Cloning and expression of a membrane antigen of Entamoeba histolytica possessing multiple tandem repeats.

Abstract

Entamoeba histolytica causes amebic dysentery and amebic liver abscess, major causes of morbidity and mortality worldwide. We have used differential hybridization screening to isolate an E. histolytica-speciflic cDNA clone. The cDNA was found to encode a serine-rich E. histolytica protein (SREHP) containing multiple tandem repeats. The structural motif of SREHP resembles some of the repetitive antigens of malarial species, especially the circumsporozoite proteins. A recombinant trpE fusion protein containing the tandem repeats of SREHP was recognized by immune serum from a patient with amebiasis, demonstrating that SREHP is a naturally immunogenic protein. An antiserum raised against the recombinant fusion protein specifically bound to two distinct bands with apparent molecular masses of 46 and 52 kDa in a crude preparation of E. histolytica trophozoite membranes. This antiserum also inhibited E. histolytica trophozoite adhesion to Chinese hamster ovary cells in vitro. The ability to isolate E. histolytica-specific genes, and to express those genes in Escherichia coli, may be important in studying the molecular basis ofE. histolytica pathogenesis and for the future development of vaccines.The protozoan pathogen Entamoeba histolytica is a major cause of debilitating illness and death worldwide, infecting 500 million people and causing an estimated 50 million cases of diarrhea and 50,000 deaths yearly (1). There is an urgent need for a vaccine that could prevent the establishment of E. histolytica infection or the development of invasive disease. Studies in animal models have demonstrated that immunity to E. histolytica infection can be produced by immunization with E. histolytica lysates (2)(3)(4). However, the difficulty in obtaining large quantities of trophozoites, and the relatively crude nature of the immunizing preparations, have severely limited the scope of these studies. To approach this problem, we isolated genes expressed in a pathogenic strain ofE. histolytica but not in the nonpathogenic E. histolytica-like Laredo strain.

The Most Abundant Glycoprotein of Amebic Cyst Walls (Jacob) Is a Lectin with Five Cys-Rich, Chitin-Binding Domains

“…Jacob repeat domains also contained conserved aromatic amino acids (Tyr, Phe, and Trp), which may be involved in binding sugars, as described for WGA (52). Between the Jacob repeat domains were acidic domains, which were similar in their composition but not their sequence to repeats in amebic chitinases, Ser-rich E. histolytica proteins, or Ariel surface protein (11,28,41). There were no proteins homologous to amebic Jacob in GenBank, EST, or unfinished microbial genome databases, although BLAST with Jacob identified the large subunit of the E. histolytica Gal/GalNAc lectin and Gi- ardia lamblia variable surface proteins, which are also Cys rich (1,29,33,44).…”

A severe combined immunodeficient (SCID) mouse model for infection with Entamoeba histolytica.

“…Immune serum was obtained from a rabbit vaccinated with HMI:IMSS trophozoites (18), and stored at -20~ until use. SCID mice were injected intraperitoneally with 300 #1 immune rabbit serum or an equivalent amount ofpreimmune serum can be seen in areas of necrosis within the abscess (A).…”

Isolation and Characterization of Polymorphic DNA from Entamoeba histolytica

“…At present, the most polymorphic gene of E. histolytica is that encoding the serine-rich E. histolytica protein (SREHP or K2) a surface antigen with tandem 8-and 12-amino-acid repeats (17,28). Repeat number, sequence, and restriction enzyme site polymorphisms have been reported among different E. histolytica isolates (8,14).…”