Transcriptional Gene Silencing Reveals Two Distinct Groups of Entamoeba histolytica Gal/GalNAc-Lectin Light Subunits

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Abstract
The Entamoeba histolytica cell surface Gal/GalNAc-inhibitable lectin is a heterodimer between a heavy (170 kDa) subunit linked via a disulfide bond to a light (31 to 35 kDa) subunit. Five light subunit genes with high homology have been identified (Eh lgl1 to - 5 ). We have previously shown that silencing of the expression of Eh lgl1 , in the G3 trophozoites which had already been silenced in the amoebapore gene (Eh ap-a ), also suppressed the transcription of Eh lgl2 and - 3 (strain RBV). The total absence of the lgl1 to -3 subunits in the RBV trophozoites affected their ability to cap the surface Gal-lectin molecules to the uroid region. We have now found that in the RBV trophozoites, the lgl4 and -5 subunits (31 kDa) are overexpressed and appear to compensate for the missing lgl1 to -3 in the heterodimer complex. Transcriptional silencing of Eh lgl5 was achieved by transfection of G3 trophozoites with a plasmid containing the open reading frame of Eh lgl5 ligated to the 5′ promoter region of the Eh ap-a gene. The transfected trophozoites (strain L5) were silenced in Eh lgl5 and the closely related Eh lgl4 , while the expression of the larger lgl1 to -3 subunits was upregulated. L5 trophozoites retained their ability to cap the Gal-lectin molecules. Attempts to simultaneously silence all of the Eh lgl genes have failed so far, possibly due to their crucial importance to the Gal-lectin functions. Our ability to silence part of the genes belonging to the same family can serve as a tool to study the relationships and functions of the members of other gene families.

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