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Rapid detachment of Giardia lamblia trophozoites as a mechanism of antimicrobial action of the isoflavone formononetin.

ABSTRACT: Attachment to the small intestinal mucosa is crucial for initiating and maintaining Giardia infection. We tested the effect of isoflavones on Giardia attachment.We evaluated the effect of formononetin on trophozoite attachment to glass, to intestinal epithelial cell layers in vitro and to murine small intestinal explants, and on the intestinal load in mice.We found that the isoflavone formononetin inhibits both attachment and flagellar motility within minutes and reduces the trophozoite load of Giardia in mice within 1.5 h after treatment.The antigiardial activity of formononetin is at least partially due to its capacity to rapidly detach trophozoites.

SUBMITTER: Lauwaet T

PROVIDER: S-EPMC2818108 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Rapid detachment of Giardia lamblia trophozoites as a mechanism of antimicrobial action of the isoflavone formononetin.

Lauwaet Tineke T Andersen Yolanda Y Van de Ven Liesbeth L Eckmann Lars L Gillin Frances D FD

The Journal of antimicrobial chemotherapy 20100112 3

<h4>Objectives</h4>Attachment to the small intestinal mucosa is crucial for initiating and maintaining Giardia infection. We tested the effect of isoflavones on Giardia attachment.<h4>Methods</h4>We evaluated the effect of formononetin on trophozoite attachment to glass, to intestinal epithelial cell layers in vitro and to murine small intestinal explants, and on the intestinal load in mice.<h4>Results</h4>We found that the isoflavone formononetin inhibits both attachment and flagellar motility ...[more]

PMID: 20067984

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Project description:The cell membrane of a Giardia lamblia trophozoite is covered with a single species of variant-specific surface protein (VSP) that is replaced by another VSP every 6 to 13 generations of cell growth, possibly for an evasion of host immunity. Experimentally, only six VSP species have been verified to localize to the cell membrane thus far. By assuming that VSP contains multiple CXXC motifs, 219 vsp genes were annotated in GiardiaDB of the WB isolate. By further assuming that VSP possesses both CXXC motifs and a CRGKA tail at the C terminus, Adam et al. (BMC Genomics 11:424, 2010) identified a total of 303 potential vsp genes in Giardia WB. The discrepancies between these two assumed VSP identities have caused some confusion. Here, we used experimental approaches to further verify what is required of the structures of a VSP to localize to the surface of cell membrane. The data led to the following conclusions. (i) The C-terminal CRGKA sequence is not essential for localizing VSPs to the cell membrane. (ii) A "motif 1" of 45 residues, consisting of two CXXCs separated by 12 to 15 amino acid residues, located close to the C terminus and a hydrophobic "motif 2" of 38 residues at the C terminus are both essential and sufficient for localizing the protein to the cell membrane. (ii) An N-terminal sequence upstream from motif 1 is not required for targeting VSPs to the cell membrane. By these criteria, we are able to identify 73 open reading frames as the putative vsp genes in Giardia. IMPORTANCE The intestinal pathogen Giardia lamblia expresses only one variant-specific surface protein (VSP) on the cell membrane surface at a given time, but it changes spontaneously every 6 to 13 generations of growth, presumably for evading the host immunity. Only 6 VSPs have been empirically shown to localize to the cell membrane surface thus far. Here, we used mutations of VSPs and methods of identifying their locations in Giardia cells and found that a "motif 1" of 45 residues, consisting of two CXXCs separated by 12 to 15 amino acid residues, located close to the C terminus and a hydrophobic "motif 2" of 38 residues at the C terminus are the only essential and sufficient structural requirements for localizing a protein to the cell membrane. By these criteria, 73 genes are identified in the Giardia WB strain genome database as the putative repertoire of VSPs.

Project description:BackgroundDiarrheal diseases caused by protozoa have a great impact on human health around the world. Giardia lamblia is one of the most common flagellates in the intestinal tract. Factors such as adverse effects to first-line drugs or the appearance of drug-resistant strains, make it necessary to identify new treatment alternatives. Agroindustry waste, like pomegranate peel, are a source of phenolic compounds, which possess antiparasitic activities. In vivo studies demonstrated antigiardiasic potential by reducing cyst shedding and protecting intestinal cells; however, they did not identify the compounds or elucidate any mechanism of action in the parasite. The objective of this study is to identify potential molecular targets and to test the in vitro effects of polyphenols from Punica granatum on Giardia lamblia.MethodsThe in vitro antigiardial potential of polyphenolic extract from pomegranate peel (Punica granatum L.) obtained using microwave-ultrasound methodology was evaluated on Giardia lamblia trophozoites. Extract phytochemical identification was performed by HPLC/MS analysis. The effect of polyphenolic extract on growth and adhesion capacity was determined by parasite kinetics; morphological damage was evaluated by SEM, alteration on α-tubulin expression and distribution were analyzed by western blot and immunofluorescence, respectively.ResultsThe pomegranate peel extract showed the presence of ellagitannins (punicalin and punicalagin, galloyl-dihexahydroxydiphenoyl-hexoside), flavones (luteolin), and ellagic acid, that caused an inhibitory effect on growth and adhesion capacity, particularly on cells treated with 200 µg/mL, where growth inhibition of 74.36%, trophozoite adherence inhibition of 46.8% and IC50 of 179 µg/mL at 48 h were demonstrated. The most important findings were that the extract alters α-tubulin expression and distribution in Giardia trophozoites in a concentration-independent manner. Also, an increase in α-tubulin expression at 200 µg/mL was observed in western blot and diffuse or incomplete immunolabeling pattern, especially in ventral disk. In addition, the extract caused elongation, disturbance of normal shape, irregularities in the membrane, and flagella abnormalities.DiscussionThe pomegranate peel extract affects Giardia trophozoites in vitro. The damage is related to the cytoskeleton, due to expression and distribution alterations in α-tubulin, particularly in the ventral disk, a primordial structure for adhesion and pathogenesis. Microtubule impairment could explain morphological changes, and inhibition of adhesion capacity and growth. Besides, this is the first report that suggests that ellagic acid, punicalin, punicalagin and luteolin could be interactioning with the rich-tubulin cytoskeleton of Giardia. Further investigations are needed in order to elucidate the mechanisms of action of the isolated compounds and propose a potential drug alternative for the giardiasis treatment.

Dataset Information

Rapid detachment of Giardia lamblia trophozoites as a mechanism of antimicrobial action of the isoflavone formononetin.

Publications

Rapid detachment of Giardia lamblia trophozoites as a mechanism of antimicrobial action of the isoflavone formononetin.

Similar Datasets

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