title: 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.
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.
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi-110067, India. alok0200@mail.jnu.ac.in.
authors.forename: Divya
authors.initials: D
authors.lastname: Vats
authors.forename: Ram A
authors.initials: RA
authors.lastname: Vishwakarma
authors.forename: Sudha
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: Alok
authors.initials: A
authors.lastname: Bhattacharya
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D049109:Cell Proliferation; D004705:Endocytosis / Q000235:genetics; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D017125:Genes, Protozoan; D018503:Genome, Protozoan; D017261:Glycosylphosphatidylinositols / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016372:RNA, Antisense / Q000235:genetics / Q000494:pharmacology; D022161:Transport Vesicles / Q000201:enzymology
chemical_list: D017261:Glycosylphosphatidylinositols; D015800:Protozoan Proteins; D016372:RNA, Antisense; D000581:Amidohydrolases; C087696:N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase