EhCP112 is an Entamoeba histolytica secreted cysteine protease that may be involved in the parasite-virulence

Abstract

SummaryEhCP112 is an Entamoeba histolytica protease that together with the EhADH112 protein forms the EhC-PADH complex involved in trophozoite virulence. Here, we produced the recombinant EhCP112 and studied its relationships with extracellular matrix components and with target cells. A DNA fragment containing the pro-peptide and the mature enzyme was expressed in bacteria as an active enzyme (rEhCP112), whereas the full gene containing the signal peptide, the pro-peptide and the mature enzyme expressed a non-active protein. The fragment only with the mature enzyme was not expressed. rEhCP112 purified by affinity columns digested azocasein and had a strong autoproteolytic activity. Four hours after purification the protein appeared degraded. Anti-tag antibodies, monoclonal antibodies against the EhCP112 and sera from human patients with amoebiasis recognized rEhCP112. rEhCP112 digested gelatin, collagen type I, fibronectin and haemoglobin; it destroyed MDCK cell monolayers and bound to red blood cells. The native EhCP112 was poorly expressed in a virulence-deficient mutant, and in the wild-type clone it was located in secreted vesicles, forming the EhCPADH complex. Altogether these results show that EhCP112 is a molecule able to disrupt cell monolayers and digest proteins of the extracellular matrix and haemoglobin, and it is secreted by the trophozoites.

Molecular Basis of Pathogenesis in Amoebiasis

“…Among these, the EhCP-ADH complex has been studied most extensively [62][63][64][65][66][67]. EhCP-ADH complex encodes two proteins, a cysteine protease, EhCP112, and an adhesion protein, EhADH112 [68].…”

Conformational changes induced by detergents during the refolding of chemically denatured cysteine protease ppEhCP-B9 from Entamoeba histolytica

“…Proteinase activity was determined by substrate-gel electrophoresis using 10% gelatine as a substrate as previously reported [2,5]. Briefly, 50 μL samples were mixed with equal volumes of 2 × Laemmli buffer and incubated for 20 min at 37°C.…”

“…E. histolytica trophozoites secrete EhCP-B9, which degrades collagen and fibronectin. EhCP-B9 also binds to and lyses red blood cells and degrades haemoglobin, and virulence-deficient mutant poorly expressed EhCP-B9 [2]. Furthermore, overexpression of the ehcp-b9 gene restores the pathogenic phenotype of a non-pathogenic E. histolytica clone, generating amoebic liver abscesses in both gerbil and mouse models [3].…”

“…As a potential target for drug design, the recombinant EhCP-B9 proenzyme has been expressed as an active enzyme in heterologous systems, albeit with very low yields because of strong autoproteolytic activity and instability [2]. To overcome this problem, ppEhCP-B9 was overexpressed in Escherichia coli inclusion bodies, which enabled high-yield purification of the denatured enzyme but required a refolding step.…”

Crystal structure of the cysteine protease inhibitor 2 from Entamoeba histolytica: Functional convergence of a common protein fold

“…The genome of E. histolytica contains open reading frames for 86 proteases, including at least 50 cysteine proteases (CP) (Clark et al, 2007). CPs are wellcharacterized virulence factors of E. histolytica (Bruchhaus et al, 1996;Ocadiz et al, 2005). Furthermore, amebic colitis and liver abscess are dependent on the ability of E. histolytica to damage host tissues (Stanley, 2003).…”