Protease activity in Giardia duodenalis trophozoites of axenic strains isolated from symptomatic and asymptomatic patients

Abstract

We have examined by gelatin-SDS-PAGE the protease activity in cell lysates of Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic patient and an asymptomatic carrier , and the reference strain Portland 1 (P1). The proteolysis band patterns showed differences among strains isolated from asymptomatic and symptomatic individuals. The lysate of the strain BTU-10, showed only five hydrolysis bands, while a greater number of bands (10-11 bands) Giardia duodenalis (syn. G. lamblia, G. intestinalis) is a flagellated protozoan found in the small intestine of man and other mammals. Giardiasis is currently considered to be an important public health problem in developing countries, where it is one of the most common causes of diarrhea among children.Recent advances in the understanding of the biochemistry and molecular biology of protozoa have permitted the study of molecules that directly participate in the parasites life cycle or in the pathogenicity (North et al. 1990). Many important parasite species such as Entamoeba histolytica, Trypanosoma cruzi, Leshmania spp., Trichomonas vaginalis and Plasmodium spp. are actively proteolytic and contain multiple proteases that play a role in the life cycle, morphogenesis and infectivity (McKerrow et al. 1993). With respect to Giardia, despite evidence that proteolysis is involved in its nutrition, development and pathogenicity, relatively little is known. Initially, the intracelular activity of proteases in Giardia trophozoites was attributed to two major proteases corresponding to 105 and 40 kDa bands detected by SDS-PAGE using gelatin as a substrate (Hare et al. 1989, Parenti 1991 on the protease activity in trophozoites of other axenic strains. Moreover, there is increasing interest to conduct studies in attempting to learn more about strains isolated and axenized in endemic areas, in order to know the biological, clinical and epidemiological inter-strain differences in these areas where the search for control measures is still frequent.In the present investigation, we analyzed the protease activities in lysates of G. duodenalis trophozoites of two strains from a symptomatic (BTU-11) and an asymptomatic patient (BTU-10) isolated and axenized in Brazil, and the reference strain P1. The objectives were to analyse the protease activity in trophozoites and to determine if there are differences in such activity among the strains. MATERIALS AND METHODSParasites and culture -Trophozoites of three axenic strains of G. duodenalis were studied. Two of them were isolated in Brazil, at the Giardiasis Laboratory (IB/Unesp) in Botucatu, São Paulo: BTU-10 from a 21 year-old asymptomatic male and BTU-11 from a 40 year-old symptomatic male presenting diarrhea, flatulence and abdominal cramps. Both strains were obtained from cysts in the patient's feces. The third strain P1, originally isolated by Meyer (1976) from a 36 year-old symptomatic female from Portland, Oregon (USA), was obtained from HW Pillai (Division of Geographic Medicine Tufts University...

SGLT-1-mediated glucose uptake protects human intestinal epithelial cells against Giardia duodenalis-induced apoptosis

“…Together, the data suggest that a soluble heat-labile protein fraction in G. duodenalis trophozoites triggers this SGLT-1-mediated host response. Giardia duodenalis trophozoite proteases play an important role in the microbial-host epithelial interaction during infection ( [Farthing, 1993] and [Guimaraes et al, 2003]). Moreover, protease-activated receptors have been identified on intestinal epithelial cells and are involved in the regulation of cell apoptosis and inflammatory responses ( [Chin et al, 2003] and [Hansen et al, 2005]).…”

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

“…At this moment, few studies have identified and characterized trophozoite proteases of strains isolated in endemic areas. Proteolytic activity in trophozoites of two Brazilian axenic strains and the predominance of cysteine proteases were firstly reported by GUIMARÃES et al 3 . More recently, CORADI & GUIMARÃES 2 , employing cell lysates of five strains isolated and axenized in Brazil observed differences in the Giardia proteases ability to hydrolyse different protein substrates and the inhibition of enzymatic activity was substrate dependent.…”

“…In relation to the intestinal protozoan Giardia, proteases have been characterized and may play role in metabolism and physiologic process such as nutrition, excystation, pathogenesis and evasion of the host immune response [2][3][4][5][9][10][11]15 .…”

“…Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. [2][3][4][5][9][10][11]15 , it is clear that the protozoan Giardia contains many proteins with proteolytic activities and that these molecules may be involved in the host-parasite relationship. Most of the reported observations have focused on proteolysis in the trophozoites of the standard strain Portland 1, whereas few studies have been identified and characterized proteases of other axenic strains.…”

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.…”