Premium
Expression and Functional Characterization of Endosomal Nucleases of the Human Enteric Pathogen, Entamoeba histolytica
Author(s) -
McGugan Glen Chapman,
Joshi Manju B,
Dwyer Dennis M
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a277-c
Subject(s) - entamoeba histolytica , biology , gene , endosome , entamoeba , nuclease , pathogen , microbiology and biotechnology , genetics , intracellular
The protozoan parasite, Entamoeba histolytica , is the causative agent of potentially fatal amebic abscesses in humans. These parasites are totally dependent on their human host for nutrients, including preformed nucleotides, as they are incapable of synthesizing either purines or pyrimidines de novo . Presumably, Entamoeba accesses host nucleotides using its endogenous nucleases. Following analysis of the E. histolytica genome database, we identified several nuclease genes, some of which appear to code for endosomal or secretory enzymes. A PCR‐based approach was used to clone and sequence three of these genes, designated EhRNuc1 , EhRNuc2 , and EnRNuc3 . Using RT‐PCR, we demonstrated that Entamoeba expressed each of these genes. To further characterize the products of these genes, epitope‐tagged constructs were episomally expressed in Entamoeba and found to localize in endosomes by immunofluorescence microscopy. All three nucleases were DTT‐sensitive and cleaved polyA, polyI, polyC, polyU, and ssRNA, but showed no activity with polyG, ssDNA, or dsDNA. In addition, these enzymes showed higher activity at acidic pH, a property consistent with endosomal enzymes. Presumably, these nucleases are critical for the survival of this pathogen, and may provide new chemotherapeutic targets. This research was supported by the intramural research program of the DIR, NIAID, NIH.