Characterization of an Entamoeba histolytica High-Mobility-Group Box Protein Induced during Intestinal Infection

Abstract

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...

Using differential gene expression to study Entamoeba histolytica pathogenesis

“…Of these, 20 were also modulated in the mouse model of intestinal amebiasis at either day 1 or day 29 (when EhHMGB1 transcripts were increased sixfold). However, this change, which was possibly due to a dominant-negative effect from the overexpression of EhHMGB1 (at least 100-fold over the basal level), was in the converse direction of the change in vivo [30]. …”

The R2R3 Myb protein family in Entamoeba histolytica

“…For instance, DNA-binding proteins that recognize specific sequences in promoter regions have been identified and characterized in E. histolytica. These include the TATA-binding protein (TBP) (Luna-Arias et al, 1999), the upstream regulatory element 3 binding protein (URE3-BP) (Gilchrist et al, 2001), the enhancer binding proteins 1 and 2 (EhEBP1 and EhEBP2) (Schaenman et al, 2001), a p53-like protein (Mendoza et al, 2003), and a highmobility-group box protein (EhHMGB1) (Abhyankar et al, 2008). Proteins that resemble transcription factors C/EBP and STAT were also identified using human heterologous antibodies (Marchat et al, 2002;Cruz-Vera et al, 2003).…”

Deciphering molecular mechanisms of mRNA metabolism in the deep‐branching eukaryote Entamoeba histolytica

“…Its overexpression led to the modulation of 20 transcripts that were previously reported as regulated in an animal model of amoebiasis. 25 Notably, transcripts corresponding to the light subunit of the Gal/GalNAc lectin, an important virulence factor in E. histolytica, were upregulated, confirming the relevance of EhHMGB1 in modulating the virulence properties of trophozoites. 25…”