Giardia Cysteine Proteases: The Teeth behind the Smile.

Giardia duodenalis is one of the most prevalent human enteropathogens and a major cause of diarrheal disease worldwide. Cysteine proteases (CPs) have been identified as major virulence factors in protozoan parasites, playing important roles in disease pathogenesis and in parasitic life cycles. G. duodenalis exhibits high proteolytic activity, and CPs play significant roles in giardiasis. Giardia CPs are directly involved in intestinal epithelial junctional complex disruption, intestinal epithelial cell apoptosis, and degradation of host immune factors, including chemokines and immunoglobulins. Giardia CPs have also been implicated in mucus depletion and microbiota dysbiosis induced by the parasite. This review discusses the most recent advances in characterization of Giardia Assemblage A and B CPs, including cathepsin B (catB)-like proteases.

[DETERMINATION OF GIARDIA DUODENALIS GENOTYPES IN LAMBLIA-INFESTED PEOPLE IN THE CITY OF MOSCOW].

Our first experience in genotyping Giardia from Moscow residents, has shown that 4 and 2 of seven samples belong to G. duodenalis genotype A and genotype B, respectively; one sample was negative during amplification with two types of primers. Genotyping was Carried out using the specific primers TPIA and TPIB to the gene encoding for the enzyme triosephosphate isomerase from the parasite. Thus, further such investigations using a larger number of samples will be able to complement the epidemiology of Lamblia infection in Moscow residents.

Structural basis for inactivation of Giardia lamblia carbamate kinase by disulfiram.

Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC50 values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.

Host immunity and pathogen strain contribute to intestinal disaccharidase impairment following gut infection.

Infection or other inflammatory insults in the small intestine often result in reduced disaccharidase enzyme levels. Using a mouse model of giardiasis, we examined the role of host immunity and pathogen virulence in mediating disaccharidase deficiency postinfection (p.i.). C57BL/6J mice were infected with two strains, WB and GS, of the human parasite Giardia duodenalis. The levels of sucrase, maltase, and lactase decreased in wild-type mice p.i. with the GS strain but not with the WB strain. Both CD4-deficient and SCID mice failed to eliminate the infection and did not exhibit disaccharidase deficiency. ß(2)-Microglobulin knockout animals controlled infections similar to wild-type mice but exhibited no decrease in disaccharidase activity. Analysis of cytokine production by spleen and mesenteric lymph node cells showed production of IL-4, IL-10, IL-13, IL-17, IL-22, TNF-α, and IFN-γ p.i. with both WB and GS, with IFN-γ being the dominant cytokine for both parasite strains. Mesenteric lymph node cells produced lower levels of cytokines compared with splenocytes in response to parasite extract, although the overall pattern was similar. These data suggest that T cell responses mediate parasite clearance whereas also contributing to pathogenesis. They also demonstrate that differences in pathogen strain can also determine the outcome of infection and further our understanding of the clinical variation seen in human giardiasis.

Inducible nitric oxide synthase in duodenum of children with Giardia lamblia infection.

The investigation were performed on children with Giardia lamblia infection, diagnosed on the basis of positive stool tests for Giardia antigen (Elisa) or by microscopical detection of trophozoites in duodenal fluid. In duodenal biopsies morphological studies and immunohistochemical reaction for inducible nitric oxide synthase (iNOS) were performed. The control group was made up of duodenal tissue of children with excluded giardiasis and inflammation of the upper part of gastrointestinal tract. The duodenal biopsies from children without Giardia lamblia infection were found to have a high immunoreactivity for iNOS in enterocytes, the cells of intestinal crypts, endothelial cells of vessels and connective tissue cells of lamina propria. In children with giardiasis: in some biopsies the expression of iNOS was as high as in control group, in others was weaker detectable and the shortening of intestinal villi was seen. There were also duodenal biopsies with the lack of immunoreactivity for iNOS, with shorter villi and a large amount of mucus in the intestinal epithelium. Beside of goblet cells, also enterocytes were loaded with mucus. The pathological changes may cause malabsorption and also may have a negative influence on the defense of the intestinal wall against Giardia lamblia infection. The different morphological and immunohistochemical results in the duodenum of children with giardiasis can elucidate a variety of clinical symptoms from asymptomatic to severe infection.

Characterization, kinetics, and crystal structures of fructose-1,6-bisphosphate aldolase from the human parasite, Giardia lamblia.

Class I and class II fructose-1,6-bisphosphate aldolases (FBPA), glycolytic pathway enzymes, exhibit no amino acid sequence homology and utilize two different catalytic mechanisms. The mammalian class I FBPA employs a Schiff base mechanism, whereas the human parasitic protozoan Giardia lamblia class II FBPA is a zinc-dependent enzyme. In this study, we have explored the potential exploitation of the Giardia FBPA as a drug target. First, synthesis of FBPA was demonstrated in Giardia trophozoites by using an antibody-based fluorescence assay. Second, inhibition of FBPA gene transcription in Giardia trophozoites suggested that the enzyme is necessary for the survival of the organism under optimal laboratory growth conditions. Third, two crystal structures of FBPA in complex with the transition state analog phosphoglycolohydroxamate (PGH) show that the enzyme is homodimeric and that its active site contains a zinc ion. In one crystal form, each subunit contains PGH, which is coordinated to the zinc ion through the hydroxamic acid hydroxyl and carbonyl oxygen atoms. The second crystal form contains PGH only in one subunit and the active site of the second subunit is unoccupied. Inspection of the two states of the enzyme revealed that it undergoes a conformational transition upon ligand binding. The enzyme cleaves d-fructose-1,6-bisphosphate but not d-tagatose-1,6-bisphosphate, which is a tight binding competitive inhibitor. The essential role of the active site residue Asp-83 in catalysis was demonstrated by amino acid replacement. Determinants of catalysis and substrate recognition, derived from comparison of the G. lamblia FBPA structure with Escherichia coli FBPA and with a closely related enzyme, E. coli tagatose-1,6-bisphosphate aldolase (TBPA), are described.

Impact of helicobacter pylori infection on the activities of urease and lipase enzymes in patients with giardiasis.

The study of the effect of Giardia lamblia and Helicobacter pylori organisms coexistence on the activities of urease and lipase enzymes was the aim of this work which was done through choosing 50 cases of giardiasis in addition to 10 normal individuals chosen as a control group (free from giardiasis). It is well known that H. pylori is considered one of the most important causes of gastric and duodenal inflammations which could predispose to ulcers and hypochlorhydria leading to increased susceptibility to giardiasis as it is known that HCl acts as a chemical barrier to microbes. The biochemical tests were done to investigate the activity of both urease and lipase enzymes extracted from the gastric juice of patients and controls. A significant increase in urease activity in the group having combined infection (giardiasis and H.pylori) than the group infected with G.lamblia alone and the control group was found. The same findings were obtained regarding the lipase activity. In the present work, both infections H. pylori and G. lamblia coexisted in 75% of epigastric pain cases which could be explained on the basis that both organisms predispose to each other.

Neuronal nitric oxide synthase is necessary for elimination of Giardia lamblia infections in mice.

NO produced by inducible NO synthase (NOS2) is important for the control of numerous infections. In vitro, NO inhibits replication and differentiation of the intestinal protozoan parasite Giardia lamblia. However, the role of NO against this parasite has not been tested in vivo. IL-6-deficient mice fail to control Giardia infections, and these mice have reduced levels of NOS2 mRNA in the small intestine after infection compared with wild-type mice. However, NOS2 gene-targeted mice and wild-type mice treated with the NOS2 inhibitor N-iminoethyl-L-lysine eliminated parasites as well as control mice. In contrast, neuronal NOS (NOS1)-deficient mice and wild-type mice treated with the nonspecific NOS inhibitor NG-nitro-L-arginine methyl ester and the NOS1-specific inhibitor 7-nitroindazole all had delayed parasite clearance. Finally, Giardia infection increased gastrointestinal motility in wild-type mice, but not in SCID mice. Furthermore, treatment of wild-type mice with NG-nitro-L-arginine methyl ester or loperamide prevented both the increased motility and the elimination of parasites. Together, these data show that NOS1, but not NOS2, is necessary for clearance of Giardia infection. They also suggest that increased gastrointestinal motility contributes to elimination of the parasite and may also contribute to parasite-induced diarrhea. Importantly, this is the first example of NOS1 being involved in the elimination of an infection.

Giardia intestinalis: molecular characterization of UDP-N-acetylglucosamine pyrophosphorylase.

The flagellated protozoan Giardia intestinalis is one of the most prevalent human-infective parasites with a worldwide distribution. This parasite must encyst to complete the life cycle and N-acetylgalactosamine is produced from endogenous glucose for cyst wall synthesis during the transformation. UDP-N-acetylglucosamine pyrophosphorylase in G. intestinalis (GiUAP, EC 2.7.7.23) is the fourth enzyme in the inducible pathway of N-acetylgalactosamine biosynthesis, catalysing the conversion of N-acetylglucosamine-1-P to UDP-N-acetylglucosamine. In this study the gene GiUAP was cloned and sequenced from the Portland 1 strain using PCR techniques. It has an ORF of approximately 1.3 kb and contains no introns. BLAST and ClustalW analysis of the deduced amino acid sequence revealed significant similarities to other eukaryotic UAPs with putative active sites identified. Southern hybridization showed that GiUAP exists as a single-copy gene and it was shown to have two transcripts by RT-PCR and Northern hybridization. RLM-RACE identified both 5' and 3' untranslated regions and suggested the transcripts exist as a 5'-capped mRNA, with the use of two tandem polyadenylation sites to generate two unusually long giardial 3' untranslated regions of approximately 522 bp and approximately 3 kb. Moreover, a recombinant protein (rGiUAP) was expressed in E. coli and subjected to physical characterizations. Surprisingly the results obtained in this study were significantly different from those reported for the GiUAP in MR4 strain, suggesting this gene is under different transcription control in different strains of G. intestinalis. This report describes the molecular characterization of GiUAP and provides an opportunity to explore the control of gene expression during encystation of the parasite.

Giardia lamblia: expression of alkaline phosphatase activity in infected rat intestine.

Alkaline phosphatase (IAP) is a marker of intestinal microvillus membrane. Changes in IAP activity have been studied as a function of Giardia lamblia (G. lamblia) infection using rat as the experimental model. At day 11 and 15 post-infection, enzyme activity was reduced (p<0.01) compared to controls. The enzyme levels were essentially similar to control values by day 30 post-infection. Analysis of the enzyme activity in cell fractions across crypt-villus axis revealed a marked decrease in enzyme activity in the villus tip and mid villus regions but a considerable increase (p<0.01) in enzyme activity in the crypt base of 11 day post-infected animals compared to that in controls. The observed changes in IAP activity were confirmed by assaying the enzyme activity in acrylamide gels using bromo-chloro-indolyl phosphate staining under non-denaturing conditions. These findings indicate differential changes across the crypt-villus axis, but impaired alkaline phosphatase levels in G. lamblia infected rat intestine.

Fecal elastase-1 is decreased in villous atrophy regardless of the underlying disease.

BACKGROUND: Fecal elastase-1 (E1) is a sensitive and reliable test in the assessment of exocrine pancreatic function in cystic fibrosis (CF). In patients with celiac disease (CD), different E1 values have been reported. E1 levels in other malabsorption conditions are unknown. Therefore, the aim of the present study was to evaluate E1 concentrations in various malabsorption syndromes. MATERIAL AND METHODS: The study was carried out in 54 patients, selected from patients referred with suspicion of CF, who had been diagnosed as celiac disease (CD; n = 16), secondary malabsorption syndrome (SMS, giardiasis- or cow milk-related enteropathy; n = 18) and food allergy (FA; n = 20). 70 age-matched healthy children (HC) and 131 cystic fibrosis (CF) patients served as control groups. In CD and SMS patients, a gluten-free diet was introduced. In addition, SMS patients were treated appropriately to underlying disease. In all subjects, E1 concentrations were measured. In CD and SMS patients, E1 concentrations were repeatedly measured after one year of the treatment. RESULTS: With a cut-off level of 200 microg g-1, abnormal E1 concentrations were found in 87.2% of the CF group and in 56.2% and 50.0% of the CD and SMS subgroups, respectively. In none of FA patients, were E1 values below the normal range. After mucosal recovery, E1 concentrations in patients with CD and SMS increased, suggesting that villous atrophy can diminish exocrine pancreatic secretion. In 18 out of 19 CD and SMS patients with abnormal E1 concentrations, monitored for at least 12 months of a gluten-free diet, abnormal E1 concentrations increased above the cut-off value to normal range. Two out of the 54 referred patients were finally diagnosed as having CF, one with stable low E1 levels and the second with finally normal values. CONCLUSIONS: The exocrine pancreatic function is decreased in villous atrophy regardless of underlying disease. The specificity of the fecal elastase-1 test in the differentiation between 'primary' exocrine pancreatic insufficiency and intestinal malabsorption with mucosal atrophy is low. After mucosal regeneration, fecal elastase-1 specificity is high.

Effect of Giardia lamblia on duodenal disaccharidase levels in humans.

The study was conducted to detect the effect of giardiasis on human disaccharidase levels. Forty patients attending the medical outpatient department of PGIMER, Chandigarh were enrolled. Twenty patients, positive for Giardia lamblia comprised the study group while 20 patients negative for Giardia lamblia were taken as controls. Upper gastrointestinal endoscopy was performed in all patients. Estimation of lactase, sucrase, maltase and trehalase was done in biopsies. Histopathological investigation was carried out in all biopsy specimens after Haematoxylin and Eosin staining. Complaints of pain abdomen and bloating occurred commonly in giardiasis. Four biopsy samples in study group showed mild increase in lymphomononuclear infiltrate. Giardia lamblia was detected in 7 biopsies. Lactase levels were decreased significantly (p < 0.05) in giardiasis. Rest of the enzymes were comparable to the controls. No differences in the enzyme activities were observed between males and females in either group and with the duration of symptoms.

Purification of a fraction of Giardia lamblia trophozoite extract associated with disaccharidase deficiencies in immune Mongolian gerbils (Meriones unguiculatus).

The effects on disaccharidase activities of challenging gerbils previously exposed to Giardia lamblia with fradions of the crude trophozoite extract were examined. Gel filtration of the soluble extract on a Sephacryl S-200 HR column resulted in 3 fradions: F1, F2 and F3. Only a challenge with fraction F1 (0.1 mg total dose) was found to induce disaccharidase deficiencies. Boiling F1 prior to challenge did not change this effect on the enzyme activities. However, the decreases were not obtained when the total F1 dose was reduced to 0.05 mg. Column chromatography of fraction F1 under dissociating and reducing conditions resulted in 2 further fractions: F1a and F1b. Challenging immune gerbils with F1b led to impairments of disaccharidose activity similar to those obtained with F1. Protein analysis of the crude extract, as well as the fractions of the extract, revealed several high and low molecular weight bonds. These findings indicate that a constituent(s) of fraction F1b is the portion of the parasite which induces disaccharidase deficiencies in immune gerbils. This fraction consists of proteins ranging in molecular weight from 32 to 200 kDa. In addition the G. lamblia fraction involved in the decreases in enzyme activity is heat-stable.

Disaccharidase activity in male and female C57BL/6 mice infected with Giardia muris.

To elucidate the contribution of host and parasite factors in induction of small-intestinal disaccharidase deficiency in giardiasis, we determined the activity of four enzymes in male and female C57BL/6 mice infected with Giardia muris. Both male and female mice exhibited significant disaccharidase deficiency as shown by decreases in the activities of lactase, sucrase, trehalase and maltase on day 10 after infection. However, by 20 days after infection the females had normal enzyme activities, whereas those in males remained significantly reduced. Prolonged disaccharidase deficiency in the males was related to the course of the primary infection where males had higher parasite loads in the small intestine than did females on day 20 after infection. By day 40 after the primary infection the enzyme activities had returned to normal levels and were similar in male and female mice. Secondary exposure of mice to either the infective cysts or a crude extract of the trophozoites caused disaccharidase deficiency. The females had lower activities of sucrase and trehalase as compared with males after the challenge. Thus, during the primary infection, disaccharidase deficiency was strongly associated with parasite number, whereas after challenge infections the more resistant females had lower enzyme activities in the small intestine than did males.

Disaccharidase activity in the small intestine of susceptible and resistant mice after primary and challenge infections with Giardia muris.

The activities of four disaccharidases were examined in resistant (C57Bl/6) and susceptible (C3H/HeN) mice during the primary infection with Giardia muris and after challenge with either trophozoite extract or cysts. Significant decreases in lactase, sucrase, trehalase, and maltase activities in C57Bl/6 mice and lactase and sucrase activities in C3H/HeN mice in the anterior 25% of the small intestine were observed on day 10 after infection. The activities of maltase, sucrase, trehalase, and lactase in the jejunum of C3H/HeN mice were significantly reduced after challenge with trophozoite extract, when compared with the uninfected or infected, but not challenged animals. Decreases in enzyme activities of C3H/HeN mice were evident as early as 12 hours after challenge with the extract. The resistant C57Bl/6 mice showed little change in disaccharidase activity after challenge with trophozoite extract. On the other hand, challenge with cysts resulted in a few decreases in disaccharidase activities in both strains of mice: C57Bl/6 mice showed decreases in the duodenum, while disaccharidases of C3H/HeN mice had lower activity more posteriorly. Thus, challenge with parasite antigen results in a more severe disaccharidase deficiency in susceptible hosts when compared with resistant ones.

Alterations in enzymatic activities of the intestinal mucosa during the course of Giardia lamblia infection in mice.

Decline in the specific activities of intestinal cytosolic glucose-6-phosphate dehydrogenase (G6PD) and isocitrate dehydrogenase (ICDH); brush border glucoamylase, and isomaltase; and basolateral (Na+, K+)-ATPase activities were observed during the establishment, acute phase and decline phase of infection in Giardia lamblia-infected mice. The degree of decline in the activities of various enzymes correlated well with the number of trophozoites counted in the jejunum. There appeared to be a gradual recovery of enzymatic activities during the decline phase of infection, when the number of trophozoites also declined. The decline in activities of these enzymes may contribute to malabsorption of nutrients during giardiasis.

Growth, activities of enzymes in the small intestine, and ultrastructure of microvillous border in gerbils infected with Giardia duodenalis.

The aim of this study was to assess and correlate changes in weight gain, food intake, small intestinal disaccharidase activities and microvillous border surface area over the course of a primary Giardia duodenalis infection in weanling Mongolian gerbils (Meriones unguiculatus). Weight gain in infected animals was significantly impaired between days 8 and 20 postinoculation when compared to age- and weight-matched controls. No difference in food intake was observed between groups. Trophozoite population in the small intestine was maximal on day 4 and 6 of infection, and colonization persisted in the duodenum throughout the experiment (30 days). In infected gerbils, mucosal sucrase and maltase activities were significantly depressed in the duodenum and jejunum on day 4 and in all areas of the small intestine by day 6. Eight and 25 days postinoculation, disaccharidase activities had recovered in the jejunum and distal small intestine but remained depressed in the duodenum, the area where trophozoite colonization persisted. Diffuse loss of microvillous border surface area was observed in the duodenum and jejunum after 6 days of infection. Eight days postinoculation, microvillus surface area had returned to normal in the jejunum, but not in the duodenum. Our findings demonstrate that acute giardiasis in weanling gerbils impairs weight gain, depresses disaccharidase activities, and diffusely reduces mucosal microvillous border surface area.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of murine giardiasis on growth, intestinal morphology , and disaccharidase activity.

The aim of this study was to assess the effects of Giardia muris on host growth and food intake, small intestinal morphometrics, mucosal enzyme activities, and brush border ultrastructure. Weanling mice infected with 1,000 G. muris cysts were compared to control and pair-fed sham-treated animals. Infection with G. muris resulted in decreased food intake and retarded growth. In infected animals, villus atrophy was observed in the duodenum throughout the study period and in the jejunum on days 8 and 50. On day 30, whereas jejunal architecture returned to normal in infected animals, malnourished pair-fed animals exhibited a compensatory increase in villus height. Sucrase and maltase were depressed in infected animals on days 2-24. On day 8 jejunal disaccharidases in pair-fed animals were also decreased but to a lesser extent than in infected animals. On day 24, disaccharidase values for control and infected mice were similar, whereas values in pair-fed animals were increased. On day 8, jejunal microvilli were shorter in infected animals than in control and pair-fed animals. This brush border injury was present throughout the jejunum and was also observed in pair-fed animals, but to a lesser extent. These findings suggest that G. muris retards growth in weanling mice, results in small intestinal injury, and interferes with the compensatory response to malnutrition of the infected host. Villus atrophy and brush border enzyme deficiencies associated with the disease mainly occur in the duodenum and jejunum, where trophozoites are most numerous. In infected and in pair-fed animals, the decrease in jejunal disaccharidase activities correlated with a diffuse shortening of brush border microvilli.

[Acute hepatic lesion caused by Giardia lamblia]. / Lesión hepática aguda por Giardia lamblia.

A study was made of 20 rats infested by Giardia muris in which a histologic study was made of the liver, as well as of 25 patients with giardiasis and elevated alanine-aminotransferase levels. Patients with positive A or B hepatitis markers, cholelithiasis or history of drug or alcohol use were excluded. Tests of liver function and liver biopsy were performed and antiparasite therapy was given during three months of follow-up, after which the liver biopsy was repeated. Humoral alterations were compared to those of 30 patients with acute viral hepatitis (15 type A and 15 type B) over the same periods of time. In 20% of the rats, nonspecific liver lesions were found. In the patients liver enzymes and the thymol test normalized a month after treatment and serum bile acids became normal in the third month. The liver biopsy demonstrated hepatic damage in 94% of the patients (in 20 cases cell lesions and in 12 cases inflammatory lesions) which regressed in the third month, the follow-up biopsy being normal after eradication of the parasite was confirmed. The comparative study with viral hepatitis showed highly significant differences in all the variables studied during the follow-up stage. Emphasis is placed on the importance of this lesion and its differential diagnosis to prevent its progression to chronic liver disease.