Progress 10/01/15 to 09/30/16
Outputs
Target Audience:The target audience would be other scientists who study eukaryotic pathogens such as parasites or pathogenic fungi. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Ms. Hollie Hendrick, Ms. Michelle Rock and Ms. Natacha Karambizi, graduate students in the laboratory, are involved in these projects. Several training and professional development opportunities have been provided. First, Ms. Hendrick presented her data in poster presentation format at a local scientific meeting, the 3nd Annual Cell Biology of Eukaryotic Pathogens Symposium (Clemson University; October 2015). Given that this symposium took place at our home institution, Ms. Hendrick also participated in the organization of this meeting. She assisted with registration, and management of poster sessions. Second, Ms. Rock presented a poster presentation for the Clemson Biological Sciences Annual Student Symposium (CBASS). Third, both Ms. Hendrick Ms. Rock are members of EPIC scholars, which is the graduate student association in our research center (Eukaryotic Pathogens Innovation Center (EPIC)). As EPIC scholars they are involved in a number of professional activities including journal club and hosting an outside speaker annually. Both Ms. Hendrick and Ms. Rock graduated during the project period. Ms. Karambizi is now characterizing the kinases responsible for phosphorylating eIF2α during stress or encystation. Three undergraduate students, Ms. Julianna Hoover, Mr. Drew Gianos, and Mr. Matthew Hapstack, have also been involved in the research. They were trained in a variety of biochemical, molecular and cellular techniques. Mr. Hapstack was awarded a prestigious American Society for Cell Biology Undergraduate Research Fellowship. He presented his data at the Annual Meeting of the ASM (June 2016). Two postdoctoral fellows, Drs. Suchitra Chavan and Steve Sykes, participated on the project. How have the results been disseminated to communities of interest?The results of a subset of our studies on stress were presented at the 3rd Annual Cell Biology of Eukaryotic Pathogens Symposium (Clemson University; October 2015) and at ASM Microbe 2016 (Boston, MA; May 2016). A manuscript describing these data is in revision for PLoS Pathogens. The results of the studies on recombinases were published in two manuscripts (PLoS ONE and Molecular and Biochemical Parsitology). What do you plan to do during the next reporting period to accomplish the goals?A final report is also prepared.
Impacts
What was accomplished under these goals?
IMPACT: Amoebic dysentery is a food- and water-borne illness that is prevalent in the developing world. It is acquired by ingesting the cyst form of the parasite, Entamoeba histolytica. Globally, it is the 2nd leading cause of morbidity and mortality attributable to parasitic infections. The prevalence of E. histolytica infection has been estimated to range from 1% to 40% of the population in Central and South America, Africa and Asia and from 0.2% to 10% in developed nations such as the USA. Interestingly, many infections are asymptomatic or go unreported. Thus, the magnitude of the problem is difficult to discern. None-the-less, as of 2010, it is estimated that 2.6 billion people worldwide do not use modern sanitation practices, and 886 million do not have access to clean drinking water sources. Thus, there is considerable global risk for acquiring E. histolytica infection. The pathogen can bind to human cells via its lectin, a surface structure made up of 3 proteins. Adhesion to human cells is an important virulence function, without which infection cannot be established. In the human host, the parasite resides in the large intestine, but can also escape the intestine and set up infection in the liver, lungs or brain. Many of the deaths associated with this pathogen are the result of these extra-intestinal complications. During the course of infection in the host, E. histolytica likely confronts stress brought on by ever-changing environments in the host. In addition to adhering to host cells, the parasite must also circumvent stress to survive. Stress is also thought to induce the formation of cysts, the infectious form of the parasite. Thus, it may be useful to target E. histolytica's response to stress for therapy. Over the reporting period our studies have provided significant insight into how the parasites binds to human cells and how the parasite responds to stress. Thus, we have gained insight into the mechanisms of infection (see below), which represents a change in knowledge. SPECIFIC ACCOMPLISHMENTS: Summary of project on lectin: E. histolytica binds to host cells, in part, through the action of a cell surface adhesion molecule known as the Gal/GalNAc lectin (GGL). GGL is made up of three subunits, the heavy subunit (Hgl), which is covalently attached to the light subunit (Lgl), and a non-covalently interacting intermediate subunit (Igl). Igl is constitutively localized to lipid rafts, cholesterol-rich membrane domains on the parasite. Hgl-Lgl, which is mostly found in non-raft membrane, transiently localizes to rafts when the parasite interacts with host. To determine the role of Igl in the localization of Hgl-Lgl to rafts, we generated a cell line with reduced expression of Igl and characterized the submembrane location of the Hgl-Lgl dimer. Interestingly, the submembrane distribution of the dimer was unchanged suggesting that Igl is not necessary for Hgl-Lgl localization. To gain further insight into the molecular mechanisms that regulate the location of GGL, we are characterizing a cell line with reduced expression of a rhomboid protease (EhROM1). Rhomboid proteases regulate cell surface transmembrane proteins in other systems. During the reporting period we discovered the EhROM1 localizes to rafts. Furthermore, in the EhROM1-knockdown cell line, there is more Hgl-Lgl dimer in lipid rafts, as compared to control and the molecular mass of Hgl is larger. Together, these data suggest that EhROM1 regulates the submembrane location of GGL by some undetermined cleavage event. A manuscript describing these findings is in preparation for the International Journal of Parasitology. Summary of project on stress: In other parasites, stress can activate eIF2α kinases that phosphorylate the α-subunit of eukaryotic initiation factor-2 (eIF2α). eIF2α is part of a protein complex that delivers Met-tRNA to ribosomes for translation initiation. Phosphorylation of eIF2α inhibits this activity which, in turn, leads to a decline in protein synthesis. This allows cells to conserve resources and reconfigure gene expression to effectively manage stress. Genome analyses reveal that E. histolytica possesses all of the components of this system including eIF2α with a conserved phosphorylatable serine at position 59 (Ser59). However, the premise that phospho-eIF2α controls stress and translation in this pathogen has not been tested. To test the stress-response system, we exposed cells to different stress conditions and measured viability, and the level of total and phospho-EheIF2α. Long-term serum starvation, long-term heat shock, and oxidative stress induced an increase in the level of phospho-EheIF2α, while short-term serum starvation, short-term heat shock, or glucose deprivation did not. Long-term serum starvation also caused a decrease in polyribosome abundance, which is in accordance with the observation that this condition induces phosphorylation of EheIF2α. We generated transgenic cells that overexpress wildtype EheIF2α, a non-phosphorylatable variant of eIF2α in which Ser59 was mutated to alanine (EheIF2α-S59A), or a phosphomimetic variant of eIF2α in which Ser59 was mutated to aspartic acid (EheIF2α-S59D). Consistent with the known functions of eIF2α, cells expressing wildype or EheIF2α-S59D exhibited increased or decreased protein translation, respectively. Surprisingly, cells expressing EheIF2α-S59A also exhibited reduced protein translation. The mechanism regulating this unusual phenotype is yet to be discerned. Cells expressing EheIF2α-S59D were better able to survive long-term serum starvation illustrating a protective nature of phospho-eIF2α during stress. Together, these data demonstrate that the eIF2α-dependent stress response system is operational in E. histolytica. A manuscript describing these findings is in revision for PLoS Pathogens. In many systems, DNA damage can induce a stress-response that includes activation of homology-based DNA repair enzymes. We collaborated with Dr. Michael Sehorn (Clemson) to characterize two DNA recombinases in E. histolytica. The first, ehRad51, preferentially binds ssDNA, forms a presynaptic filament and possesses ATP hydrolysis activity that is stimulated by the presence of DNA. Evidence is provided that ehRad51 catalyzes robust DNA strand exchange over at least 5.4 kilobase pairs. The second, ehDmc1, forms presynaptic filaments and catalyzes ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. In both cases, the DNA pairing and strand exchange activities are enhanced by the presence of calcium and the meiosis-specific recombination accessory factor, Hop2-Mnd1. The results of these studies were published in two manuscripts (PLoS ONE and Molecular and Biochemical Parsitology). Summary of project on encystation: During the reporting period we were also awarded an NIH grant (R21 AI108287) to characterize the role of eIF2α in encystation in E. histolytica. Latent food- and water-borne cysts are the infectious form of this parasite. E. histolytica does not readily encyst in axenic culture. Thus, E. invadens, a reptilian parasite that encysts in vitro, has been used as a model. The inability to reproducibly induce E. histolytica encystation in vitro represents a significant gap in the research tool-set and has hampered studies of E. histolytica latency. In other parasites latency is regulated, in part, by the eIF2α translational control system. During the project period, we have acquired data that demonstrate that levels of phospho-eIF2α increase during encystation in E. invadens cells. This is the first demonstration that protein translational control may play a role in encystation in the Entamoebae. This information is included in the PLoS Pathogens manuscript.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Kelso, A.A. Goodson, S.D., Chavan, S., Say, A.F., Turchik, A., Sharma, D., Ledford, L.L., Ratterman, E., Leskoske, E., King, A.V., Attaway, C.C., Bandera, Y., Foulger, S.H., Temesvari, L.A., Sehorn, M.G. Characterization of the recombination activities of the Entamoebahistolytica Rad51 recombinase. Int. J. Parasitol. (In press).
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Kelso, A.A., Say, A.F., Sharma, D., Ledford, L.L., Turchick, A., Saski, C., King, A.V., Attaway, C.C., Temesvari, L.A., and Sehorn, M.G. Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange that is Stimulated by Calcium and Hop2-Mnd1. PLoS One http://dx.doi.org/10.1371/journal.pone.0139399 (September 2015).
- Type:
Journal Articles
Status:
Under Review
Year Published:
2016
Citation:
Holland, H.M., Haptstack, M.A., Welter, B.H., Sykes, S.E., Sullivan, W. and Temesvari, L.A. Phosphorylation of Eukaryotic Initiation Factor-2alpha Regulates Stress in the Human Protozoan Parasite, Entamoeaba histolytica. PLoS Pathogens (in revision)
|
Progress 07/01/12 to 09/30/16
Outputs
Target Audience:The target audience would be other scientists who are studying the molecular mechanisms regulating pathogenesis of eukaryotes, cellular stress responses, and/or cell-cell interactions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Five graduate students (Dr. Amrita Koushik, Dr. Amanda Goldston, Ms. Hollie Hendrick, Ms. Michelle Rock and Ms. Natacha Karambizi), have been involved in these projects. Several training and professional development opportunities have been provided. First, these students presented the results of their research in at least 6 local, national or international conferences. Ms. Hendrick also participated in the organization of the local meeting. She assisted with registration, and management of poster sessions. Several of these graduate students were members of EPIC scholars, which is the graduate student association in our research center (Eukaryotic Pathogens Innovation Center (EPIC)). As EPIC scholars they are involved in a number of professional activities including journal club and hosting an outside speaker annually. Seven undergraduate students have also been involved in the research. They were trained in a variety of biochemical, molecular and cellular techniques. One undergraduate student, Mr. Matthew Hapstack was awarded a prestigious American Society for Cell Biology Undergraduate Research Fellowship. He presented his data at the Annual Meeting of the ASM (June 2016). Two postdoctoral fellows, Drs. Suchitra Chavan and Steve Sykes, participated on the project. How have the results been disseminated to communities of interest?The results of our studies on stress have been published in six high-impact peer-reviewed journal articles and were presented in at least six local, national or international symposia. We are currently revising one additional manuscript and preparing one additional manuscript. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT: Amoebic dysentery is a food- and water-borne illness that is prevalent in the developing world. It is acquired by ingesting the cyst form of the parasite, Entamoeba histolytica. Globally, it is the 2nd leading cause of morbidity and mortality attributable to parasitic infections. The prevalence of E. histolytica infection has been estimated to range from 1% to 40% of the population in Central and South America, Africa and Asia and from 0.2% to 10% in developed nations such as the USA. Interestingly, many infections are asymptomatic or go unreported. Thus, the magnitude of the problem is difficult to discern. None-the-less, as of 2010, it is estimated that 2.6 billion people worldwide do not use modern sanitation practices, and 886 million do not have access to clean drinking water sources. Thus, there is considerable global risk for acquiring E. histolytica infection. The pathogen can bind to human cells via its lectin, a surface structure made up of 3 proteins. Adhesion to human cells is an important virulence function, without which infection cannot be established. In the human host, the parasite resides in the large intestine, but can also escape the intestine and set up infection in the liver, lungs or brain. Many of the deaths associated with this pathogen are the result of these extra-intestinal complications. During the course of infection in the host, E. histolytica likely confronts stress brought on by ever-changing environments in the host. In addition to adhering to host cells, the parasite must also circumvent stress to survive. Stress is also thought to induce the formation of cysts, the infectious form of the parasite. Thus, it may be useful to target E. histolytica's response to stress for therapy. Over the reporting period our studies have provided significant insight into how the parasites binds to human cells and how the parasite responds to stress. Thus, we have gained insight into the mechanisms of infection (see below), which represents a change in knowledge. SPECIFIC ACCOMPLISHMENTS: Summary of project on lectin: E. histolytica binds to host cells, in part, through the action of a cell surface adhesion molecule known as the Gal/GalNAc lectin (GGL). GGL is made up of 3 subunits, the heavy subunit (Hgl), which is covalently attached to the light subunit (Lgl), and a non-covalently interacting intermediate subunit (Igl). Igl is constitutively localized to lipid rafts, cholesterol-rich membrane domains on the parasite. Hgl-Lgl, which is mostly found in non-raft membrane, transiently localizes to rafts when the parasite interacts with host. Over the course of the project period we demonstrated that interaction of GGL with a variety of its ligands regulates the sub-membrane distribution. For example, binding of host red blood cells or collagen induced enrichment of GGL in lipid rafts. Although physical attachment was necessary to induce raft-enrichment is was not sufficient. We, thus hypothesized that signaling must be involved. In support of this hypothesis, we demonstrated that PI(4,5)P2, an important signaling lipid, was localized to lipid rafts and may participate in regulating the submembrane distribution of GGL. To gain further insight into PI(4,5)P2-based signaling, we conducted genome-wide screens to identify genes that functionally interact with the signaling lipid. To gain further insight into the molecular mechanisms that regulate the location of GGL, we characterized a cell line with reduced expression of a rhomboid protease (EhROM1). Rhomboid proteases regulate cell surface transmembrane proteins in other systems. During the reporting period we discovered the EhROM1 localizes to rafts. Furthermore, in the EhROM1-knockdown cell line, there is more Hgl-Lgl dimer in lipid rafts, as compared to control and the molecular mass of Hgl is larger. Together, these data suggest that EhROM1 regulates the submembrane location of GGL by some undetermined cleavage event. Summary of project on stress: In other parasites, stress can activate eIF2α kinases that phosphorylate the α-subunit of eukaryotic initiation factor-2 (eIF2α). eIF2α is part of a protein complex that delivers Met-tRNA to ribosomes for translation initiation. Phosphorylation of eIF2α inhibits this activity which, in turn, leads to a decline in protein synthesis. This allows cells to conserve resources and reconfigure gene expression to effectively manage stress. Genome analyses reveal that E. histolytica possesses all of the components of this system including eIF2α with a conserved phosphorylatable serine at position 59 (Ser59). However, the premise that phospho-eIF2α controls stress and translation in this pathogen has not been tested. To test the stress-response system, we exposed cells to different stress conditions and measured viability, and the level of total and phospho-EheIF2α. Long-term serum starvation, long-term heat shock, and oxidative stress induced an increase in the level of phospho-EheIF2α, while short-term serum starvation, short-term heat shock, or glucose deprivation did not. Long-term serum starvation also caused a decrease in polyribosome abundance, which is in accordance with the observation that this condition induces phosphorylation of EheIF2α. We generated transgenic cells that overexpress wildtype EheIF2α, a non-phosphorylatable variant of eIF2α in which Ser59 was mutated to alanine (EheIF2α-S59A), or a phosphomimetic variant of eIF2α in which Ser59 was mutated to aspartic acid (EheIF2α-S59D). Consistent with the known functions of eIF2α, cells expressing wildype or EheIF2α-S59D exhibited increased or decreased protein translation, respectively. Surprisingly, cells expressing EheIF2α-S59A also exhibited reduced protein translation. The mechanism regulating this unusual phenotype is yet to be discerned. Cells expressing EheIF2α-S59D were better able to survive long-term serum starvation illustrating a protective nature of phospho-eIF2α during stress. Together, these data demonstrate that the eIF2α-dependent stress response system is operational in E. histolytica. A manuscript describing these findings is in revision for PLoS Pathogens. In many systems, DNA damage can induce a stress-response that includes activation of homology-based DNA repair enzymes. We collaborated with Dr. Michael Sehorn (Clemson) to characterize two DNA recombinases in E. histolytica. The first, ehRad51, preferentially binds ssDNA, forms a presynaptic filament and possesses ATP hydrolysis activity that is stimulated by the presence of DNA. Evidence is provided that ehRad51 catalyzes robust DNA strand exchange over at least 5.4 kilobase pairs. The second, ehDmc1, forms presynaptic filaments and catalyzes ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. In both cases, the DNA pairing and strand exchange activities are enhanced by the presence of calcium and the meiosis-specific recombination accessory factor, Hop2-Mnd1. The results of these studies were published in two manuscripts (PLoS ONE and Molecular and Biochemical Parsitology). Summary of project on encystation: Latent food- and water-borne cysts are the infectious form of this parasite. E. histolytica does not readily encyst in axenic culture. Thus, E. invadens, a reptilian parasite that encysts in vitro, has been used as a model. The inability to reproducibly induce E. histolytica encystation in vitro represents a significant gap in the research tool-set and has hampered studies of E. histolytica latency. In other parasites latency is regulated, in part, by the eIF2α translational control system. We demonstrated that levels of phospho-eIF2α increase during encystation in E. invadens cells. This is the first demonstration that protein translational control may play a role in encystation in the Entamoebae.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Kelso, A.A. Goodson, S.D., Chavan, S., Say, A.F., Turchik, A., Sharma, D., Ledford, L.L., Ratterman, E., Leskoske, E., King, A.V., Attaway, C.C., Bandera, Y., Foulger, S.H., Temesvari, L.A., Sehorn, M.G. Characterization of the recombination activities of the Entamoebahistolytica Rad51 recombinase. Int. J. Parasitol. (In press).
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Kelso, A.A., Say, A.F., Sharma, D., Ledford, L.L., Turchick, A., Saski, C., King, A.V., Attaway, C.C., Temesvari, L.A., and Sehorn, M.G. Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange that is Stimulated by Calcium and Hop2-Mnd1. PLoS One http://dx.doi.org/10.1371/journal.pone.0139399 (September 2015).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
Hendrick, H., Welter, B.H., Hapstack, M., Sykes, S.E., Sullivan, W., Temesvari, L.A. Translational control of stress in Entamoeba histolytica. Submitted to PLoS Pathogens
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Koushik, A.B., Welter, B. H., Rock, M.L., and Temesvari, L.A. A Genome-wide Over-Expression Screen Identifies Genes Involved in the Phosphatidylinositol 3-kinase Pathway in the Human Protozoan Parasite, Entamoeba histolytica. Eukaryotic Cell 13:401-411.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Koushik, A.B.*, Powell, R.R., and Temesvari, L.A. Localization of phosphatidylinositol 4,5-bisphosphate to lipid rafts and uroids in the human protozoan parasite, Entamoeba histolytica. Infect Immun. Infect Immun. 81, 2145-2155.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
King, A.V., Welter, B.H., Koushik, A.B.*, and Temesvari, L.A. A Genome-Wide Over-Expression Screen Identifies Genes Involved in Phagocytosis in the Human Protozoan Parasite, Entamoeba histolytica, PloS ONE 7, e43025.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Goldston A.M.*, Powell R.R., Koushik A.B.*, and Temesvari L.A. Exposure to Host Ligands Correlates with Co-localization of Gal/GalNAc Lectin Subunits in Lipid Rafts and PIP2 Signaling in Entamoeba histolytica. Eukaryot. Cell. 11, 743-751.
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Ms. Hollie Hendrick and Ms. Michelle Rock, graduate students in the laboratory, are involved in these projects. Several training and professional development opportunities have been provided. First, Ms. Hendrick presented her data in poster presentation format at a local scientific meeting, the 3nd Annual Cell Biology of Eukaryotic Pathogens Symposium (Clemson University; October 2015). Given that this symposium took place at our home institution, Ms. Hendrick also participated in the organization of this meeting. She assisted with registration, and management of poster sessions. Second, Ms. Rock is preparing a poster presentation for the Clemson Biological Sciences Annual Student Symposium (CBASS). Third, both Ms. Hendrick Ms. Rock are members of EPIC scholars, which is the graduate student association in our research center (Eukaryotic Pathogens Innovation Center (EPIC)). As EPIC scholars they are involved in a number of professional activities including journal club and hosting an outside speaker annually. Three undergraduate students, Ms. Julianna Hoover, Mr. Drew Gianos, and Mr. Matthew Hapstack, have also been involved in the research. They are being trained in a variety of biochemical, molecular and cellular techniques. Mr. Hapstack was awarded a prestigious American Society for Cell Biology Undergraduate Research Fellowship. He will be presenting his data at the Annual Meeting of the ASM (June 2016). Finally, a new postdoctoral fellow, Dr. Suchitra Chavan, has joined the group. Her focus is the encystation project. How have the results been disseminated to communities of interest?The results of a subset of our studies were presented at the 3rd Annual Cell Biology of Eukaryotic Pathogens Symposium (Clemson University; October 2015). A manuscript describing these data is in preparation for PLoS Pathogens. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we will focus on gaining insight into mechanism by which ROM1 regulates the GGL in E. histolytica. Specifically, to discover the reason that Hgl is bigger in the ROM1-knockdown cells, we will track the biosynthesis and recycling of this protein by biotinylation. We will also continue to explore the role of Igl in Hgl-Lgl localization. We will attempt to knockdown expression using a different system, known as the "trigger" system. Characterizaiton of this cell line will reveal if Igl is necessary for a variety of virulence functions (adhesion, phagocytosis, host cell cytolysis). We will also continue to characterize the role of translational control in encystation using the E. invadens model. Encystation will tracked in E. invadens cell lines expressing non-phosphorylatable and phosphomimetic mutant versions of eIF2α. This will reveal if phospho-eIF2α is necessary and sufficient for stage conversion. The levels of phospho-eIF2α will be manipulated genetically in E. histolytica in an attempt to induce encystation- and excystation-like events in the human pathogen. If successful, it would represent the first protocol for reproducibly recapitulating the life cycle of this parasite in vitro. Establishing an in vitro system for encystation and excystation of E. histolytica would provide an innovative tool to the community of researchers that would inspire numerous novel studies of E. histolytica latency.
Impacts
What was accomplished under these goals?
Summary of project on lectin: E. histolytica binds to host cells, in part, through the action of a cell surface adhesion molecule known as the Gal/GalNAc lectin (GGL). GGL is made up of three subunits, the heavy subunit (Hgl), which is covalently attached to the light subunit (Lgl), and a non-covalently interacting intermediate subunit (Igl). Igl is constitutively localized to lipid rafts, cholesterol-rich membrane domains on the parasite. Hgl-Lgl, which is mostly found in non-raft membrane, transiently localizes to rafts when the parasite interacts with host. To determine the role of Igl in the localization of Hgl-Lgl to rafts, we generated a cell line with reduced expression of Igl and characterized the submembrane location of the Hgl-Lgl dimer. Interestingly, the submembrane distribution of the dimer was unchanged suggesting that Igl is not necessary for Hgl-Lgl localization. To gain further insight into the molecular mechanisms that regulate the location of GGL, we are characterizing a cell line with reduced expression of a rhomboid protease (EhROM1). Rhomboid proteases regulate cell surface transmembrane proteins in other systems. During the reporting period we discovered the EhROM1 localizes to rafts. Furthermore, in the EhROM1-knockdown cell line, there is more Hgl-Lgl dimer in lipid rafts, as compared to control and the molecular mass of Hgl is larger. Together, these data suggest that EhROM1 regulates the submembrane location of GGL by some undetermined cleavage event. Summary of project on stress: During the course of infection in the host, E. histolytica likely confronts stress brought on by ever-changing environments in the host. To survive, the parasite must circumvent these exogenous pressures. Thus, it may be useful to target E. histolytica's response to stress for therapy. In other parasites, stress can activate eIF2α kinases that phosphorylate the α-subunit of eukaryotic initiation factor-2 (eIF2α). eIF2α is part of a protein complex that delivers Met-tRNA to ribosomes for translation initiation. Phosphorylation of eIF2α inhibits this activity which, in turn, leads to a decline in protein synthesis. This allows cells to conserve resources and reconfigure gene expression to effectively manage stress. Genome analyses indicate that E. histolytica possesses all of the components of this stress response system; however, the premise that phospho-eIF2α controls stress and translation in this pathogen has not been tested. This represents a profound gap in knowledge. Thus, the goal of this project is to characterize this stress response system in E. histolytica. To gain insight into the function of phospho-eIF2α we monitored the level of the phospho-form of eIF2α during a variety of stress conditions including serum-starvation, heat shock, glucose deprivation, and oxidative stress. Only serum-starvation induced phosphorylation of eIF2α in E. histolytica. Next, we generated mutants that over-express wild-type, non-phosphorylatable (S59A), or phosphomimietic (S59D) forms of eIF2α. Predictably, cells that were unable to phosphorylate eIF2α (S59A), were also more sensitive to stress than the other cell lines. Since eIF2α regulates protein translation we also tracked protein translation in the mutants using two different methods. As expected, mutants over-expressing the phosphomimetic form (S59D) exhibited a reduction in global protein translation and the mutants over-expressing the wild-type version exhibited an increase in global protein translation. A manuscript describing these data is in preparation for PLoS Pathogens. Summary of project on encystation: During the reporting period we were also awarded an NIH grant (R21 AI108287) to characterize the role of eIF2α in encystation in E. histolytica. Latent food- and water-borne cysts are the infectious form of this parasite. E. histolytica does not readily encyst in axenic culture. Thus, E. invadens, a reptilian parasite that encysts in vitro, has been used as a model. The inability to reproducibly induce E. histolytica encystation in vitro represents a significant gap in the research tool-set and has hampered studies of E. histolytica latency. In other parasites latency is regulated, in part, by the eIF2α translational control system. During the project period, we have acquired data that demonstrate that levels of phospho-eIF2α increase during encystation in E. invadens cells. This is the first demonstration that protein translational control may play a role in encystation in the Entamoebae.
Publications
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The target audience are those scientists whose research may be classified as parasitology, cellular signal transduction, stress biology and/or lipid biology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Ms. Hollie Hendrick, a graduate student in the laboratory, is involved in this project. Several training and professional development opportunities have been provided. First, Ms. Hendrick presented her data in oral presentation format at a local scientific meeting, the 2nd Annual Cell Biology of Eukaryotic Pathogens Symposium (Clemson University; October 2014). Given that this symposium took place at our home institution, Ms. Hendrick also participated in the organization of this meeting. She assisted with registration, management of poster sessions, and was one of the attendees at the student dinner with the keynote speaker, Dr. Heidi Elmendorf (Georgetown University). Second, Ms. Hendrick made an oral presentation at the Clemson Biological Sciences Annual Student Symposium (CBASS). Third, Ms. Hendrick became a member of EPIC scholars, which is the graduate student association in our research center (Eukaryotic Pathogens Innovation Center (EPIC)). As an EPIC scholar Ms. Hendrick is involved in a number of professional activities including journal club and hosting an outside speaker annually. Finally, in addition to laboratory research meetings, Ms. Hendrick meets one-on-one with the PI on a bi-weekly basis to discuss research progress. A new graduate student, Ms. Michelle Rock, and 3undergraduate students,Ms. Victoria Tanenbaum, Ms. Lindsey Stegall, and Mr. Matthew Hapstack, have been involved in the research. They are being trained in a variety of biochemical, molecular and cellular techniques. Ms. Tanenbaum and Ms. Stegall will each present posters describing their researchat the American Association for the Advancement of Science (AAAS) in San Jose, CA in February (2015). These abstracts have been accepted by conference organizers. How have the results been disseminated to communities of interest? The results of a subset of our studies related to the cell signaling portion of our project were published in the journal, Eukaryotic Cell. The results of our work on the E. histolytica stress response were presented at the 2nd Annual Cell Biology of Eukaryotic Pathogens Symposium (Clemson University; October 2014). What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period we will focus on gaining insight into the function of the intermediate subunit (Igl) of the Gal/GalNac lectin (GGL) in E. histolytica adhesion and virulence. To this end we have generated a mutant cell line in which expression of Igl is knocked down by short hairpin RNA technology. Characterizaiton of this cell line will reveal if Igl is necessary for a variety of virulence functions (adhesion, phagocytosis, host cell cytolysis). We will submit a grant proposal to the NIH R21 program in February 2015. We will also continue to characterize the stress response in E. histolytica. We will survey the phosphorylation status of eIF2α during 6 additional stresses. These include ER stress (1 mM sodium nitroprusside, 1 hr), glucose deprivation (glucose free medium, 12 hr), iron deprivation (iron-free medium, 16 hr), oxidative stress (1 mM hydrogen peroxide, 1 hr), nitrosative stress (200 uM DPTA-NONOate or 350 uM S-nitrosoglutathione, 1hr). In all cases, we will use Western blotting with our new specific antibodies to track total and phospho-eIF2α during stress and we will monitor cell viability. We will also determine if phosphorylation of eIF2α is necessary for the stress response by over-expression a mutant form that cannot be phosphorylated. Finally, since phosphorylation of eIF2α down-regulates global protein translation, we will monitor protein translation during stress by polyribosome profiling.
Impacts
What was accomplished under these goals?
IMPACT: Amoebic dysentery is a food- and water-borne illness that is prevalent in the developing world. It is acquired by ingesting the parasite, Entamoeba histolytica. Globally, it is the 2nd leading cause of morbidity and mortality attributable to parasitic infections. The prevalence of E. histolytica infection has been estimated to range from 1% to 40% of the population in Central and South America, Africa and Asia and from 0.2% to 10% in developed nations such as the USA. Interestingly, many infections are asymptomatic or go unreported. Thus, the magnitude of the problem is difficult to discern. None-the-less, as of 2010, it is estimated that 2.6 billion people worldwide do not use modern sanitation practices, and 886 million do not have access to clean drinking water sources. Thus, there is considerable global risk for acquiring E. histolytica infection. The pathogen can bind to human cells via its lectin, a surface structure made up of 3 proteins. Adhesion to human cells is an important virulence function, without which infection cannot be established. In the human host, the parasite resides in the large intestine, but can also escape the intestine and set up infection in the liver, lungs or brain. Many of the deaths associated with this pathogen are the result of these extra-intestinal complications. During the course of infection in the host, E. histolytica likely confronts stress brought on by ever-changing environments in the host. In addition to adhering to host cells,the parasite must also be able to circumvent stress to survive. Thus, it may be useful to target E. histolytica's response to stress for therapy. Over the reporting period our studies have provided significant insight into how the parasites binds to human cells and how the parasite responds to stress. Thus, we have gained insight into the mechanisms of infection (see below), which represents a change in knowledge. SPECIFIC ACCOMPLISHMENTS: During the funding period we have characterized the role of phosphatidylinositol-containing lipids in virulence, and the role of PI 3-kinase signaling in drug resistance. We published a paper in a high-impact journal Eukaryotic Cell. Summary of project published to be published in Eukaryotic Cell: Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated, in part, by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were over-expressing genes from a cDNA library, with a near-lethal dose of the PI3K inhibitor, wortmannin. This screen was based on the rationale that survivors would be over-expressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the over-expressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells over-expressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of PIP3, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen. This study was published in Eukaryotic Cell in March 2014 and was featured by the editors as a publication of significance. During the reporting period we were also awarded an NIH grant (1R03AI107950) to characterize the stress response in E. histolytica. During the course of infection in the host, E. histolytica likely confronts stress brought on by ever-changing environments in the host. To survive, the parasite must circumvent these exogenous pressures. Thus, it may be useful to target E. histolytica's response to stress for therapy. In other parasites, stress can activate eIF2α kinases that phosphorylate the α-subunit of eukaryotic initiation factor-2 (eIF2α). eIF2α is part of a protein complex that delivers Met-tRNA to ribosomes for translation initiation. Phosphorylation of eIF2α inhibits this activity which, in turn, leads to a decline in protein synthesis. This allows cells to conserve resources and reconfigure gene expression to effectively manage stress. Genome analyses indicate that E. histolytica possesses all of the components of this stress response system; however, the premise that phospho-eIF2α controls stress and translation in this pathogen has not been tested. This represents a profound gap in knowledge. Thus, the goal of this proposal is to characterize this stress response system in E. histolytica. Specifically, we will define the function of phospho-eIF2α. This involves tracking phospho-eIF2α during a variety of stressful conditions. During the reporting period we generated specific antibody that recognizes phospho-eIF2α and total eIF2α and monitored the phospho-form during two different stress conditions (serum-starvation and heat shock). While serum-starvation induces phosphorylation of eIF2α heat shock does not. This suggests that this cellular response system is stress-specific.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Koushik, A.B., Welter, B.H., Rock, M.L., Temesvari, L.A. 2014. A genomewide overexpression screen identifies genes involved in the phosphatidylinositol 3-kinase pathway in the human protozoan parasite Entamoeba histolytica. Eukaryot. Cell 13:401-411.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The target audience are those scientists whose research may be classified as parasitology, cellular signal transduction, and/or lipid biology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Ms. Brenda Welter, a Research Associate in my group, is integral to the project as evidenced by her authorship on our 2013 and 2014 publications. Over the past year, we have adopted a new technique in our lab, polyribosome profiling. Ms. Welter conducted literature searches and adapted the protocol to our needs. As such, her repertoire of skills has increased. Two graduate students, Drs. Amanda Goldston and Amrita Koushik, were involved in the project over the previous funding period. Both of these students graduated during the past funding period. Two undergraduate students joined my research group in the last funding period.: Ms. Michelle Rock and Ms. Lindsey Stegall. They are being trained in a variety of biochemical, molecular and cellular techniques. Ms. Rock has just been accepted to the MS graduate program and will begin in the Fall 2014. How have the results been disseminated to communities of interest? The results of a subset of our studies related to our cell signaling project were published in the journal, Infection and Immunity. A second manuscript describing our gene discovery work was submitted to the journal, Eukaryotic Cell. It has been accepted and will be published in the next funding period. A review paper for the journal, Eukaryotic Cell, is in preparation. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period we plan to focus on the following milestones: (i) Gain insight into the role of post-translational modification in receptor-raft interactions and (ii) Gain insight into the role of protein domains in receptor-raft interactions. To this end we have generated 2 mutant cell lines in the laboratory. In the first cell line, expression of a key enzyme in the biosynthesis of GPI-anchors, an important post-translation modification, has been reduced. Characterizaiton of this cell line will reveal the role for GPI anchors in receptor-raft interactions. In the second cell line, a truncated cell surface receptor is being expressed. Characterization of this cell line will tell us the role of the ectodomain of this receptor in receptor-raft interactions. Successful completion of these goals will greatly advance the field as they will provide significant insight into E. histolytica virulence.
Impacts
What was accomplished under these goals?
Grant Submissions: We submitted an NIH R03 proposal to fund our research. We received a fundable score and we are awaiting a notice of award. We also submitted an NIH COBRE proposal. This is a $7.5 million center grant to fund the research of 5 investigators. This proposal was scored and is still pending. Summary of a project published in Infection and Immunity: Entamoeba histolytica is an intestinal protozoan parasite and is the causative agent of amoebiasis. During invasive infection, highly motile amoebae destroy the colonic epithelium, enter the blood circulation, and disseminate to other organs such as liver, causing liver abscess. Motility is a key factor in E. histolytica pathogenesis, and this process relies on a dynamic actomyosin cytoskeleton. In other systems, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is known to regulate a wide variety of cellular functions, including signal transduction, actin remodeling, and cell motility. Little is known about the role of PI(4,5)P2 in E. histolytica pathogenicity. In this study, we demonstrate that PI(4,5)P2 is localized to cholesterol-rich microdomains, lipid rafts, and the actin-rich fractions of the E. histolytica membrane. Microscopy revealed that the trailing edge of polarized trophozoites, uroids, are highly enriched in lipid rafts and their constituent lipid, PI(4,5)P2. Polarization and enrichment of uroids and rafts with PI(4,5)P2 were enhanced upon treatment of E. histolytica cells with cholesterol. Exposure to cholesterol also increased intracellular calcium, which is a downstream effector of PI(4,5)P2, with a concomitant increase in motility. Together, our data suggest that in E. histolytica, PI(4,5)P2 may signal from lipid rafts and cholesterol may play a role in triggering PI(4,5)P2-mediated signaling to enhance the motility of this pathogen. Summary of project to be published in Eukaryotic Cell: Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated, in part, by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were over-expressing genes from a cDNA library, with a near-lethal dose of the PI3K inhibitor, wortmannin. This screen was based on the rationale that survivors would be over-expressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the over-expressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells over-expressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of PIP3, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Koushik, A.B., Powell, R.R., and Temesvari, L.A. Localization of phosphatidylinositol 4,5-bisphosphate to lipid rafts and uroids in the human protozoan parasite, Entamoeba histolytica. Infect Immun. 81(6):2145.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: During the funding period we continued to carry out research to gain insight into the cell and molecular mechanisms regulating virulence in the human protozoan parasite, Entamoeba histolytica. This food-borne pathogen is the causative agent of amoebic dysentery and ranks second in the world for deaths due to parasitic disease. In particular, we have characterized the role of cholesterol and phosphatidylinositol-containing lipids in virulence, and the role of PI 3-kinase signaling in drug resistance. We have submitted NIH R03 and R21 grant proposals to fund our research. These proposals are pending review at the NIH. Dissemination: The results of our studies related to our gene discovery project were published in PLoS ONE. The results of our studies on the role of host cell components in the submembrane distribution of the Gal/GalNAc lectin were published in Eukaryotic Cell. It was subsequently awarded Editor's Choice Recognition by the journal editorial board. We also published a review article in Trends in Parasitology on lipid rafts in parasites. The results of our studies were also presented at 4 international conferences. The results of our studies on the relationships between phosphatidylinositol, cholesterol and virulence of E. histolytica were recently submitted as a manuscript to Infection and Immunity. Our findings provide significant insight into the cell biology of this parasite. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Summary of a project published in PLoS ONE: Entamoeba histolytica is a protozoan parasite for which forward genetics approaches have not been extensively applied. Given that the E. histolytica genome has been sequenced, it should be possible to apply genomic approaches to discover gene function. We used a genome-wide over-expression screen to uncover genes regulating an important virulence function of E. histolytica, namely phagocytosis. We developed an episomal E. histolytica cDNA over-expression library, transfected the collection of plasmids into trophozoites, and applied a high-throughput screen to identify phagocytosis mutants in the population of over-expressing cells. The screen was based on the phagocytic uptake of human red blood cells loaded with the metabolic toxin, tubercidin. Expression plasmids were isolated from trophozoites that survived exposure to tubercidin-charged erythrocytes (phagocytosis mutants), and the cDNAs were sequenced. We isolated the gene encoding profilin, a well-characterized cytoskeleton-regulating protein with a known role in phagocytosis. This supports the validity of our approach. We assigned a phagocytic role to several genes not previously known to function in this manner. To our knowledge, this is the first genome-wide forward genetics screen to be applied to this pathogen. The study demonstrates the power of forward genetics in revealing genes regulating virulence in E. histolytica. The study validates an E. histolytica cDNA over-expression library as a valuable tool for functional genomics. Summary of project published in Eukaryotic Cell: Entamoeba histolytica cell surface receptors, such as the Gal/GalNAc lectin, facilitate attachment to host cells and extracellular matrix. The Gal/GalNAc lectin binds to galactose or N-acetylgalactosamine residues on host components and is composed of heavy (Hgl), intermediate (Igl), and light (Lgl) subunits. Although Igl is constitutively localized to lipid rafts (cholesterol-rich membrane domains), Hgl and Lgl transiently associate with this compartment in a cholesterol-dependent fashion. In this study, trophozoites were exposed to biologically relevant ligands to determine if ligand binding influences the submembrane distribution of the subunits. Exposure to human red blood cells (hRBCs) or collagen was correlated with enrichment of Hgl and Lgl in rafts. This enrichment was abrogated in the presence of galactose, suggesting that direct lectin-ligand interactions are necessary to influence subunit location. Using a cell line that is able to attach to, but not phagocytose, hRBCs, it was shown that physical attachment to ligands was not sufficient to induce the enrichment of lectin subunits in rafts. Finally, intracellular calcium levels increased upon attachment to collagen; this increase was essential for the enrichment of lectin subunits in rafts. Together, these data provide evidence that ligand-induced enrichment of lectin subunits in rafts may be the first step in a signaling pathway that involves both PIP(2) and calcium signaling.
Publications
- King, A.V., Welter, B.H., Koushik, A.B., and Temesvari, L.A. (2012). A Genome-Wide Over-Expression Screen Identifies Genes Involved in Phagocytosis in the Human Protozoan Parasite, Entamoeba histolytica, PloS ONE 7, e43025.
- Goldston A.M., Powell R.R., Koushik A.B., and Temesvari L.A. (2012).Exposure to Host Ligands Correlates with Co-localization of Gal/GalNAc Lectin Subunits in Lipid Rafts and PIP2 Signaling in Entamoeba histolytica. Eukaryot. Cell. 11, 743-751.
- Goldston, A. M., Powell, R.R., and Temesvari, L.A. (2012) Sink or swim: lipid rafts in parasite pathogenesis. Trends parasitol. 28, 417-426.
- ABSTRACT-Goldston, A.M., Koushik, A.B., Welter, B.H., Powell, R.R., and Temesvari, L.A. (2012) Lipid Rafts and Virulence in Entamoeba histolytica. Presented at the International Conference on Anaerobic Protists. (September 2012, Los Angeles, CA).
- ABSTRACT-A. B. Koushik, R. R. Powell, A. M. Goldston, B. H. Welter, L. A. Temesvari. (2012) Effect of an Extracellular Cholesterol Source on Localization of Phosphatidylinositol-4,5- bisphosphate and Virulence Functions in Entamoeba histolytica. Presented at the 112th General Meeting of the American Society for Microbiology (San Francisco, CA).
- ABSTRACT-Lee, J.S., Reeder, W., Lau, J., Reed, T., and Temesvari, L.A. (2012)Effect of Colon-specific Prodrugs on the amoeba-host cell interaction. Presented at the Biomaterials and Engineering Sciences Annual meeting, Atlanta, GA.
- ABSTRACT-Goldston, A., Koushik, A., Powell, R., Temesvari, L.A. (2012) Exposure to Host Ligands Correlates with Co-localization of Gal/GalNAc Lectin Subunits in Lipid Rafts and Phosphatidylinositol Signaling in Entamoeba histolytica. Presented at the International Parasitology Meeting. University of Georgia, Athens, GA.
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