The unicellular eukaryote Entamoeba histolytica is a human parasite that causes amebic dysentery and liver abscess. A genome-wide analysis of gene expression modulated by intestinal colonization and invasion identified an upregulated transcript that encoded a putative high-mobility-group box (HMGB) protein, EhHMGB1. We tested if EhHMGB1 encoded a functional HMGB protein and determined its role in control of parasite gene expression. Recombinant EhHMGB1 was able to bend DNA in vitro, a characteristic of HMGB proteins. Core conserved residues required for DNA bending activity in other HMGB proteins were demonstrated by mutational analysis to be essential for EhHMGB1 activity. EhHMGB1 was also able to enhance the binding of human p53 to its cognate DNA sequence in vitro, which is expected for an HMGB1 protein. Confocal microscopy, using antibodies against the recombinant protein, confirmed its nuclear localization. Overexpression of EhHMGB1 in HM1:IMSS trophozoites led to modulation of 33 transcripts involved in a variety of cellular functions. Of these, 20 were also modulated at either day 1 or day 29 in the mouse model of intestinal amebiasis. Notably, four transcripts with known roles in virulence, including two encoding Gal/GalNAc lectin light chains, were modulated in response to EhHMGB1 overexpression. We concluded that EhHMGB1 was a bona fide HMGB protein with the capacity to recapitulate part of the modulation of parasite gene expression seen during adaptation to the host intestine.Entamoeba histolytica is the causative agent of amebiasis and prevails in areas of poor sanitation. The organism is estimated to be responsible for 50 million infections and 100,000 deaths each year. Infection can lead to amebic dysentery, resulting from trophozoites invading the intestinal wall. Amebic liver abscess and other extraintestinal lesions can result through the spread of trophozoites from the intestine into the bloodstream. The host and parasite factors determining infection outcome have yet to be fully elucidated.We hypothesize that alteration in transcription of certain crucial genes may contribute to the expression of the virulence phenotype. Distinct patterns of E. histolytica gene expression have been observed under a variety of experimental conditions (3,6,14,18,19,23,24,27,33,43,44,67). Previously we catalogued the gene expression profile of E. histolytica associated with amebic colitis in the murine model (27). mRNA transcripts more abundant in vivo (mouse colon) than in vitro (laboratory culture) included putative DNA/RNA regulatory factors, representing a pool of potential phenotype-specific transcription factors. The transcript of the locus XM_652200/EHI_093800 was upregulated more than twofold at day 1 of amebic colitis. This gene showed significant sequence similarity to the high-mobility-group box chromosomal protein 1 (HMGB1).HMGB1 is an abundant nonhistone nuclear protein and is a member of the HMG superfamily. The protein is highly conserved in all metazoans, plants, and yeasts and in the parasites Tr...