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.

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.

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.

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.

Changes in the incidence of intestinal giardiosis in Mexican population during five years (2011-2015).

Giardiosis is a parasitic disease caused by the protozoan Giardia intestinalis, which is distributed worldwide. Most of the data on the prevalence of giardiosis in Mexico comes from research, but it is also necessary to study the data provided by the Mexican Health Ministry and issued by the General Directorate of Epidemiology. The aim of this work was analyse the national surveillance data for human giardiosis in order to update the epidemiological data of this disease in Mexico. A retrospective observational analysis of giardiosis (from January 2011 to December 2015) was performed in the annual reports emitted by the GDE in Mexico. The cases were classified by year, state, age group, gender and seasons of the year. During the period of 2011-2015, a reduction of 38.51% was observed in the total number of new cases of giardiosis reported in the whole country The states of Sinaloa, Yucatan, and Chiapas presented the highest number of new cases reported during the analysed period. Giardiosis rates were always higher among women in all age groups, but the maximum incidence was observed in both sexes in the age group of 1-4 years old (the most susceptible group). On the other hand, the number of cases increased dramatically in southern states during warmer months. Giardiosis is influenced by ambient temperature changes along the year, although this study suggests that tends to decrease in all the analysed states and could be related to the overall improvement of hygienic practices within the Mexican population.

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.

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.

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.

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.

Participation of actin on Giardia lamblia growth and encystation.

BACKGROUND: Microfilaments play a determinant role in different cell processes such as: motility, cell division, phagocytosis and intracellular transport; however, these structures are poorly understood in the parasite Giardia lamblia. METHODOLOGY AND PRINCIPAL FINDINGS: By confocal microscopy using TRITC-phalloidin, we found structured actin distributed in the entire trophozoite, the label stand out at the ventral disc, median body, flagella and around the nuclei. During Giardia encystation, a sequence of morphological changes concurrent to modifications on the distribution of structured actin and in the expression of actin mRNA were observed. To elucidate whether actin participates actively on growth and encystation, cells were treated with Cytochalasin D, Latrunculin A and Jasplakinolide and analyzed by confocal and scanning electron microscopy. All drugs caused a growth reduction (27 to 45%) and changes on the distribution of actin. Besides, 60 to 80% of trophozoites treated with the drugs, exhibited damage at the caudal region, alterations in the flagella and wrinkles-like on the plasma membrane. The drugs also altered the cyst-yield and the morphology, scanning electron microscopy revealed diminished cytokinesis, cysts with damages in the wall and alterations in the size and on the intermembranal space. Furthermore, the drugs caused a significant reduction of the intensity of fluorescence-labeled CWP1 on ESV and on cyst wall, this was coincident with a reduction of CWP1 gene expression (34%). CONCLUSIONS AND SIGNIFICANCE: All our results, indicated an important role of actin in the morphology, growth and encystation and indirectly suggested an actin role in gene expression.