Entamoeba histolytica expressing a dominant negative N-truncated light subunit of its gal-lectin are less virulent.
The 260-kDa heterodimeric Gal/GalNAc-specific Lectin (Gal-lectin) of Entamoeba histolytica dissociates under reducing conditions into a heavy (hgl, 170 kDa) and a light subunit (lgl, 35 kDa). We have previously shown that inhibition of expression of the 35-kDa subunit by antisense RNA causes a decrease in virulence. To further understand the role of the light subunit of the Gal-lectin in pathogenesis, amoebae were transfected with plasmids encoding intact, mutated, and truncated forms of the light subunit lgl1 gene. A transfectant in which the 55 N-terminal amino acids of the lgl were removed, overproduced an N-truncated lgl protein (32 kDa), which replaced most of the native 35-kDa lgl in the formation of the Gal-lectin heterodimeric complex and exerted a dominant negative effect. Amoebae transfected with this construct showed a significant decrease in their ability to adhere to and kill mammalian cells as well as in their capacity to form rosettes with and to phagocytose erythrocytes. In addition, immunofluorescence confocal microscopy of this transfectant with anti-Gal-lectin antibodies showed an impaired ability to cap. These results indicate that the light subunit has a role in enabling the clustering of Gal-lectin complexes and that its N-truncation affects this function, which is required for virulence.
Katz
Uriel
U
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Ankri
Serge
S
Stolarsky
Tamara
T
Nuchamowitz
Yael
Y
Mirelman
David
D
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002448:Cell Adhesion; D002460:Cell Line; D002469:Cell Separation; D002846:Chromatography, Affinity; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000235:genetics* / Q000378:metabolism*; D005434:Flow Cytometry; D015723:Gene Library; D005799:Genes, Dominant; D005822:Genetic Vectors; D037102:Lectins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010587:Phagocytosis; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; C434499:Igl1 protein, Entamoeba histolytica; D037102:Lectins; D015800:Protozoan Proteins; D012333:RNA, Messenger