Repurposing Terfenadine as a Novel Antigiardial Compound.

Giardia lamblia is a highly infectious protozoan that causes giardiasis, a gastrointestinal disease with short-term and long-lasting symptoms. The currently available drugs for giardiasis treatment have limitations such as side effects and drug resistance, requiring the search for new antigiardial compounds. Drug repurposing has emerged as a promising strategy to expedite the drug development process. In this study, we evaluated the cytotoxic effect of terfenadine on Giardia lamblia trophozoites. Our results showed that terfenadine inhibited the growth and cell viability of Giardia trophozoites in a time-dose-dependent manner. In addition, using scanning electron microscopy, we identified morphological damage; interestingly, an increased number of protrusions on membranes and tubulin dysregulation with concomitant dysregulation of Giardia GiK were observed. Importantly, terfenadine showed low toxicity for Caco-2 cells, a human intestinal cell line. These findings highlight the potential of terfenadine as a repurposed drug for the treatment of giardiasis and warrant further investigation to elucidate its precise mechanism of action and evaluate its efficacy in future research.

Curcumin Stimulates the Overexpression of Virulence Factors in Salmonella enterica Serovar Typhimurium: In Vitro and Animal Model Studies.

Salmonella spp. is one of the most common food poisoning pathogens and the main cause of diarrheal diseases in humans in developing countries. The increased Salmonella resistance to antimicrobials has led to the search for new alternatives, including natural compounds such as curcumin, which has already demonstrated a bactericidal effect; however, in Gram-negatives, there is much controversy about this effect, as it is highly variable. In this study, we aimed to verify the antibacterial activity of curcumin against the Salmonella enterica serovar Typhimurium growth rate, virulence, and pathogenicity. The strain was exposed to 110, 220 or 330 µg/mL curcumin, and by complementary methods (spectrophotometric, pour plate and MTT assays), we determined its antibacterial activity. To elucidate whether curcumin regulates the expression of virulence genes, Salmonella invA, fliC and siiE genes were investigated by quantitative real-time reverse transcription (qRT-PCR). Furthermore, to explore the effect of curcumin on the pathogenesis process in vivo, a Caenorhabditis elegans infection model was employed. No antibacterial activity was observed, even at higher concentrations of curcumin. All concentrations of curcumin caused overgrowth (35−69%) and increased the pathogenicity of the bacterial strain through the overexpression of virulence factors. The latter coincided with a significant reduction in both the lifespan and survival time of C. elegans when fed with curcumin-treated bacteria. Our data provide relevant information that may support the selective antibacterial effects of curcumin to reconsider the indiscriminate use of this phytochemical, especially in outbreaks of pathogenic Gram-negative bacteria.

Drug repositioning: antiprotozoal activity of terfenadine against Entamoeba histolytica trophozoites.

The infection caused by Entamoeba histolytica is still a serious public health problem, especially in developing countries. The goal of this study was to evaluate the effect of terfenadine against Entamoeba histolytica. The trophozoites were exposed to 1, 2, 3, and 4 µM of terfenadine, for 24 and 48 h. Consequently, the viability of cells was determined by trypan blue exclusion test. The effect of terfenadine on adhesion of Entamoeba histolytica was evaluated in Caco-2 cells. In addition, the effect of terfenadine on the erythrophagocytic capacity of the parasite was investigated. The results show that terfenadine affects the growth and cell viability in a time- and dose-dependent manner. The higher inhibitory effects were observed with 4 µM at 48 h; 91.6% of growth inhibition and only 22.5% of trophozoites were viable. Additionally, we demonstrate that terfenadine is highly selective for the parasite and has low toxicity on Caco-2 cells. Furthermore, adhesion to Caco-2 cells and erythrophagocytic capacity were significantly inhibited. These findings demonstrate that terfenadine exerts significant effects on the virulence of Entamoeba histolytica. This is the first study demonstrating the amoebicidal activity of terfenadine and the results suggest it may be effective in the treatment of amoebiasis.

MDR1 protein (ABC-C1) Over Expression in Giardia Intestinalis Incubated with Albendazole and Nitazoxanide.

BACKGROUND: Giardia intestinalis is a worldwide parasite. Drugs used for the treatment of giardiasis are metronidazole, albendazole and nitazoxanide. The development of drug resistance is an obstacle to the effective treatment. Resistance mechanisms in some parasites involve the participation of ATP-binding cassette (ABC) transporter superfamily. PURPOSE: To find if the ATP-binding cassette genes are overexpressed in trophozoites treated with albendazole or nitazoxanide. METHODS: A search for ATP-binding cassette genes in Giardia sequence database (GiardiaDB) was done and six genes were selected. Trophozoites treated with albendazole or nitazoxanide and the expression of these six ABC genes was quantitated by real-time RT-PCR. The ABC-C1 gene was selected, and a fragment cloned. The ABC-C1 protein was expressed, and polyclonal antibodies were elicited in mice to detect the protein in treated trophozoites, finally a docking analysis was performed for ABC-C1 and tizoxanide interaction. RESULTS: Bioinformatics analysis showed that the ATP-binding cassette (ABC) topology is present in the six proteins. The qRT-PCR revealed that the ABC-C1 gene was overexpressed in cells incubated with nitazoxanide or albendazole. Confocal analysis showed that ABC-C1 protein levels increased in trophozoites with both treatments but was higher with nitazoxanide. The mark was detected heavily in the periphery of the cells. Using a docking analysis, it was found that the nitazoxanide metabolite, tizoxanide was docked close to the ATP-binding region as well as in the exit tunnel, located in the transmembrane region. CONCLUSION: These findings in Giardia intestinalis, support the possible role of ABC-C1 in drug efflux.

Giardia intestinalis coiled-coil cytolinker protein 259 interacts with actin and tubulin.

Giardia intestinalis is a human parasite that causes a diarrheal disease in developing countries. G. intestinalis has a cytoskeleton (CSK) composed of microtubules and microfilaments, and the Giardia genome does not code for the canonical CSK-binding proteins described in other eukaryotic cells. To identify candidate actin and tubulin cross-linking proteins, we performed a BLAST analysis of the Giardia genome using a spectraplakins consensus sequence as a query. Based on the highest BLAST score, we selected a 259-kDa sequence designated as a cytoskeleton linker protein (CLP259). The sequence was cloned in three fragments and characterized by immunoprecipitation, confocal microscopy, and mass spectrometry (MS). CLP259 was located in the cytoplasm in the form of clusters of thick rods and colocalized with actin at numerous sites and with tubulin in the median body. Immunoprecipitation followed by mass spectrometry revealed that CLP259 interacts with structural proteins such as giardins, SALP-1, axonemal, and eight coiled-coils. The vesicular traffic proteins detected were Mu adaptin, Vacuolar ATP synthase subunit B, Bip, Sec61 alpha, NSF, AP complex subunit beta, and dynamin. These results indicate that CLP259 in trophozoites is a CSK linker protein for actin and tubulin and could act as a scaffold protein driving vesicular traffic.

Antigiardial Activity of Acetylsalicylic Acid Is Associated with Overexpression of HSP70 and Membrane Transporters.

Giardia lamblia is a flagellated protozoan responsible for giardiasis, a worldwide diarrheal disease. The adverse effects of the pharmacological treatments and the appearance of drug resistance have increased the rate of therapeutic failures. In the search for alternative therapeutics, drug repositioning has become a popular strategy. Acetylsalicylic acid (ASA) exhibits diverse biological activities through multiple mechanisms. However, the full spectrum of its activities is incompletely understood. In this study we show that ASA displayed direct antigiardial activity and affected the adhesion and growth of trophozoites in a time-dose-dependent manner. Electron microscopy images revealed remarkable morphological alterations in the membrane, ventral disk, and caudal region. Using mass spectrometry and real-time quantitative reverse transcription (qRT-PCR), we identified that ASA induced the overexpression of heat shock protein 70 (HSP70). ASA also showed a significant increase of five ATP-binding cassette (ABC) transporters (giABC, giABCP, giMDRP, giMRPL and giMDRAP1). Additionally, we found low toxicity on Caco-2 cells. Taken together, these results suggest an important role of HSPs and ABC drug transporters in contributing to stress tolerance and protecting cells from ASA-induced stress.

GABAergic system's Injuries Induced by Sodium Sulfite in Caenorhabditis elegans Were Prevented by the Anti-Oxidative Properties of Dehydroepiandrosterone Sulfate.

Several pathophysiological processes involve Hypoxia conditions, where the nervous system is affected as well. We postulate that the GABAergic system is especially sensitive. Furthermore, drugs improving the resistance to hypoxia have been investigated, such as the neurosteroid dehydroepiandrosterone sulfate (DHEAS) which has shown beneficial effects in hypoxic processes in mammals; however, at the cellular level, its exact mechanism of action has yet to be fully elucidated. Here, we used a chemical hypoxia model through sodium sulfite (SS) exposure in Caenorhabditis elegans (C. elegans), a nematode whose response to hypoxia involves pathways and cellular processes conserved in mammals, and that allows study the direct effect of DHEAS without its conversion to sex hormones. This work aimed to determine the effect of DHEAS on damage to the GABAergic system associated with SS exposure in C. elegans. Worms were subjected to nose touch response (Not Assay) and observed in epifluorescence microscopy. DHEAS decreased the shrinkage response of Not Assay and the level of damage in GABAergic neurons on SS-exposed worms. Also, the enhanced nuclear localization of DAF-16 and consequently the overexpression of chaperone HSP-16.2 by hypoxia were significantly reduced in SS + DHEAS exposed worms. As well, DHEAS increased the survival rate of worms exposed to hydrogen peroxide. These results suggest that hypoxia-caused damage over the GABAergic system was prevented at least partially by DHEAS, probably through non-genomic mechanisms that involve its antioxidant properties related to its chemical structure.

Encapsulation of curcumin into layered double hydroxides improve their anticancer and antiparasitic activity.

OBJECTIVES: Curcumin (CUR) has well-known activity against cancer cells and parasites; however, its applications are limited since this is an unstable molecule, which may suffer degradation by light and temperature, also, the low water solubility reduce its bioavailability. Layered double hydroxides (LDH) are well-known materials owing to the excellent anion exchange capacity, good biocompatibility and low toxicity. METHODS: Layered double hydroxides nanoparticles prepared with zinc and magnesium cations were used as a vehicle for CUR in Caco-2, Giardia lamblia and Entamoeba histolytica cultures. The physicochemical properties of Mg-LDH-CUR and Zn-LDH-CUR were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectrometry (FTIR) and X-ray powder diffraction (XRD). Additionally, the load efficiency, release profiles and photostability of CUR were quantified by high-performance liquid chromatography (HPLC) and UV-Vis spectrometry. Then, Mg-LDH-CUR and Zn-LDH-CUR were tested on Caco-2, G. lamblia and E. histolytica cultures. KEY FINDINGS: The experiments demonstrated that Zn-LDH-CUR protects better against photodegradation by UV light, while Mg-LDH-CUR showed increased toxicity against Caco-2 cell, G. lamblia and E. histolytica, in comparison with free CUR. CONCLUSIONS: Layered double hydroxides are good vehicles to improve stability, resistance to degradation of CUR, also they are useful to improve solubility, provide a controlled release and improve the cytotoxic activity. Additionally, it was shown that the composition of the M+2 cation of LDH affects its properties and structure and that this directly influences its biological activity. The findings are important to select the composition of the encapsulation vehicle for a specific activity.

Curcumin Blocks Cytotoxicity of Enteroaggregative and Enteropathogenic Escherichia coli by Blocking Pet and EspC Proteolytic Release From Bacterial Outer Membrane.

Pet and EspC are toxins secreted by enteroaggregative (EAEC) and enteropathogenic (EPEC) diarrheagenic Escherichia coli pathotypes, respectively. Both toxins are members of the Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) family. Pet and EspC are important virulence factors that produce cytotoxic and enterotoxic effects on enterocytes. Here, we evaluated the effect of curcumin, a polyphenolic compound obtained from the rhizomes of Curcuma longa L. (Zingiberaceae) on the secretion and cytotoxic effects of Pet and EspC proteins. We found that curcumin prevents Pet and EspC secretion without affecting bacterial growth or the expression of pet and espC. Our results show that curcumin affects the release of these SPATEs from the translocation domain, thereby affecting the pathogenesis of EAEC and EPEC. Curcumin-treated EAEC and EPEC did not induce significant cell damage like the ability to disrupt the actin cytoskeleton, without affecting their characteristic adherence patterns on epithelial cells. A molecular model of docking predicted that curcumin interacts with the determinant residues Asp1018-Asp1019 and Asp1029-Asp1030 of the translocation domain required for the release of Pet and EspC, respectively. Consequently, curcumin blocks Pet and EspC cytotoxicity on epithelial cells by preventing their release from the outer membrane.

Identification and molecular characterization of the tubulin-podophyllotoxin-type lignans binding site on Giardia lamblia.

There are several drugs for the treatment of giardiasis; however, there is a tendency for patients to abandon treatment because of drug-related adverse effects, resulting in relapses, acquired resistance, and higher rates of treatment failure. Recently, we reported some podophyllotoxin-type lignans from Bursera fagaroides var. fagaroides showing antigiardial activity. In the present work, we demonstrated that 5'-desmethoxy-peltatin-A-methylether (5-DES), acetylpodophyllotoxin (APOD), and podophyllotoxin (POD) affect the distribution and staining pattern of microtubular structures on Giardia trophozoites. Virtual screening results revealed that the lignans act via binding in a hydrophobic pocket in the heterodimer interface of tubulin in Giardia. This study provides useful insight to understand the action mechanism of 5DES, APOD, and POD on Giardia lamblia. The optimization of these podophyllotoxin-type lignans will lead to promising candidates for antigiardial drugs.

Curcumin Attenuates the Pathogenicity of Entamoeba histolytica by Regulating the Expression of Virulence Factors in an Ex-Vivo Model Infection.

Infection with the enteric protozoan Entamoeba histolytica is still a serious public health problem, especially in developing countries. Amoebic liver abscess (ALA) is the most common extraintestinal manifestation of the amoebiasis, and it can lead to serious and potentially life-threatening complications in some people. ALA can be cured by metronidazole (MTZ); however, because it has poor activity against luminal trophozoites, 40-60% of treated patients get repeated episodes of invasive disease and require repeated treatments that can induce resistance to MTZ, this may emerge as an important public health problem. Anti-virulence strategies that impair the virulence of pathogens are one of the novel approaches to solving the problem. In this study, we found that low doses of curcumin (10 and 50 µM) attenuate the virulence of E. histolytica without affecting trophozoites growth or triggering liver injury. Curcumin (CUR) decreases the expression of genes associated with E. histolytica virulence (gal/galnac lectin, ehcp1, ehcp5, and amoebapore), and is correlated with significantly lower amoebic invasion. In addition, oxidative stress is critically involved in the etiopathology of amoebic liver abscess; our results show no changes in mRNA expression levels of superoxide dismutase (SOD) and catalase (CAT) after E. histolytica infection, with or without CUR. This study provides clear evidence that curcumin could be an anti-virulence agent against E. histolytica, and makes it an attractive potential starting point for effective treatments that reduce downstream amoebic liver abscess.

Effect of Telmisartan in the Oxidative Stress Components Induced by Ischemia Reperfusion in Rats.

The therapeutic effects of telmisartan, an angiotensin II receptor antagonist and a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, have been demonstrated in several disorders. It has antioxidant and immune response modulator properties and has shown promising results in the treatment of an ischemia/reperfusion (I/R) lesion. In this study, a skeletal muscle (right gastrocnemius muscle) I/R lesion was induced in rats and different reperfusion times (1 h, 24 h, 72 h, 7-day, and 14-day subgroups) were assessed. Furthermore, levels of oxidative markers such as enzymatic scavengers (catalase (CAT) and superoxide dismutase (SOD)) and metabolites (nitrates and 8-oxo-deoxyguanosine) were determined. The degree of tissue injury (total lesioned fibers and inflammatory cell count) was also evaluated. We observed an increase in CAT and SOD expression levels under telmisartan treatment, with a decrease in injury and oxidative biomarker levels in the 72 h, 7-day, and 14-day subgroups. Telmisartan reduced oxidative stress and decreased the damage of the I/R lesion.

Identification of a novel potassium channel (GiK) as a potential drug target in Giardia lamblia: Computational descriptions of binding sites.

BACKGROUND: The protozoan Giardia lamblia is the causal agent of giardiasis, one of the main diarrheal infections worldwide. Drug resistance to common antigiardial agents and incidence of treatment failures have increased in recent years. Therefore, the search for new molecular targets for drugs against Giardia infection is essential. In protozoa, ionic channels have roles in their life cycle, growth, and stress response. Thus, they are promising targets for drug design. The strategy of ligand-protein docking has demonstrated a great potential in the discovery of new targets and structure-based drug design studies. METHODS: In this work, we identify and characterize a new potassium channel, GiK, in the genome of Giardia lamblia. Characterization was performed in silico. Because its crystallographic structure remains unresolved, homology modeling was used to construct the three-dimensional model for the pore domain of GiK. The docking virtual screening approach was employed to determine whether GiK is a good target for potassium channel blockers. RESULTS: The GiK sequence showed 24-50% identity and 50-90% positivity with 21 different types of potassium channels. The quality assessment and validation parameters indicated the reliability of the modeled structure of GiK. We identified 110 potassium channel blockers exhibiting high affinity toward GiK. A total of 39 of these drugs bind in three specific regions. DISCUSSION: The GiK pore signature sequence is related to the small conductance calcium-activated potassium channels (SKCa). The predicted binding of 110 potassium blockers to GiK makes this protein an attractive target for biological testing to evaluate its role in the life cycle of Giardia lamblia and potential candidate for the design of novel antigiardial drugs.

Amoebicidal activity of curcumin on Entamoeba histolytica trophozoites.

OBJECTIVES: This study was undertaken to investigate the amoebicidal potential of curcumin on Entamoeba histolytica, as well as its synergistic effect with metronidazole. METHODS: Entamoeba histolytica trophozoites were exposed to 100, 200 and 300 µm of curcumin, for 6, 12 and 24 h. Consequently, the viability of cells was determined by trypan blue exclusion test. All specimens were further analysed by scanning electron microscopy. For drug combination experiment, the Chou-Talalay method was used. KEY FINDINGS: Curcumin affected the growth and cell viability in a time- and dose-dependent manner. The higher inhibitory effects were observed with 300 µm at 24 h; 65.5% of growth inhibition and only 28.8% of trophozoites were viable. Additionally, curcumin also altered adhesion and the morphology of the trophozoites. Scanning electron microscopy revealed treated trophozoites with damages on the membrane, size alterations and parasites with loss of cellular integrity. In addition, the combination of curcumin + metronidazole exhibited a synergistic effect; the activity of both drugs was improved. CONCLUSIONS: This is the first report evaluating the effectiveness of curcumin against E. histolytica. Our results suggest that CUR could be considered for evaluation in future pharmacological studies as a promising amoebicidal agent or as complementary therapy.

Curcumin alters the cytoskeleton and microtubule organization on trophozoites of Giardia lamblia.

Giardia lamblia is a worldwide protozoan responsible for a significant number of intestinal infections. There are several drugs for the treatment of giardiasis, but they often cause side effects. Curcumin, a component of turmeric, has antigiardial activity; however, the molecular target and mechanism of antiproliferative activity are not clear. The effects of curcumin on cellular microtubules have been widely investigated. Since tubulin is the most abundant protein in the cytoskeleton of Giardia, to elucidate whether curcumin has activity against the microtubules of this parasite, we treated trophozoites with curcumin and the cells were analyzed by scanning electron microscopy and confocal microscopy. Curcumin inhibited Giardia proliferation and adhesion in a time-concentration-dependent mode. The higher inhibitory concentrations of curcumin (3 and 15µM) disrupted the cytoskeletal structures of trophozoites; the damage was evident on the ventral disk, flagella and in the caudal region, also the membrane was affected. The immunofluorescence images showed altered distribution of tubulin staining on ventral disk and flagella. Additionally, we found that curcumin caused a clear reduction of tubulin expression. By docking analysis and molecular dynamics we showed that curcumin has a high probability to bind at the interface of the tubulin dimer close to the vinblastine binding site. All the data presented indicate that curcumin may inhibit Giardia proliferation by perturbing microtubules.

Antigiardial Activity of Podophyllotoxin-Type Lignans from Bursera fagaroides var. fagaroides.

Giardiasis, a diarrheal disease, is highly prevalent in developing countries. Several drugs are available for the treatment of this parasitosis; unfortunately, all of them have variable efficacies and adverse effects. Bursera fagaroides has been known for its anti-inflammatory and antidiarrheal properties in Mexican traditional medicine. We investigated the in vitro anti-giardial activities of four podophyllotoxin-type lignans from Bursera fagaroides var. fagaroides, namely, 5'-desmethoxy-ß-peltatin-A-methylether (5-DES), acetylpodophyllotoxin (APOD), burseranin (BUR), and podophyllotoxin (POD). All lignans affected the Giardia adhesion and electron microscopy images revealed morphological alterations in the caudal region, ventral disk, membrane, and flagella, to different extents. Only 5-DES, APOD, and POD caused growth inhibition. Using the Caco-2 human cell line as a model of the intestinal epithelium, we demonstrated that APOD displayed direct antigiardial killing activity and low toxicity on Caco-2 cells. This finding makes it an attractive potential starting point for new antigiardial drugs.

Alternative Interventions to Prevent Oxidative Damage following Ischemia/Reperfusion.

Ischemia/reperfusion (I/R) lesions are a phenomenon that occurs in multiple pathological states and results in a series of events that end in irreparable damage that severely affects the recovery and health of patients. The principal therapeutic approaches include preconditioning, postconditioning, and remote ischemic preconditioning, which when used separately do not have a great impact on patient mortality or prognosis. Oxidative stress is known to contribute to the damage caused by I/R; however, there are no pharmacological approaches to limit or prevent this. Here, we explain the relationship between I/R and the oxidative stress process and describe some pharmacological options that may target oxidative stress-states.

Albendazole and its derivative JVG9 induce encystation on Giardia intestinalis trophozoites.

In the present study, we evaluated the effect of an albendazole (ABZ) derivative JVG9 on cultured Giardia intestinalis. To assess the JVG9 effects, we evaluated the tubulin cytoskeleton by confocal microscopy, and we found that the characteristic staining was modified. The scanning electron microscopy images revealed extremely damaged trophozoites and cyst-like cells. The confocal images revealed that this drug triggered the expression of cyst wall protein 1 and encystation. We also found that at low doses, AL triggered the encystation process too.

Importance of enolase in Giardia lamblia differentiation.

The ability of Giardia to differentiate into cysts which survive in the environment and release the virulent trophozoites after ingestion in the small intestine is essential for transmission and disease. We examined the role of enolase, a glycolytic enzyme, in Giardia differentiation. The sequence of Giardia lamblia enolase (gEno) is most similar to enolases in Homo sapiens and Leishmania mexicana, and shows the conserved catalytic and metal-binding residues. We used an integration vector to stably express wild type and mutant gEno. In trophozoites, wild type gEno localized to the cell membrane, caudal flagella and cytosol. gEno is present on the wall of mature cysts, but not in encystation secretory vesicles (ESV). The expression of gEno with a deletion of residues G167-K169, or mutations H389Q/R390S significantly inhibited excystation while mutation of residue D257K had no effect. These results suggest a role for enolase in regulation of Giardia excystation.

Rab11 and actin cytoskeleton participate in Giardia lamblia encystation, guiding the specific vesicles to the cyst wall.

BACKGROUND: Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol. CONCLUSIONS AND SIGNIFICANCE: Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and suggest that Rab11 and actin may be useful as novel pharmacological targets.