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ABSTRACT: Host defense to the apicomplexan parasite Toxoplasma gondii is critically dependent on CD8(+) T cells, whose effector functions include the induction of apoptosis in target cells following the secretion of granzyme proteases. Here we demonstrate that T. gondii induces resistance of host cells to apoptosis induced by recombinant granzyme B. Granzyme B induction of caspase-independent cytochrome c release was blocked in T. gondii-infected cells. Prevention of apoptosis could not be attributed to altered expression of the Bcl-2 family of apoptotic regulatory proteins, but was instead associated with reduced granzyme B-mediated, caspase-independent cleavage of procaspase 3 to the p20 form in T. gondii-infected cells, as well as reduced granzyme B-mediated cleavage of the artificial granzyme B substrate, GranToxiLux. The reduction in granzyme B proteolytic function in T. gondii-infected cells could not be attributed to altered granzyme B uptake or reduced trafficking of granzyme B to the cytosol, implying a T. gondii-mediated inhibition of granzyme B activity. Apoptosis and GranToxiLux cleavage were similarly inhibited in T. gondii-infected cells exposed to the natural killer-like cell line YT-1. The endogenous granzyme B inhibitor PI-9 was not up-regulated in infected cells. We believe these findings represent the first demonstration of granzyme B inhibition by a cellular pathogen and indicate a new modality for host cell protection by T. gondii that may contribute to parasite immune evasion.
International journal for parasitology 02/2011; 41(6):595-607. · 3.39 Impact Factor
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ABSTRACT: The apicomplexan Toxoplasma gondii replicates by endodyogeny, in which replicated organelles assemble into nascent daughter buds within the maternal parasite. The mechanisms governing this complex sequence are not understood. We now report that the kinase inhibitor 3-methlyadenine (3-MA) efficiently blocks T. gondii replication. The inhibition could not be attributed to the effects of 3-MA on mammalian phosphatidylinositol 3-kinase and host cell autophagy. Furthermore, we show that accumulation of host lysosomes around the parasitophorous vacuoles was unaffected. Most 3-MA-treated parasites failed to form daughter buds or replicate DNA, indicating arrest in G1 or early S-phase. Some 3-MA-treated parasites displayed abortive cell division, in which nuclear segregation to malformed daughter buds was incomplete or asymmetrical. Electron microscopy revealed the presence of residual body-like structures in many vacuoles, even in the absence of daughter buds. Most treated parasites had otherwise normal morphology and were able to resume replication upon drug removal. 3-MA-treated and control parasites were similar with respect to the extent of Golgi body division and apicoplast elongation; however, treated parasites rarely possessed replicated centrosomes or apicoplasts. These data are suggestive of a generalized blockade of T. gondii cell cycle progression at stages preceding centrosome replication, rather than arrest at a specific checkpoint. We hypothesize that 3-MA treatment triggers a cell cycle pause program that may serve to protect parasites during periods, such as subsequent to egress, when cell cycle progression might be deleterious. Elucidation of the mechanism of 3-MA inhibition may provide insight into the control of parasite growth.
Molecular and Biochemical Parasitology 10/2010; 173(2):142-53. · 2.55 Impact Factor
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ABSTRACT: The invasion of host cells by Toxoplasma gondii is accompanied by a reorganization of host cell structure, in which the host centrosome and Golgi apparatus are localized to the vacuole, and mitochondria, microtubules, and endolysosomes are recruited to the vacuole perimeter. The mechanism and functional significance of this process have not been well defined. Here, we report that the centrosome-vacuole association was abolished in mammalian target of rapamycin complex 2 (mTORC2)-deficient cells, which also displayed a disordered distribution of perivacuolar host mitochondria and lysosomes. Infection of fibroblasts led to stable, mTORC2-dependent activation of Akt, and Akt inhibition mimicked the effect of mTORC2 ablation on centrosome, mitochondria, and lysosome localization. Mobilization of the centrosome by Akt inhibition was abrogated by inhibitors of glycogen synthase kinase 3 (GSK3), implying that the centrosome is constrained to the vacuole through an mTORC2-Akt-GSK3 pathway. Infected cells were incapable of migration in a wounded monolayer model, and this effect was associated with the inability of centrosomes to reorient in the direction of migration. Both migration and centrosome reorientation were fully restored upon ablation of mTORC2. These findings provide the first linkage of host signals to parasite-mediated host cell reorganization and demonstrate migratory suppression as a novel functional consequence of this process that is associated with mTORC2-mediated centrosome constraint.
Journal of Biological Chemistry 03/2010; 285(20):15611-8. · 4.77 Impact Factor
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Lin Deng,
Jin-Feng Zhou,
Rani S Sellers,
Jiu-Feng Li,
Andrew V Nguyen,
Yubao Wang, Amos Orlofsky,
Qiang Liu,
David A Hume,
Jeffrey W Pollard,
Leonard Augenlicht,
Elaine Y Lin
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ABSTRACT: Inflammatory bowel disease (IBD) is a high-risk condition for human colorectal cancer. However, our mechanistic understanding of the link between inflammation and tumorigenesis in the colon is limited. Here we established a novel mouse model of colitis-associated cancer by genetically inactivating signal transducer and activator of transcription 3 (Stat3) in macrophages, with partial deletion in other myeloid and lymphoid cells. Inflammation developed in the colon of mutant mice spontaneously, and tumor lesions, including invasive carcinoma, arose in the inflamed region of the intestine with a frequency similar to that observed in human IBD patients. The development of both inflammation and tumors in the mutant mice required the presence of microflora. Indeed, inflammation was associated with disruption of colonic homeostasis, fulminant epithelial/tumor cell proliferation, and activation of the mammalian target of rapamycin (mTOR)-Stat3 pathway in epithelial and tumor cells. The activation of this pathway was essential for both the excess proliferation of epithelial/tumor cells and the disruption of colonic homeostasis in the mutant mice. Notably, a similar abnormal up-regulation of mTOR-Stat3 signaling was consistently observed in the colonic epithelial cells of human IBD patients with active disease. These studies demonstrate a novel mouse model of IBD-colorectal cancer progression in which disrupted immune regulation, mTOR-Stat3 signaling, and epithelial hyperproliferation are integrated and simultaneously linked to the development of malignancy.
American Journal Of Pathology 02/2010; 176(2):952-67. · 4.89 Impact Factor
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ABSTRACT: The apicomplexan parasite Toxoplasma gondii expands during acute infection via a cycle of invasion, intracellular replication, and lytic egress. Physiological regulation has not yet been demonstrated for either invasion or egress. We now report that, in contrast to cell culture systems, in which egress occurs only after five or more parasite divisions (2-3 days), intracellular residence is strikingly abbreviated in inflammatory cells in vivo, and early egress (after zero to two divisions) is the dominant parasite fate in acutely infected mice. Adoptive transfer experiments demonstrate rapid, reciprocal, kinetically uniform parasite transfer between donor and recipient compartments, with a t(1/2) of approximately 3 h. Inflammatory macrophages are major participants in this cycle of lytic egress and reinfection, which drives rapid macrophage turnover. Inflammatory triggering cells, principally macrophages, elicit egress in infected target macrophages, a process we term externally triggered egress (ETE). The mechanism of ETE does not require reactive oxygen or nitrogen species, the mitochondrial permeability transition pore, or a variety of signal transduction mediators, but is dependent on intracellular calcium and is highly sensitive to SB203580, an inhibitor of p38 MAPK as well as a related parasite-encoded kinase. SB203580 both inhibited the initiation of ETE and altered the progression of egress. Parasites recently completing a cycle of egress and reinfection were preferentially restricted in vivo, supporting a model in which ETE may favor host defense by a process of haven disruption. ETE represents a novel example of interaction between a parasite infectious cycle and host microenvironment.
The Journal of Immunology 11/2009; 183(10):6667-80. · 5.79 Impact Factor
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Amos Orlofsky
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ABSTRACT: The regulation and function of autophagy in response to metabolic signals is not yet well understood. A recent study from our laboratory indicates that an intracellular parasite, Toxoplasma gondii, derives nutritive benefit from the upregulation of host cell autophagy. We discuss this and related findings suggesting that autophagy in infected cells functions as part of a metabolic futile cycle. The hypothesis is presented that endogenous autophagy-based futile cycles may operate in normal mammalian cells, providing a substrate for manipulation by pathogens.
Autophagy 05/2009; 5(3):404-6. · 7.45 Impact Factor
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ABSTRACT: The Ser/Thr kinase mammalian-target-of-rapamycin (mTOR) is a central regulator of anabolism, growth and proliferation. We investigated the effects of Toxoplasma gondii on host mTOR signalling. Toxoplasma invasion of multiple cell types rapidly induced sustained mTOR activation that was restricted to infected cells, as determined by rapamycin-sensitive phosphorylation of ribosomal protein S6; however, phosphorylation of the growth-associated mTOR substrates 4E-BP1 and S6K1 was not detected. Infected cells still phosphorylated S6K1 and 4E-BP1 in response to insulin, although the S6K1 response was blunted. Parasite-induced S6 phosphorylation was independent of S6K1 and did not require activation of canonical mTOR-inducing pathways mediated by phosphatidylinositol 3-kinase-Akt and ERK. Host mTOR was localized in a vesicular pattern surrounding the parasitophorous vacuole, suggesting potential activation by phosphatidic acid in the vacuolar membrane. In spite of a failure to phosphorylate 4E-BP1 and S6K1, intracellular T. gondii triggered host cell cycle progression in an mTOR-dependent manner and progression of infected cells displayed increased sensitivity to rapamycin. Moreover, normal cell growth was maintained during parasite-induced cell cycle progression, as indicated by total cellular S6 levels. The Toxoplasma-infected cell provides a unique example of non-canonical mTOR activation supporting growth that is independent of signalling through either S6K1 or 4E-BP1.
Cellular Microbiology 03/2009; 11(6):983-1000. · 5.46 Impact Factor
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ABSTRACT: Autophagy has been shown to contribute to defense against intracellular bacteria and parasites. In comparison, the ability of such pathogens to manipulate host cell autophagy to their advantage has not been examined. Here we present evidence that infection by Toxoplasma gondii, an intracellular protozoan parasite, induces host cell autophagy in both HeLa cells and primary fibroblasts, via a mechanism dependent on host Atg5 but independent of host mammalian target of rapamycin suppression. Infection led to the conversion of LC3 to the autophagosome-associated form LC3-II, to the accumulation of LC3-containing vesicles near the parasitophorous vacuole, and to the relocalization toward the vacuole of structures labeled by the phosphatidylinositol 3-phosphate indicator YFP-2xFYVE. The autophagy regulator beclin 1 was concentrated in the vicinity of the parasitophorous vacuole in infected cells. Inhibitor studies indicated that parasite-induced autophagy is dependent on calcium signaling and on abscisic acid. At physiologically relevant amino acid levels, parasite growth became defective in Atg5-deficient cells, indicating a role for host cell autophagy in parasite recovery of host cell nutrients. A flow cytometric analysis of cell size as a function of parasite content revealed that autophagy-dependent parasite growth correlates with autophagy-dependent consumption of host cell mass that is dependent on parasite progression. These findings indicate a new role for autophagy as a pathway by which parasites may effectively compete with the host cell for limiting anabolic resources.
Journal of Biological Chemistry 12/2008; 284(3):1694-701. · 4.77 Impact Factor
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ABSTRACT: While reactive oxygen species (ROS) can kill Toxoplasma gondii in vitro the role these molecules play in vivo is not known. We used a flow cytometry-based assay to investigate the relationship between intracellular infection and ROS production during acute peritoneal toxoplasmosis in mice. A distinct population of ROS(+) inflammatory macrophages, detected by the oxidation of hydroethidine, was observed to increase progressively in frequency during the course of infection, and to be inversely correlated with the degree of cell parasitization. These data imply that either intracellular parasites inhibit ROS synthesis or, alternatively, ROS-producing cells contain anti-Toxoplasma activity. The latter interpretation was supported by the finding that uninfected ROS-producing inflammatory macrophages were resistant to infection in vivo. However, in the same animals, ROS-producing macrophages that had previously been parasitized could readily be infected with additional parasites, suggesting that the difference in ROS production between highly infected and less infected cells was not due to ROS-associated killing of parasites within these cells. In addition, macrophages infected with T. gondii in vitro and then briefly transferred to acutely infected mice upregulated ROS production in a manner that was again inversely correlated with the degree of intracellular parasitization. Taken together, these findings suggest that both ROS-associated anti-Toxoplasma activity and parasite-driven inhibition of ROS production underlie the observed pattern of ROS production. ROS function and parasite evasion of this function may contribute significantly to the balance between host defense and disease progression during acute infection.
International Journal for Parasitology 05/2006; 36(4):433-41. · 3.39 Impact Factor
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ABSTRACT: B lymphocyte stimulator (BLyS), a TNF family protein essential for peripheral B cell development, functions primarily through attenuation of B cell apoptosis. In this study, we show that BLyS activates NF-kappaB through both classical and alternative pathways with distinct kinetics in quiescent mature B cells. It rapidly and transiently enhances the p50/p65 DNA binding activity and induces phosphorylation of IkappaBalpha characteristic of the classical NF-kappaB pathway, albeit maintaining IkappaBalpha at a constant level through ongoing protein synthesis and proteasome-mediated destruction. With delayed kinetics, BLyS promotes the processing of p100 to p52 and sustained formation of p52/RelB complexes via the alternative NF-kappaB pathway. p50 is dispensable for p100 processing. However, it is required to mediate the initial BLyS survival signals and concomitant activation of Bcl-x(L) in quiescent mature B cells ex vivo. Although also a target of BLyS activation, at least one of the A1 genes, A1-a, is dispensable for the BLyS survival function. These results suggest that BLyS mediates its survival signals in metabolically restricted quiescent B cells, at least in part, through coordinated activation of both NF-kappaB pathways and selective downstream antiapoptotic genes.
The Journal of Immunology 08/2003; 171(2):761-8. · 5.79 Impact Factor
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ABSTRACT: The regulation of cell death in activated naive T cells is not well understood. We examined the expression of A1, an antiapoptotic member of the Bcl-2 family, following activation of naive mouse splenocytes. A1 gene expression was strongly but transiently induced during the first day of activation, with a peak at 2 to 6 hours, whereas Bcl-2 mRNA was simultaneously transiently down-regulated. Transgenic (Tg) overexpression of A1-a in T cells via the lck distal promoter resulted in decreased apoptosis following activation either with concanavalin A or with antibodies to CD3 and CD28 and led to a doubling of T-cell yield by 5 days. Tg A1-a also partially protected thymocytes from several proapoptotic stimuli but did not protect T-cell blasts from cell death induced by reactivation via the T-cell receptor. Tg Bcl-2 and Tg A1-a showed a similar ability to reduce apoptosis in both resting and activated T cells. However, in activated splenocyte cultures, the increase in 5-day T-cell yield observed with Tg Bcl-2 was only half that produced by Tg A1-a. This difference could be attributed at least in part to the fact that A1, unlike Bcl-2, did not inhibit S-phase entry of activated cells. The A1 protein may represent an adaptation of the Bcl-2 gene family to the need for survival regulation in the context of a proliferative stimulus.
Blood 05/2003; 101(7):2679-85. · 9.90 Impact Factor
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ABSTRACT: A1 is an anti-apoptotic member of the Bcl-2 family that is up-regulated in inflammatory myeloid cells. In the present study, we investigated the role of A1 in the maintenance of acute inflammation in mice. Mice possess three genes encoding highly related isoforms of A1. A1-a isoform mRNA was minimally expressed in resident peritoneal macrophages, but was present at a 300-fold higher level in inflammatory macrophages elicited by i.p. infection with Toxoplasma gondii. In comparison, A1-b and A1-d levels were 3- and 10-fold higher, respectively. Peritoneal leukocytosis was decreased in infected A1-a-deficient mice compared with wild-type, and this reduction was associated with a small but reproducible enhancement of survival. These effects could not be explained by an alteration in peritoneal parasite load, nor by increased apoptosis of infected inflammatory cells, which were protected from cell death by an A1-a-independent mechanism. Increased apoptosis in inflammatory neutrophils was observed sporadically in A1-a-deficient mice. Regulation of apoptosis by A1-a may be an important proinflammatory event in acute host responses.
The Journal of Immunology 03/2002; 168(4):1840-6. · 5.79 Impact Factor
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Mammalian Genome 03/1997; 8(4):293-294. · 2.89 Impact Factor
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ABSTRACT: Chemokines are typically found as products of acute stimulation of host defence cells. In contrast, the mouse CC chemokine C10 was previously shown to be a delayed, stably induced product of macrophages treated with interleukin 3 (IL-3), IL-4 or GM-CSF. We investigated the possibility that C10 is differentially regulated by cytokines associated with Th1and Th2cells. Northern blot analysis of bone marrow-derived macrophages showed that, in addition to IL-4, the Th2-specific cytokines IL-10 and IL-13 upregulated C10 over a 48-h period in a dose-dependent manner. In contrast, MIP-1α and MCP-1/JE were induced by IL-3 or GM-CSF at 48 h and this induction was inhibited by IL-4. Interferon γ, a Th1-specific product, abolished the induction of C10 mRNA and protein by either IL-3 or granulocyte–macrophage colony-stimulating factor (GM-CSF) in either bone marrow-derived or peritoneal macrophages. The inhibition of C10 production by interferon γ was not NO dependent. Finally the GM-CSF-mediated induction of C10 in peritoneal macrophages was eliminated when these cells presented antigen to established T cells of Th1phenotype. The findings are consistent with a potential role for C10 in the modulation of immune reactions of Th2type.
Cytokine.
