Ultrastructural and proapoptotic-like effects of kaempferol in Giardia duodenalis trophozoites and bioinformatics prediction of its potential protein target

BACKGROUND Kaempferol (KPF) is a flavonoid with antiparasitic activity including experimental giardiasis which mechanism of action is unknown. OBJECTIVE To analyse the cytotoxic effects of KPF on Giardia duodenalis trophozoites and to identify a likely parasite target of this compound. METHODS We used inhibitory concentrations of KPF (IC25, IC50 and IC100) and albendazole (ABZ) as reference drug. The ultrastructure of the trophozoites was analysed by transmission electron microscopy (TEM) whilst apoptosis/necrosis, production of reactive oxygen species (ROS) and cell cycle progression were assessed by flow cytometry (FCM) and confocal laser microscopy (CLM). Ligand-protein docking analyses were carried out using KPF structure from a drug library and crystal structure of a G. duodenalis aldose reductase (GdAldRed) homolog. RESULTS KPF provoked appearance of perinuclear and periplasmic spaces devoid of cytosolic content and multilamellar structures. KPF induced proapoptotic death associated with partial arrest in the S phase without ROS production. Bioinformatics approaches predicted that GdAldRed is a viable KPF target (ΔG = -7.09 kCal/mol), exhibiting 92% structural identity and a similar coupling pattern as its human homolog. CONCLUSIONS KPF exerted a proapoptotic effect on G. duodenalis trophozoites involving partial interruption of DNA synthesis without oxidative stress or structure damage to chromatin and cytoskeletal structures. GdAldRed is a likely target underlying its antigiardial activity.

Giardia duodenalis: analysis of secreted proteases upon trophozoite-epithelial cell interaction in vitro

Protease secretion by Giardia duodenalis trophozoites upon interaction with epithelial cells and its association with the parasite adhesion was studied in co-cultures of parasites with IEC6 epithelial cell monolayers in the presence or absence of protease inhibitors. Proteolytic activity in supernatants from trophozoites was enhanced when they were co-cultured with IEC6 cells. This activity was strongly inhibited by pre-incubation of live trophozoites with E-64 and TPCK and a concomitant inhibition of parasite adhesion to IEC6 cells was observed. These data suggest that trophozoites secrete cysteine-type proteases that play a role in the adhesion of G. duodenalis to epithelial cells.

Giardia duodenalis: Analysis of humoral immune response in experimentally infected gerbils (Meriones unguiculatus)

In this work, we have analyzed the humoral immune response in Mongolian gergils infected with Giardia duodenalis trophozoites of strains P-1 ad WB. The course of infection in the animals was assessed by monitoring cyst shedding in feces, and serum samples were collected at weekly intervals to measure antibody levels by ELISA. Parallel studies were carried out to determine the patterns of total and surface antigens of the parasite recognized by antibodies using Western blot and radioimmunoprecipitation (RIP) assays with the use of homospecific enzyme conjugates. Typical patterns of cyst shedding were observed in the infected animals and cyst numbers per grams of feces were consistently higher in gerbils infected with WB strain. Antibody levels to G. duodenalis antigens were observed by week 2 post-infection and were still detectable 4 months after infection. G. duodenalis antigen showed a complex but quantitatively and qualitatively different recongnition pattern by infection-induced antibodies in Western blot assays which related to infecting strain. However, RIP assays showed a more restricted and common pattern of recognition of surface antigens from either strain. Taken together, the data obtained in this study provides further information regardin direct comparisons among infecting strain, pattern of infectivity, and host immune response toward G duodenalis antigens in the gerbil model