scholarly journals A rhomboid protease, EhROM1, regulates the submembrane distribution and size of the Gal/GalNAc lectin subunits in the human protozoan parasite, Entamoeba histolytica

2019 ◽  
Author(s):  
Brenda H. Welter ◽  
Lesly A. Temesvari
Keyword(s):  
Cell Surface ◽  
Entamoeba Histolytica ◽  
Molecular Mechanisms ◽  
Mutant Cell ◽  
Protozoan Parasite ◽  
Rhomboid Protease ◽  
Host Interactions ◽  
Rhomboid Proteases ◽  
Lipid Environment ◽  
Insight Into

AbstractEntamoeba histolytica is a food- and waterborne parasite that is the causative agent of amebic dysentery and amoebic liver abscesses. Adhesion is one of the most important virulence functions as it facilitates motility, colonization of host, destruction of host tissue, and uptake of nutrients by the parasite. One well-characterized parasite cell surface adhesin is the Gal/GalNAc lectin, which binds to galactose or N-acetylgalactosamine residues on host components and is composed of heavy (Hgl), intermediate (Igl), and light (Lgl) subunits. Igl has been shown to be constitutively localized to lipid rafts (cholesterol-rich membrane domains), whereas Hgl and Lgl transiently associate with rafts. When all three subunits are localized to rafts there is an increase in galactose-sensitive adhesion. Thus, submembrane location may regulate the function of this adhesion. Rhomboid proteases are a conserved family of intramembrane proteases that also participate in the regulation of parasite-host interactions. In E. histolytica, one rhomboid protease, EhROM1, cleaves Hgl as a substrate, and knockdown of its expression inhibits parasite-host interactions. Since rhomboid proteases are found within membranes, it is not surprising that lipid composition regulates their activity and enzyme-substrate binding. Given the importance of the lipid environment for both rhomboid proteases and the Gal/GalNAc lectin, we sought to gain insight into the relationship between rhomboid proteases and submembrane location of the lectin in E. histolytica. We demonstrated that EhROM1, itself, is enriched in rafts. Reducing rhomboid protease activity, either pharmacologically or genetically, correlated with an enrichment of Hgl and Lgl in rafts. In a mutant cell line with reduced EhROM1 expression, there was also a significant augmentation of the level of all three Gal/GalNAc subunits on the cell surface and an increase in the molecular weight of Hgl and Lgl. Overall, the study provides insight into the molecular mechanisms governing parasite-host adhesion for this pathogen.

scholarly journals Network-based Virus-Host Interaction Prediction with Application to SARS-CoV-2

2020 ◽  
Author(s):  
Hangyu Du ◽  
Feng Chen ◽  
Hongfu Liu ◽  
Pengyu Hong
Keyword(s):  
Molecular Mechanisms ◽  
Interaction Network ◽  
Spectrum Prediction ◽  
Health Crisis ◽  
Host Interactions ◽  
Multi Scale ◽  
Host Interaction ◽  
Human Proteins ◽  
Potential Risks ◽  
Insight Into

COVID-19, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has quickly become a global health crisis since the first report of infection in December of 2019. However, the infection spectrum of SARS-CoV-2 and its comprehensive protein-level interactions with hosts remain unclear. There is a massive amount of under-utilized data and knowledge about RNA viruses highly relevant to SARS-CoV-2 and their hosts’ proteins. More in-depth and more comprehensive analyses of that knowledge and data can shed new insight into the molecular mechanisms underlying the COVID-19 pandemic and reveal potential risks. In this work, we constructed a multi-layer virus-host interaction network to incorporate these data and knowledge. A machine learning-based method, termed Infection Mechanism and Spectrum Prediction (IMSP), was developed to predict virus-host interactions at both protein and organism levels. Our approach revealed five potential infection targets of SARS-CoV-2, which deserved public health attention, and eight highly possible interactions between SARS-CoV-2 proteins and human proteins. Given a new virus, IMSP can utilize existing knowledge and data about other highly relevant viruses to predict multi-scale interactions between the new virus and potential hosts.

scholarly journals A Genomewide Overexpression Screen Identifies Genes Involved in the Phosphatidylinositol 3-Kinase Pathway in the Human Protozoan Parasite Entamoeba histolytica

2014 ◽  
Vol 13 (3) ◽  
pp. 401-411 ◽  
Author(s):  
Amrita B. Koushik ◽  
Brenda H. Welter ◽  
Michelle L. Rock ◽  
Lesly A. Temesvari
Keyword(s):  
Entamoeba Histolytica ◽  
Mutant Cell ◽  
Lethal Dose ◽  
Protozoan Parasite ◽  
Pi3k Pathway ◽  
Forward Genetics ◽  
Pi3k Inhibitor ◽  
Phosphatidylinositol 3 Kinase ◽  
Content Type ◽  
Phosphatidylinositol 3

ABSTRACTEntamoeba histolyticais a protozoan parasite that causes amoebic dysentery and liver abscess.E. histolyticarelies 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 inE. histolytica. We treated a population of trophozoites that were overexpressing 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 overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed 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-richE. histolyticaprotein (SREHP) were also identified. Cells overexpressing 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 phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K inE. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen.

scholarly journals An internally quenched peptide as a new model substrate for rhomboid intramembrane proteases

2018 ◽  
Vol 399 (12) ◽  
pp. 1389-1397 ◽  
Author(s):  
Elena Arutyunova ◽  
Zhenze Jiang ◽  
Jian Yang ◽  
Ayodeji N. Kulepa ◽  
Howard S. Young ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Catalytic Mechanism ◽  
Mass Spectrometry Analysis ◽  
Aqueous Environment ◽  
Growth Factor Signaling ◽  
Rhomboid Protease ◽  
Spectrometry Analysis ◽  
Intramembrane Proteases ◽  
Rhomboid Proteases ◽  
Lipid Environment

AbstractRhomboids are ubiquitous intramembrane serine proteases that cleave transmembrane substrates. Their functions include growth factor signaling, mitochondrial homeostasis, and parasite invasion. A recent study revealed that theEscherichia colirhomboid protease EcGlpG is essential for its extraintestinal pathogenic colonization within the gut. Crystal structures of EcGlpG and theHaemophilus influenzaerhomboid protease HiGlpG have deciphered an active site that is buried within the lipid bilayer but exposed to the aqueous environment via a cavity at the periplasmic face. A lack of physiological transmembrane substrates has hampered progression for understanding their catalytic mechanism and screening inhibitor libraries. To identify a soluble substrate for use in the study of rhomboid proteases, an array of internally quenched peptides were assayed with HiGlpG, EcGlpG and PsAarA fromProvidencia stuartti. One substrate was identified that was cleaved by all three rhomboid proteases, with HiGlpG having the highest cleavage efficiency. Mass spectrometry analysis determined that all enzymes hydrolyze this substrate between norvaline and tryptophan. Kinetic analysis in both detergent and bicellular systems demonstrated that this substrate can be cleaved in solution and in the lipid environment. The substrate was subsequently used to screen a panel of benzoxazin-4-one inhibitors to validate its use in inhibitor discovery.

scholarly journals Insights into the catalytic properties of the mitochondrial rhomboid protease PARL

2020 ◽  
Author(s):  
Laine Lysyk ◽  
Raelynn Brassard ◽  
Elena Arutyunova ◽  
Verena Siebert ◽  
Zhenze Jiang ◽  
...  
Keyword(s):  
Catalytic Properties ◽  
Side Chain ◽  
Turnover Rates ◽  
Rhomboid Protease ◽  
Mitochondrial Homeostasis ◽  
Substrate Preferences ◽  
Rhomboid Proteases ◽  
Hydrolysis Of ◽  
Insight Into

AbstractThe rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac, which have crucial roles in mitochondrial quality control and apoptosis. To gain insight into the catalytic properties of the PARL protease, we expressed human PARL in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show PARL activity in detergent is enhanced by cardiolipin. Significantly higher turnover rates are observed for PARL reconstituted in proteoliposomes, with Smac being cleaved most rapidly at a rate of 1 min−1. PGAM5 is cleaved with the highest efficiency compared to PINK1 and Smac. In proteoliposomes, a truncated β-cleavage form of PARL is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5 and Smac. Multiplex substrate profiling reveals a substrate preference for PARL with a bulky side chain Phe in P1, which is distinct from small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences.

Cellular and molecular mechanisms that underlie Entamoeba histolytica pathogenesis: prospects for intervention

2005 ◽  
Vol 7 (13) ◽  
pp. 1-19 ◽  
Author(s):  
Richard C. Laughlin ◽  
Lesly A. Temesvari
Keyword(s):  
Entamoeba Histolytica ◽  
Molecular Mechanisms ◽  
Parasitic Infection ◽  
Protozoan Parasite ◽  
Disease Spread ◽  
Cellular Mechanisms ◽  
Cellular Processes ◽  
Treatment And Prevention ◽  
Histolytica Infection ◽  
Molecular Machinery

The protozoan parasite Entamoeba histolytica is the causative agent of amoebic dysentery. It is prevalent in developing countries that cannot prevent its fecal–oral spread and ranks second in worldwide causes of morbidity by parasitic infection. Improvements in sanitation would help curb disease spread. However, a lack of significant progress in this area has resulted in the need for a better understanding of the molecular and cellular biology of pathogenesis in order to design novel methods of disease treatment and prevention. Recent insight into the cellular mechanisms regulating virulence of E. histolytica has indicated that processes such as endocytosis, secretion, host cell adhesion and encystation play major roles in the infectious process. This review focuses on components of the molecular machinery that govern these cellular processes and their role in virulence, and discusses how an understanding of this might reveal opportunities to interfere with E. histolytica infection.

scholarly journals Downregulation of an Entamoeba histolytica Rhomboid Protease Reveals Roles in Regulating Parasite Adhesion and Phagocytosis

2010 ◽  
Vol 9 (8) ◽  
pp. 1283-1293 ◽  
Author(s):  
Leigh A. Baxt ◽  
Elena Rastew ◽  
Rivka Bracha ◽  
David Mirelman ◽  
Upinder Singh
Keyword(s):  
Cell Adhesion ◽  
Host Cell ◽  
Entamoeba Histolytica ◽  
Apoptotic Cells ◽  
Content Type ◽  
Rhomboid Protease ◽  
Phagocytic Ability ◽  
Complement Resistance ◽  
Rhomboid Proteases ◽  
Surface Receptor

ABSTRACT Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.

scholarly journals Reduction of Cell Surface Glycosylphosphatidylinositol Conjugates in Entamoeba histolytica by Antisense Blocking of E. histolytica GlcNAc-Phosphatidylinositol Deacetylase Expression: Effect on Cell Proliferation, Endocytosis, and Adhesion to Target Cells

2005 ◽  
Vol 73 (12) ◽  
pp. 8381-8392 ◽  
Author(s):  
Divya Vats ◽  
Ram A. Vishwakarma ◽  
Sudha Bhattacharya ◽  
Alok Bhattacharya
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Entamoeba Histolytica ◽  
Antisense Rna ◽  
Fluid Phase ◽  
Protozoan Parasite ◽  
Target Cells ◽  
Bioinformatic Tools ◽  
Fluid Phase Endocytosis ◽  
Sense Orientation

ABSTRACT Glycosylphosphatidylinositol (GPI)-anchored molecules such as cell surface Gal/GalNAc lectin and proteophosphoglycans of the protozoan parasite Entamoeba histolytica are thought to be involved in pathogenesis. Here, we report the identification of genes that may be involved in the GPI biosynthetic pathway of E. histolytica by use of bioinformatic tools applied to the recently published genome sequence. Of the genes identified, one of the early genes, GlcNAc-phosphatidylinositol deacetylase (PIG-L), was partially characterized. Cell lines deficient in E. histolytica PIG-L (EhPL-AS) or overproducing it (EhPL-S) were generated by expressing the gene in the antisense or sense orientation, respectively, in a tetracycline-inducible system. The overexpressing cells showed higher EhPIG-L activity and increased production of GlcN-PI. Conversely, cells expressing the antisense RNA displayed reduced GlcN-PI production. The total number of GPI-containing molecules was also reduced in these cells, as demonstrated by Alexa 488 fluorescently labeled proaerolysin labeling. The distribution of GPI-linked PPG and Gal/GalNAc lectin was altered in the tetracycline-induced EhPL-AS cell lines. Further, the antisense-blocked cells showed 36% suppression of cell growth, 50 to 60% inhibition of fluid phase endocytosis, and about 50% inhibition of adhesion to target cells. Therefore, our data suggest the importance of GPI anchors in regulating some of the events in amoebic pathogenesis. They also demonstrated the use of antisense RNA-mediated inhibition of GPI biosynthetic enzymes as an approach to decrease the amount of GPI conjugates in E. histolytica.

Scanning electron microscopy of erythrophagocytosis by Entamoeba histolytica trophozoites

1992 ◽  
Vol 50 (1) ◽  
pp. 880-881
Author(s):  
Victor Tsutsumi ◽  
Adolfo Martinez-Palomo ◽  
Kyuichi Tanikawa
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Red Blood Cells ◽  
Entamoeba Histolytica ◽  
Causative Agent ◽  
Blood Cells ◽  
Protozoan Parasite ◽  
Complex Phenomenon ◽  
Great Capacity ◽  
Scanning Electron

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis in man. The trophozoite or motile form is a highly dynamic and pleomorphic cell with a great capacity to destroy tissues. Moreover, the parasite has the singular ability to phagocytize a variety of different live or death cells. Phagocytosis of red blood cells by E. histolytica trophozoites is a complex phenomenon related with amebic pathogenicity and nutrition.

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