A Novel Entamoeba histolytica Cysteine Proteinase, EhCP4, Is Key for Invasive Amebiasis and a Therapeutic Target (original) (raw)

2010, Journal of Biological Chemistry

Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design. Entamoeba histolytica is the causative protozoan parasite of amebic colitis and liver abscesses and the second leading cause of death from parasitic diseases (1). Invasive E. histolytica trophozoites secrete 10-1,000-fold more cysteine proteinases than non-invasive Entamoeba dispar (2), whereas EhCP 2-deficient trophozoites demonstrate reduced virulence in animal models (3). During amebic invasion, EhCPs degrade the mucus barrier and destroy the extracellular matrix (4) underlying the epithelium, thereby causing epithelial detachment and ulceration (4, 5). Amebic penetration through ulcers facilitates entry into the mucosa and subsequently spread to the liver (3-5). Furthermore, EhCPs interrupt the function of the host immune system by cleaving immune molecules, including IgA and IgG (6, 7), processing complement C3 (2) and inactivating complement C3a and C5a (8). They also generate mature IL-1␤ from pro-IL-1␤ (9) but inactivate pro-IL-18 (10). These alternations of the immune response contribute to ineffective host defenses and thereby excessive inflammation and tissue damage. Genome-wide homology searches have identified 50 E. histolytica genes encoding cysteine proteases (11, 12), most of which belong to the C1 papain superfamily. These enzymes are expressed as zymogens with a hydrophobic pre-domain of 12-20 residues, a pro-domain of 55-148 residues, a catalytic domain of 190-488 residues, and six cysteine residues that form the disulfide bridges to stabilize the tertiary structure (13, 14). The EhCPs that have been characterized to date include EhCP1 (EhCP-A1) (15, 16), EhCP2 (EhCP-A2) (14), EhCP3 (EhCP-A3) (10, 14), EhCP5 (EhCP-A5) (15, 17), and EhCP112 (EhCP-B9) (18). These enzymes all undergo autocatalytic activation in which the inhibitory pro-domain is cleaved from the zymogen. They also have a cathepsin L-like structure, but like cathepsin B, these enzymes can also accommodate Arg at P2 (14). Among the remaining EhCPs,