The protozoan Entamoeba histolytica causes an estimated 50 million cases of invasive amebiasis and 40,000 to 110,000 deaths each year. The factors that control expression of virulence genes and/or promote invasive rather than commensal infection are not understood, and there is no means to prevent invasive disease. We will focus on the proteins involved in the regulation of expression of the hgl5 virulence determinant. The hgl5 gene is one of 5 genes encoding the heavy subunit of an adherence lectin required for liver abscess formation in animals. Our hypothesis is that: DNA sequence-specific binding proteins recognize upstream regulatory elements (UREs) in the hgl5 promoter, thereby controlling expression of hgl5 mRNA. Preliminary evidence (from the prior 2 1/2 years of support) is consistent with hgl5 being coordinately regulated by transcriptional activators and repressors that bind to five distinct UREs in the 5' upstream promoter. The five UREs are novel, in so far as their DNA sequences are different from those recognized by other known eukaryotic transcription factors. We will focus on URE3 and URE4 because: (1) their mutation has the greatest effect on hgl5 expression; (2) their effects on hgl5 promoter function are opposite (URE4 is a positive, and URE3 a negative, cis-acting sequence), and (3) their sequence motifs are known to be present in other E. histolytica promoters. The proteins that bind to URE3 and URE4 will be characterized. Their physiologic function in controlling expression of hgl5 will be investigated. We will also examine if these proteins regulate expression of other genes. Completion of these studies will yield the first characterization of E. histolytica transcription activators and repressors regulating expression of a virulence factor. These investigations promise new approaches to the treatment or prevention of invasive amebiasis by interfering with expression of hgl5.
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