Identification and Partial Characterization of Excretory/Secretory Products with Proteolytic Activity in Giardia intestinalis

Abstract

This report examines the presence of proteolytic activity detected in media collected from in vitro cultures of Giardia intestinalis, and the partial characterization by gelatin-substrate polyacrylamide gel electrophoresis and inhibition studies. Gelatin-substrate polyacrylamide gel electrophoresis revealed 6 bands with proteolytic activity, with estimated molecular weights of 36, 59, 63, 72, 103, and 175 kDa. These bands were not present in the control medium. On the other hand, G. intestinalis trophozoite lysates showed proteolytic bands at 16, 20, 66, 82, 108, and 120 kDa, thus indicating that intracellular proteases could be different from the excretory/secretory (E/S) products. Based on inhibition studies, 2 bands of 59 and 63 kDa were inhibited by iodoacetic acid, indicating the presence of cysteine proteases. Partial inhibition of a band of 36 kDa was found with EDTA, a metal-chelating agent, suggesting the possible presence of metalloproteases. The presence of aspartic and serine proteases were not detected under the assay conditions used. As G. intestinalis E/S may be involved in differentiation mechanisms of the parasite and also be responsible for the mucosal alterations that occur in giardiasis, the characterization of these proteases may facilitate their evaluation as targets in the therapy of the disease.

Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced by the parasiteGiardia intestinalis

“…Giardia lamblia-induced apoptosis correlated with a loss of epithelial barrier function in small intestinal monolayers, by disruption of the tight junctional ZO-1 and increasing permeability in a caspase-3-dependent process (Chin et al, 2002). In the above study, freshly sonicated trophozoites were also able to induce apoptosis, suggesting a possible role for molecules released by the parasite, particularly excretory and secretory antigens with a proteolytic activity (Jimenez et al, 2000(Jimenez et al, , 2004. However, in our study infection with live trophozoites of the WB clone C6 strain was able to induce apoptosis in enterocytes, while sonicated trophozoites of the same strain were unable to induce apoptosis in the study of Chin et al (2002).…”

In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64

“…Regarding to Giardia , advances in the study of its proteases had become clear that this protozoan is actively proteolytic and contain multiple proteases, with the predominance of the cysteine peptidases that are responsible for the main proteolytic activity 4,5,12,26 .…”

“…With respect to Giardia , notwithstanding recent studies have shed light on trophozoites proteases and their involvement in metabolism and physiologic processes 4,5,12,23 relatively little is known about the effect of protease inhibitors on vital processes of this parasite. To date, as far as we know, there are few studies that have evaluated the in vitro and in vivo performance of protease inhibitors on multiplication 8,11 and on encystation/excystation 7,23,24 of the parasite.…”

Role of scuticociliate proteinases in infection success in turbot, Psetta maxima (L.)

“…The role of proteinases in pathogenesis, including activation of inflammation and degradation of haemoglobin and other blood proteins, is well documented (11). Proteinases are not only confined within lysosomes and other similar organelles, but can also be secreted externally to act extracellularly (12)(13)(14)(15). Parasite proteinases are involved in development of the parasite, invasion of host cells and tissues, intraand extracellular digestion of nutrients, and ⁄ or destruction of host molecules involved in immune responses (12,14,16).…”