Identification of differentially expressed genes in a Giardia lamblia WB C6 clone resistant to nitazoxanide and metronidazole

Objectives The characterization of differential gene expression in Giardia lamblia WB C6 strain C4 resistant to metronidazole and nitazoxanide using microarray technology and quantitative real-time PCR. Methods In a previous study, we created and characterized the G. lamblia WB C6 clone C4 resistant to nitazoxanide and metronidazole. In this study, using a microarray-based approach, we have identified open-reading frames (ORFs) that were differentially expressed in C4 when compared with its wild-type WB C6. Using quantitative real-time PCR, we have validated the expression patterns of some of those ORFs, focusing on chaperones such as heat-shock proteins in wild-type and C4 trophozoites. In order to induce an antigenic shift, trophozoites of both strains were subjected to a cycle of en- and excystation. Expression of selected genes and resistance to nitazoxanide and metronidazole were investigated after this cycle. Results Forty of a total of 9115 ORFs were found to be up-regulated and 46 to be down-regulated in C4 when compared with wild-type. After a cycle of en- and excystation, resistance of C4 to nitazoxanide and metronidazole was lost. Resistance formation and en-/excystation were correlated with changes in expression of ORFs encoding for major surface antigens such as the variant surface protein TSA417 or AS7 (‘antigenic shift'). Moreover, expression patterns of the cytosolic heat-shock protein HSP70 B2, HSP40, and of the previously identified nitazoxanide-binding proteins nitroreductase and protein disulphide isomerase PDI4 were correlated with resistance and loss of resistance after en-/excystation. C4 trophozoites had a higher thermotolerance level than wild-type trophozoites. After en-/excystation, this tolerance was lost. Conclusions These results suggest that resistance formation in Giardia to nitazoxanide and metronidazole is correlated with altered expression of genes involved in stress response such as heat-shock protein

Identification of differentially expressed genes in a Giardia lamblia WB C6 clone resistant to nitazoxanide and metronidazole.

OBJECTIVES The characterization of differential gene expression in Giardia lamblia WB C6 strain C4 resistant to metronidazole and nitazoxanide using microarray technology and quantitative real-time PCR. METHODS In a previous study, we created and characterized the G. lamblia WB C6 clone C4 resistant to nitazoxanide and metronidazole. In this study, using a microarray-based approach, we have identified open-reading frames (ORFs) that were differentially expressed in C4 when compared with its wild-type WB C6. Using quantitative real-time PCR, we have validated the expression patterns of some of those ORFs, focusing on chaperones such as heat-shock proteins in wild-type and C4 trophozoites. In order to induce an antigenic shift, trophozoites of both strains were subjected to a cycle of en- and excystation. Expression of selected genes and resistance to nitazoxanide and metronidazole were investigated after this cycle. RESULTS Forty of a total of 9115 ORFs were found to be up-regulated and 46 to be down-regulated in C4 when compared with wild-type. After a cycle of en- and excystation, resistance of C4 to nitazoxanide and metronidazole was lost. Resistance formation and en-/excystation were correlated with changes in expression of ORFs encoding for major surface antigens such as the variant surface protein TSA417 or AS7 ('antigenic shift'). Moreover, expression patterns of the cytosolic heat-shock protein HSP70 B2, HSP40, and of the previously identified nitazoxanide-binding proteins nitroreductase and protein disulphide isomerase PDI4 were correlated with resistance and loss of resistance after en-/excystation. C4 trophozoites had a higher thermotolerance level than wild-type trophozoites. After en-/excystation, this tolerance was lost. CONCLUSIONS These results suggest that resistance formation in Giardia to nitazoxanide and metronidazole is correlated with altered expression of genes involved in stress response such as heat-shock proteins

Nitroreductase (GlNR1) increases susceptibility of Giardia lamblia and Escherichia coli to nitro drugs

Objectives The protozoan parasite Giardia lamblia causes the intestinal disease giardiasis, which may lead to acute and chronic diarrhoea in humans and various animal species. For treatment of this disease, several drugs such as the benzimidazole albendazole, the nitroimidazole metronidazole and the nitrothiazolide nitazoxanide are currently in use. Previously, a G. lamblia nitroreductase 1 (GlNR1) was identified as a nitazoxanide-binding protein. The aim of the present project was to elucidate the role of this enzyme in the mode of action of the nitro drugs nitazoxanide and metronidazole. Methods Recombinant GlNR1 was overexpressed in both G. lamblia and Escherichia coli (strain BL21). The susceptibility of the transfected bacterial and giardial cell lines to nitazoxanide and metronidazole was analysed. Results G. lamblia trophozoites overexpressing GlNR1 had a higher susceptibility to both nitro drugs. E. coli were fully resistant to nitazoxanide under both aerobic and semi-aerobic growth conditions. When grown semi-aerobically, bacteria overexpressing GlNR1 became susceptible to nitazoxanide. Conclusions These findings suggest that GlNR1 activates nitro drugs via reduction yielding a cytotoxic produc

Metabolism of nitro drugs metronidazole and nitazoxanide in Giardia lamblia: characterization of a novel nitroreductase (GlNR2)

Objectives The protozoan parasite Giardia lamblia causes giardiasis, a persistent diarrhoea. Nitro drugs such as the nitroimidazole metronidazole and the nitrothiazolide nitazoxanide are used for the treatment of giardiasis. Nitroreductases may play a role in activating these drugs. G. lamblia contains two nitroreductases, GlNR1 and GlNR2. The aim of this work was to elucidate the role of GlNR2. Methods Expression of GlNR2 was analysed by reverse transcription PCR. Recombinant GlNR2 was overexpressed in G. lamblia and drug susceptibility was analysed. Recombinant GlNR2 was subjected to functional assays. Escherichia coli expressing full-length or truncated GlNR1 and GlNR2 were grown in the presence of nitro compounds. Using E. coli reporter strains for nitric oxide and DNA damage responses, we analysed whether GlNR1 and GlNR2 elicited the respective responses in the presence, or absence, of the drugs. Results G. lamblia trophozoites overexpressing GlNR2 were less susceptible to both nitro drugs as compared with control trophozoites. GlNR2 was a functional nitroreductase when expressed in E. coli. E. coli expressing GlNR1 was more susceptible to metronidazole under aerobic and semi-aerobic and to nitazoxanide under semi-aerobic growth conditions. E. coli expressing GlNR2 was not susceptible to either drug. In reporter strains, GlNR1, but not GlNR2, elicited nitric oxide and DNA repair responses, even in the absence of nitro drugs. Conclusions These findings suggest that GlNR2 is an active nitroreductase with a mode of action different from that of GlNR1. Thus, susceptibility to nitro drugs may depend not only on activation, but also on inactivation of the drugs by specific nitroreductase

Membrane-associated proteins in giardia lamblia

The manner in which membrane-associated proteins interact with the membrane defines their subcellular fate and function. This interaction relies on the characteristics of the proteins, their journey after synthesis, and their interaction with other proteins or enzymes. Understanding these properties may help to define the function of a protein and also the role of an organelle. In the case of microorganisms like protozoa parasites, it may help to understand singular features that will eventually lead to the design of parasite-specific drugs. The protozoa parasite Giardia lamblia is an example of a widespread parasite that has been infecting humans and animals from ancestral times, adjusting itself to the changes of the environment inside and outside the host. Several membrane-associated proteins have been posted in the genome database GiardiaDB, although only a few of them have been characterized. This review discusses the data regarding membrane-associated proteins in relationship with lipids and specific organelles and their implication in the discovery of anti-giardial therapies.Fil: Touz, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Feliziani, Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Ropolo, Andrea Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin

Giardiasis treatment: an update with a focus on refractory disease

Postponed access: the file will be accessible after 2021.10.01Purpose of review Giardiasis remains a common cause of diarrhea and intestinal enteropathy globally. Here we give an overview of clinical treatment studies and discuss potential mechanisms and molecular targets for in-vitro testing of drug resistance. Recent findings Giardia is a cause of disease both in diarrheal and nondiarrheal cases. The prevalence of treatment refractory giardiasis is increasing. Recent studies reveal 5-nitroimidazole refractory infection occurs in up to 50% of cases. Mechanisms of drug resistance are not known. Placebo controlled studies of drug efficacy, taking the self-limiting course of giardiasis into account, has not been reported. No randomized controlled trials of treatment of refractory infection have been performed the last 25 years. Based on the clinical studies reported, combination treatment with a 5-nitroimidazole and a benzimidazole is more effective than repeated courses of 5-nitroimidazole or monotherapies in refractory cases. Quinacrine is effective in refractory cases, but potentially severe side effects limit its use. Summary A combination of a 5-nitroimidazole and albendazole or mebendazole, and quinacrine monotherapy, are rational choices in nitroimidazole refractory infections, but randomized controlled studies are needed. Further research into more recent clinical isolates is necessary to uncover mechanisms for the increase in metronidazole refractory giardiasis observed during the last decade.acceptedVersio

Giardia Lamblia and Giardiasis

         طفيلي الجيارديا اللامبليا من الاوالي المعوية الواسعة الانتشار للإنسان واللبائن الاخرى وقد سجلت الدراسات نسب معدل انتشار الاصابة 200مليون شخص سنويا مصاب بطفيلي الجيارديا لامبلية. اكتشف الطفيلي من قبل العالم فان ليفنهوك سنة 1961.دورة حياة الطفيلي تتضمن طورين هما الطور المتغذي المسؤول عن الامراضية والطور المتكيس المسؤول عن الاصابة وكلاهما موجود في براز الشخص المصاب .ينتقل الطفيلي بصورة رئيسية عن طريق وصول البراز من اليد الملوثة الى الفم . تتميز الاصابة بداء الجيارديا بسوء الامتصاص للامعاء الدقيقة والاسهال الدهني .امراضية الطفيلي تنتج عن الضرر المباشر للطبقة الطلائية للامعاء او الانسداد الميكانيكي لزغابات الامعاء والامتصاص . الكشف عن الطفيلي يعتمد على تمييز الطور المتغذي والطور المتكيس للطفيلي من خلال الفحص المجهري المباشر وكذلك عن طريق الكشف عن مستضدات الطفيلي باستخدام عينة البراز. هناك اختبارات اخرى مثل اختبار الاليزا واختبار الاجسام المضادة المشعة واختبار تفاعل سلسلة البلمرة. الاصابة بطفيلي الجيارديا ممكن ان يعالج بشكل فعال بالميترانيدازول كخطوة اولى للعلاج.      Giardia lamblia is widespread enteric protozoan parasite of human and other mammals . Prevalence data suggest that 200 million people are infected with Giardia lamblia. The parasite has been discovered by van Leeuwenhoek in 1961. Life cycle of G.lamblia  has two stages trophozoite and cyst of which trophozoite is the pathogenic stage and cyst is infective stage and both of them found in fecal material of patient with giardiasis. The parasite transmitted mainly by oral –fecal route .Giardia infection which is characterized by malabsorbtion of small intestine and fatty diarrhea  . Pathogenicity of Giardia trophozoite occurs due to direct damage of intestinal lining or mechanical blockage of intestinal villi and absorption. Identification of parasite is based on recognized of trophozoite and cyst microscopically and also by detection of antigen from fecal sample .ELISA test ,Direct flurescentantibody test and PCR test are also helpful ,Giardia infection can be effectively treated by metronidazole as primary stapes of treatment

A comparison of the Giardia lamblia trophozoite and cyst transcriptome using microarrays

<p>Abstract</p> <p>Background</p> <p>Compared with many protists, <it>Giardia lamblia </it>has a simple life cycle alternating between cyst and trophozoite. Most research on the molecular biology of <it>Giardia </it>parasites has focused on trophozoites and the processes of excystation and encystation, whereas cysts have attracted less interest. The striking morphological differences between the dormant cyst and the rapidly dividing and motile trophozoite implies profound changes in the metabolism as the parasite encysts in the host's intestine and excysts upon ingestion by a new host.</p> <p>Results</p> <p>To investigate the magnitude of the transcriptional changes occurring during the <it>G. lamblia </it>life cycle we compared the transcriptome of <it>G. lamblia </it>trophozoites and cysts using single-color oligonucleotide microarrays. Cysts were found to possess a much smaller transcriptome, both in terms of mRNA diversity and abundance. Genes encoding proteins related to ribosomal functions are highly over-represented. The comparison of the transcriptome of cysts generated in culture or extracted from feces revealed little overlap, raising the possibility of significant biological differences between the two types of cysts.</p> <p>Conclusions</p> <p>The comparison of the <it>G. lamblia </it>cyst and trophozoite transcriptome showed that transcripts of most genes are present at a lower level in cysts. This global view of the cyst and trophozoite transcriptome complements studies focused on the expression of selected genes during trophozoite multiplication, encystation and excystation.</p