Protease activity in extracellular products secreted in vitro by trophozoites of Giardia duodenalis

Abstract

There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in pathogenesis of giardiasis. This report describes a preliminary characterization of the proteolytic activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). The protease activity of E/S products in conditioned medium by trophozoites of each strain was analyzed using substrate (gelatin and collagen) impregnated SDS-PAGE and hemoglobin assay. The protease characterization was based on inhibition assays including synthetic inhibitors. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted degradation of the substrates and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibition assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of hemoglobin.

Giardia duodenalis Cathepsin B Proteases Degrade Intestinal Epithelial Interleukin-8 and Attenuate Interleukin-8-Induced Neutrophil Chemotaxis

“…Parasitic cysteine proteases have been shown to prevent activation of proinflammatory transcription factors (88), degrade host effector molecules (89), promote specific host immune responses (90,91), and cleave host chemokines (46,87). Prior to this study, reports had described roles for a G. duodenalis catB protease in trophozoite encystation and excystation (41), but the role of these proteases in disease pathogenesis and host immunity has remained largely speculative (43,92,93). The results presented herein demonstrated for the first time that catB proteases secreted by G. duodenalis trophozoites might be associated with the attenuation of aspects of their host's proinflammatory immune response.…”

The Immunological Enigma of Human Giardiasis

“…Such proteins were shown to stimulate a mixed Th1/Th2-like in mice [116]. Cysteine proteases may also be involved in mediating apoptosis in enterocytes and immune evasion, e.g., by cleavage of IL-8 released from enterocytes [45,115,85]. Together with secretion of metabolic enzymes such as ADI that may deplete arginine locally which may result in a nutritional stress environment, cysteine proteases may influence the outcome of the immune response in particular in newborn humans as it has been shown for bacterial infections [117].…”

“…Th2 responses have been associated with a broad variety of initiating mechanisms including protease activity, metabolic stresses induced by the pathogen or by pathogen tissue damage [73]. In this respect, it is noteworthy that G. duodenalis parasites secrete a broad variety of cysteine proteases [85,45,114,115] that may also play a part in a PRR-independent immune response. Such proteins were shown to stimulate a mixed Th1/Th2-like in mice [116].…”

“…A recent study shows lectin-binding dependent killing of Giardia trophozoites by complement, but the role of lectin-binding in the intestinal lumen is unclear [43]. The mucin Muc 2, mainly produced by Goblet cells, is the major gel-forming component of the mucus layer in the small intestine, and it has been suggested that excretory/secretory products of the parasite, likely cysteine proteases and glycosidases, degrade the mucus [44,45,42]. On the one hand, mucus may stimulate growth and protect trophozoites from toxic effects of human milk [46][47][48]; on the other hand, mucus was also shown to inhibit trophozoite attachment to culture dishes [49].…”

Genotyping of Brazilian Giardia duodenalis human axenic isolates

“…Therefore, we proposed herein the genotyping of five Brazilian axenic isolates obtained from cysts of patients from São Paulo state, for which just proteolytic activity and susceptibility to potential antigiardial agents are available yet (9)(10)(11)(12). For this purpose, we employed PCR amplification of two commonly used genetic markers, the gdh and tpi genes.…”