldolase (TcCLB.504163.40). (<b>D) Tricarboxylic Acid / Oxidative Phosphorylation (TCA/Ox-PHOS):</b> cytochrome b5 (TcCLB.506773.44), ATPase subunit 9 (TcCLB.503579.70), cytochrome c oxidase subunit V (TcCLB.510565.30), succinate dehydrogenase 11 (TcCLB.504035.84), succinate dehydrogenase 6 (TcCLB.507091.30), cytochrome c oxidase subunit VII (TcCLB.509233.150), cytochrome c oxidase subunit VI (TcCLB.511145.10), cytochrome c oxidase subunit IV (TcCLB.506529.360). (<b>E) Fatty Acid Oxidation:</b> 3-ketoacyl-CoA thiolase (TcCLB.510507.20), enoyl-CoA hydratase/isomerase (TcCLB.511529.170); enoyl-CoA hydratase, mitochondrial (TcCLB.508185.10) enoyl-CoA hydratase/isomerase (TcCLB.510997.40). <b>(F) Glutamate Dehydrogenases (DH):</b> NADP+-GlutDH (TcCLB.507875.20), NAD+ GlutDH (TcCLB.509445.39). All values are reported as log2 fold-change of difference between expression at in the trypomastigote stage and intracellular stages as reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005511#ppat.1005511.s015" target="_blank">S5 Table</a>.</p