A Fine Balance between Life and Death: Modulation of BCL-2 Family Members by Toxoplasma Gondii

A commentary on The differential effect of Toxoplasma gondii infection on the stability of BCL2-family members involves multiple activities by Carmen, J. C., and Sinai, A. P. (2011). Front. Microbiol. 2:1. doi: 10.3389/fmicb.2011.00001 Apoptosis has been shown to serve as a mechanism by which cells infected with viruses, bacteria, or intracellular parasites can be eliminated without eliciting an unwanted inflammatory response. Not surprisingly intracellular pathogens have evolved mechanisms by which to inhibit apoptosis, not only avoiding the elimination of the host cell but also extending its life span. Inhibition of apoptosis by intracellular pathogens is accomplished by affecting the fine balance between pro- and antiapoptotic factors in the infected cell. One mechanism utilized by various microbes is regulation of the pro- and anti-apoptotic proteins of the BCL-2 family (Carmen an

Identification of Fis1 Interactors in Toxoplasma gondii Reveals a Novel Protein Required for Peripheral Distribution of the Mitochondrion

Toxoplasma gondii’s single mitochondrion is very dynamic and undergoes morphological changes throughout the parasite’s life cycle. During parasite division, the mitochondrion elongates, enters the daughter cells just prior to cytokinesis, and undergoes fission. Extensive morphological changes also occur as the parasite transitions from the intracellular environment to the extracellular environment. We show that treatment with the ionophore monensin causes reversible constriction of the mitochondrial outer membrane and that this effect depends on the function of the fission-related protein Fis1. We also observed that mislocalization of the endogenous Fis1 causes a dominant-negative effect that affects the morphology of the mitochondrion. As this suggests that Fis1 interacts with proteins critical for maintenance of mitochondrial structure, we performed various protein interaction trap screens. In this manner, we identified a novel outer mitochondrial membrane protein, LMF1, which is essential for positioning of the mitochondrion in intracellular parasites. Normally, while inside a host cell, the parasite mitochondrion is maintained in a lasso shape that stretches around the parasite periphery where it has regions of coupling with the parasite pellicle, suggesting the presence of membrane contact sites. In intracellular parasites lacking LMF1, the mitochondrion is retracted away from the pellicle and instead is collapsed, as normally seen only in extracellular parasites. We show that this phenotype is associated with defects in parasite fitness and mitochondrial segregation. Thus, LMF1 is necessary for mitochondrial association with the parasite pellicle during intracellular growth, and proper mitochondrial morphology is a prerequisite for mitochondrial division

The Secreted Acid Phosphatase Domain-Containing GRA44 from Toxoplasma gondii Is Required for c-Myc Induction in Infected Cells.

During host cell invasion, the eukaryotic pathogen Toxoplasma gondii forms a parasitophorous vacuole to safely reside within the cell, while it is partitioned from host cell defense mechanisms. From within this safe niche, parasites sabotage multiple host cell systems, including gene expression, apoptosis, and intracellular immune recognition, by secreting a large arsenal of effector proteins. Many parasite proteins studied for active host cell manipulative interactions have been kinases. The translocation of effectors from the parasitophorous vacuole into the host cell is mediated by a putative translocon complex, which includes the proteins MYR1, MYR2, and MYR3. Whether other proteins are involved in the structure or regulation of this putative translocon is not known. We have discovered that the secreted protein GRA44, which contains a putative acid phosphatase domain, interacts with members of this complex and is required for host cell effects downstream of effector secretion. We have determined that GRA44 is processed in a region with homology to sequences targeted by protozoan proteases of the secretory pathway and that both major cleavage fragments are secreted into the parasitophorous vacuole. Immunoprecipitation experiments showed that GRA44 interacts with a large number of secreted proteins, including MYR1. Importantly, conditional knockdown of GRA44 resulted in a lack of host cell c-Myc upregulation, which mimics the phenotype seen when members of the translocon complex are genetically disrupted. Thus, the putative acid phosphatase GRA44 is crucial for host cell alterations during Toxoplasma infection and is associated with the translocon complex which Toxoplasma relies upon for success as an intracellular pathogen.IMPORTANCE Approximately one-third of humans are infected with the parasite Toxoplasma gondii Toxoplasma infections can lead to severe disease in those with a compromised or suppressed immune system. Additionally, infections during pregnancy present a significant health risk to the developing fetus. Drugs that target this parasite are limited, have significant side effects, and do not target all disease stages. Thus, a thorough understanding of how the parasite propagates within a host is critical in the discovery of novel therapeutic targets. Toxoplasma replication requires that it enter the cells of the infected organism. In order to survive the environment inside a cell, Toxoplasma secretes a large repertoire of proteins, which hijack a number of important cellular functions. How these Toxoplasma proteins move from the parasite into the host cell is not well understood. Our work shows that the putative phosphatase GRA44 is part of a protein complex responsible for this process

The serine/threonine phosphatases of apicomplexan parasites

The balance between phosphorylation and de-phosphorylation, which is delicately regulated by protein kinases and phosphatases, is critical for nearly all biological processes. The Apicomplexa are a large phylum which contains various parasitic protists, including human pathogens, such as Plasmodium, Toxoplasma, Cryptosporidium and Babesia species. The diverse life cycles of these parasites are highly complex and, not surprisingly, many of their key steps are exquisitely regulated by phosphorylation. Interestingly, many of the kinases and phosphatases, as well as the substrates involved in these events are unique to the parasites and therefore phosphorylation constitutes a viable target for antiparasitic intervention. Most progress on this realm has come from studies in Toxoplasma and Plasmodium of their respective kinomes and phosphoproteomes. Nonetheless, given their likely importance, phosphatases have recently become the focus of research within the apicomplexan parasites. In this review, we concentrate on serine/threonine phosphatases in apicomplexan parasites, with the focus on comprehensively identifying and naming protein phosphatases in available apicomplexan genomes, and summarizing the progress of their functional analyses in recent years

Toxoplasma gondii-positive human sera recognise intracellular tachyzoites and bradyzoites with diverse patterns of immunoreactivity

Antibody detection assays have long been the first line test to confirm infection with the zoonotic parasite Toxoplasma gondii. However, challenges exist with serological diagnosis, especially distinguishing between acute, latent and reactivation disease states. The sensitivity and specificity of serological tests might be improved by testing for antibodies against parasite antigens other than those typically found on the parasite surface during the acute stage. To this end, we analysed the reactivity profile of human sera, identified as positive for anti-Toxoplasma gondii IgG in traditional assays, by indirect immunofluorescence reactivity to acute stage intracellular tachyzoites and in vitro-induced latent stage bradyzoites. The majority of anti-Toxoplasma gondii IgG positive sera recognised both intracellularly replicating tachyzoites and in vitro-induced bradyzoites with varying patterns of immune-reactivity. Furthermore, anti-bradyzoite antibodies were not detected in sera that were IgM-positive/IgG-negative. These results demonstrate that anti-Toxoplasma gondii-positive sera may contain antibodies to a variety of antigens in addition to those traditionally used in serological tests, and suggest the need for further investigations into the utility of anti-bradyzoite-specific antibodies to aid in diagnosis of Toxoplasma gondii infection

Genetic and molecular analysis of the drosophila gene nanos

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1999.Includes bibliographical references (p. 151-161).by Gstavo A. Arrizabalaga Muñiz.Ph.D

Ionophore-resistant mutant of Toxoplasma gondii reveals involvement of a sodium/hydrogen exchanger in calcium regulation

Calcium is a critical mediator of many intracellular processes in eukaryotic cells. In the obligate intracellular parasite Toxoplasma gondii, for example, a rise in [Ca2+] is associated with significant morphological changes and rapid egress from host cells. To understand the mechanisms behind such dramatic effects, we isolated a mutant that is altered in its responses to the Ca2+ ionophore A23187 and found the affected gene encodes a homologue of Na+/H+ exchangers (NHEs) located on the parasite's plasma membrane. We show that in the absence of TgNHE1, Toxoplasma is resistant to ionophore-induced egress and extracellular death and amiloride-induced proton efflux inhibition. In addition, the mutant has increased levels of intracellular Ca2+, which explains its decreased sensitivity to A23187. These results provide direct genetic evidence of a role for NHE1 in Ca2+ homeostasis and important insight into how this ubiquitous pathogen senses and responds to changes in its environment

TgATAT-Mediated α-Tubulin Acetylation Is Required for Division of the Protozoan Parasite Toxoplasma gondii

Toxoplasma gondii is a widespread protozoan parasite that causes potentially life-threatening opportunistic disease. New inhibitors of parasite replication are urgently needed, as the current antifolate treatment is also toxic to patients. Microtubules are essential cytoskeletal components that have been selectively targeted in microbial pathogens; further study of tubulin in Toxoplasma may reveal novel therapeutic opportunities. It has been noted that α-tubulin acetylation at lysine 40 (K40) is enriched during daughter parasite formation, but the impact of this modification on Toxoplasma division and the enzyme mediating its delivery have not been identified. We performed mutational analyses to provide evidence that K40 acetylation stabilizes Toxoplasma microtubules and is required for parasite replication. We also show that an unusual Toxoplasma homologue of α-tubulin acetyltransferase (TgATAT) is expressed in a cell cycle-regulated manner and that its expression peaks during division. Disruption of TgATAT with CRISPR/Cas9 ablates K40 acetylation and induces replication defects; parasites appear to initiate mitosis yet exhibit incomplete or improper nuclear division. Together, these findings establish the importance of tubulin acetylation, exposing a new vulnerability in Toxoplasma that could be pharmacologically targeted. IMPORTANCE Toxoplasma gondii is an opportunistic parasite that infects at least one-third of the world population. New treatments for the disease (toxoplasmosis) are needed since current drugs are toxic to patients. Microtubules are essential cellular structures built from tubulin that show promise as antimicrobial drug targets. Microtubules can be regulated by chemical modification, such as acetylation on lysine 40 (K40). To determine the role of K40 acetylation in Toxoplasma and whether it is a liability to the parasite, we performed mutational analyses of the α-tubulin gene. Our results indicate that parasites cannot survive without K40 acetylation unless microtubules are stabilized with a secondary mutation. Additionally, we identified the parasite enzyme that acetylates α-tubulin (TgATAT). Genetic disruption of TgATAT caused severe defects in parasite replication, further highlighting the importance of α-tubulin K40 acetylation in Toxoplasma and its promise as a potential new drug target

A imagem e a familiaridade têm um efeito significativo na intenção de compra?

The purpose of this paper is to study the relationship between the (i) country image of Peru, (ii) cotton’s product image, (iii) familiarity with Peru, and (iv) cotton’s product familiarity with the purchase intention of Peruvian cotton. Survey techniques were used to collect the primary data, applying a closed question questionnaire in two samples that represent the consumers of two countries with different levels of familiarity with Peru: France and the United States. This study has found that there is a positive association between the studied variables, in at least one sample. One of the main managerial implications is that Peruvian cotton exporters could innovate their global marketing strategies using Peru’s country image to improve the positioning of Peruvian cotton in international markets.El objetivo de este artículo es analizar la asociación entre (i) la imagen país Perú, (ii) la imagen producto del algodón, (iii) la familiaridad con el Perú y (iv) la familiaridad con el producto algodón, con la intención de compra del algodón peruano. La técnica de encuesta se utilizó para recopilar información primaria, aplicando un cuestionario de preguntas cerradas a dos muestras que corresponden a los consumidores de dos países con diferentes niveles de familiaridad con Perú: Francia y Estados Unidos. Se encontró que existe una asociación positiva entre las variables estudiadas, en al menos una muestra. En efecto, los exportadores peruanos de algodón podrían innovar sus estrategias de marketing utilizando la imagen país Perú para mejorar el posicionamiento del algodón peruano en el extranjero.O objetivo deste artigo é analisar a relação entre a imagem do país do Peru, a imagem do produto de algodão, a familiaridade com o Peru e a familiaridade com o produto de algodão, com a intenção de comprar algodão do Peru. A técnica de pesquisa foi utilizada para coletar informações primarias, aplicando um questionário de perguntas fechadas em duas amostras que correspondem a consumidores em dois países com diferentes níveis de familiaridade com o Peru (França e Estados Unidos). Verificou-se uma associação positiva entre as variáveis estudadas, em pelo menos uma amostra. De fato, os exportadores de algodão peruano poderiam inovar suas estratégias de marketing usando do país para melhorar o posicionamento do algodão peruano