Resistance of Entamoeba histolytica to the Cysteine Proteinase Inhibitor E64 Is Associated with Secretion of Pro-enzymes and Reduced Pathogenicity

Abstract

Cysteine proteinases (CPs) have been considered suitable targets for the development of antiparasitic drugs. To assess the importance of CPs for the growth and pathogenicity of the protozoan parasite Entamoeba histolytica we have cultured amoebae in the presence of various cysteine proteinase inhibitors (CPIs). It was found that broad range CPIs, which are membrane permeable and rapidly enter the cell, are highly toxic at micromolar concentrations, and all attempts to generate E. histolytica mutants resistant to these CPIs were unsuccessful. In contrast, the broad range CPI E64, which does not permeate membranes as well, was deleterious at much higher concentrations, and amoebae rapidly developed resistance to this inhibitor. Compared with sensitive wild-type cells, E64-resistant E. histolytica were substantially reduced in the expression of various CP genes and were able to secrete unprocessed enzyme into the culture medium. Moreover, E64 resistance was associated with a significant reduction in virulence, because these cells were greatly impaired in the ability to generate liver abscesses in experimentally infected gerbils.

Entamoeba histolytica Cysteine Proteinase 5 Binds Integrin on Colonic Cells and Stimulates NFκB-mediated Pro-inflammatory Responses

“…1C) as compared with untreated controls. This finding is consistent with a previous report that showed ameba secretes both PCP5 and mature CP5 (27). Thus, to elucidate a specific functional role for EhCP5, colonic cells were exposed to secreted proteinases derived from wild-type (WT), amebapore A vector control (AP ) significantly induced the ubiquitination of NEMO, but CP5 Ϫ/Ϫ did not (Fig.…”

“…With the use of the specific CP5 enzyme inhibitor E64 to abolish CP5 enzyme activity, we were intrigued that it had no effect on PCP5-mediated NFB activation, suggesting that PCP5 may activate NFB. Although previous studies have shown that inhibiting cysteine proteinase activity decreases E. histolytica-induced liver abscess formation, it is important to note that only E64-resistant ameba cultures, rather than wildtype ameba cultured with E64, had a significant effect on liver abscess formation (27). As E64-resistant E. histolytica cultures have significantly decreased CP5 gene expression (27), this finding demonstrates precedence of CP5 as a virulent factor for E. histolytica.…”

“…histolytica cysteine proteinases are key virulence factors involved in pathogenesis (3,4). Trophozoites secrete both promature CP5 and mature CP5 (27). CP5-deficient trophozoites are known to inflict less gut inflammation and damage to the intestinal barrier and are incapable of inducing amebic liver abscesses (39).…”

“…Genetic studies suggest that NEMO is absolutely required for the activation of IKK and NFB in response to diverse stimuli (24,25), and ubiquitination of NEMO is required for the activation IKK (26). E. histolytica intrinsic and secreted proteinases contain both PCP5 and mature CP5 (27). Interestingly, when ameba was grown in the presence of E64, PCP5 levels were significantly increased (Fig.…”

The parasite Entamoeba histolytica exploits the activities of human matrix metalloproteinases to invade colonic tissue

“…Second, CP-A5 catalytically cleaves pro-MMP-3 into the active enzyme, which in turn activates other MMPs (including MMP-1) and then degrades ECM. It is noteworthy that trophozoites secrete both pro-CP-A5 and mature form 30 , which thus potentially induces and activates human MMPs at the same time. Together, the combined actions of CP-A5 contribute to the ECM destruction by human MMPs and to the acute inflammation observed during intestinal amoebiasis.…”