Dissection of Barrier Dysfunction in Organoid-Derived Human Intestinal Epithelia Induced by Giardia duodenalis.

BACKGROUND & AIMS: The protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed at overcoming these limitations and recapitulating the authentic series of pathogenic events in the primary human duodenal tissue by using the human organoid system. METHODS: We established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis-induced barrier breakdown by functional analysis of transcriptional, electrophysiological, and tight junction components. RESULTS: Organoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of the epithelial barrier upon G. duodenalis infection. Gene set enrichment analysis suggested major expression changes, including gene sets contributing to ion transport and tight junction structure. Solute carrier family 12 member 2 and cystic fibrosis transmembrane conductance regulator-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization, and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy. Functionally, barrier loss was linked to the adenosine 3',5'-cyclic monophosphate (cAMP)/protein kinase A-cAMP response element-binding protein signaling pathway. CONCLUSIONS: Data suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection during which alterations of cellular ion transport were followed by breakdown of the tight junctional complex and loss of epithelial integrity, events involving a cAMP/protein kinase A-cAMP response element-binding protein mechanism. These findings and the newly established organoid-derived model to study G. duodenalis infection may help to explore new options for intervening with disease and infection, in particular relevant for chronic cases of giardiasis.

Gastrointestinal disorders in Down syndrome.

Down syndrome is the most common human chromosomal disorder. Among clinical findings, one constant concern is the high prevalence of gastrointestinal system alterations. The aim of this study was to determine the prevalence of gastrointestinal disorders at a Down syndrome outpatient clinic during a 10-year follow-up period. Data from medical files were retrospectively reviewed from 1,207 patients. Gastrointestinal changes occurred in 612 (50.7%). The most prevalent disorder was chronic intestinal constipation. Intestinal parasite occurred in 22% (mainly giardiasis), gastroesophageal reflux disease in 14%, digestive tract malformations occurred in 5%: 13 cases of duodenal atresia, 8 of imperforate anus, 4 annular pancreases, 2 congenital megacolon, 2 esophageal atresias, 2 esophageal compression by anomalous subclavian and 1 case of duodenal membrane. We had 38/1,207 (3.1%) patients with difficulty in sucking and only three with dysphagia that resolved before the second year of life. Peptic ulcer disease, celiac disease, and biliary lithiasis were less prevalent with 3% each. Awareness of the high prevalence of gastrointestinal disorders promotes outstanding clinical follow-up as well as adequate development and greater quality of life for patients with Down syndrome and their families.

Increased Innate Lymphoid Cell 3 and IL-17 Production in Mouse Lamina Propria Stimulated with Giardia lamblia.

Innate lymphoid cells (ILCs) are key players during an immune response at the mucosal surfaces, such as lung, skin, and gastrointestinal tract. Giardia lamblia is an extracellular protozoan pathogen that inhabits the human small intestine. In this study, ILCs prepared from the lamina propria of mouse small intestine were incubated with G. lamblia trophozoites. Transcriptional changes in G. lamblia-exposed ILCs resulted in identification of activation of several immune pathways. Secretion of interleukin (IL)-17A, IL-17F, IL-1ß, and interferon-γ was increased, whereas levels of IL-13, IL-5, and IL22, was maintained or reduced upon exposure to G. lamblia. Goup 3 ILC (ILC3) was found to be dominant amongst the ILCs, and increased significantly upon co-cultivation with G. lamblia trophozoites. Oral inoculation of G. lamblia trophozoites into mice resulted in their presence in the small intestine, of which, the highest number of parasites was detected at the 5 days-post infection. Increased ILC3 was observed amongst the ILC population at the 5 days-post infection. These findings indicate that ILC3 from the lamina propria secretes IL-17 in response to G. lamblia, leading to the intestinal pathology observed in giardiasis.

Cysteine Protease-Dependent Mucous Disruptions and Differential Mucin Gene Expression in Giardia duodenalis Infection.

The intestinal mucous layer provides a critical host defense against pathogen exposure and epithelial injury, yet little is known about how enteropathogens may circumvent this physiologic barrier. Giardia duodenalis is a small intestinal parasite responsible for diarrheal disease and chronic postinfectious illness. This study reveals a complex interaction at the surface of epithelial cells, between G. duodenalis and the intestinal mucous layer. Here, we reveal mechanisms whereby G. duodenalis evades and disrupts the first line of host defense by degrading human mucin-2 (MUC2), depleting mucin stores and inducing differential gene expression in the mouse small and large intestines. Human colonic biopsy specimens exposed to G. duodenalis were depleted of mucus, and in vivo mice infected with G. duodenalis had a thinner mucous layer and demonstrated differential Muc2 and Muc5ac mucin gene expression. Infection in Muc2-/- mice elevated trophozoite colonization in the small intestine and impaired weight gain. In vitro, human LS174T goblet-like cells were depleted of mucus and had elevated levels of MUC2 mRNA expression after G. duodenalis exposure. Importantly, the cysteine protease inhibitor E64 prevented mucous degradation, mucin depletion, and the increase in MUC2 expression. This article describes a novel role for Giardia's cysteine proteases in pathogenesis and how Giardia's disruptions of the mucous barrier facilitate bacterial translocation that may contribute to the onset and propagation of disease.

Molecular Characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Captive Wildlife at Zhengzhou Zoo, China.

Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common gastrointestinal protists in humans and animals. Two hundred and three fecal specimens from 80 wildlife species were collected in Zhengzhou Zoo and their genomic DNA extracted. Three intestinal pathogens were characterized with a DNA sequence analysis of different loci. Cryptosporidium felis, C. baileyi, and avian genotype III were identified in three specimens (1.5%), the manul, red-crowned crane, and cockatiel, respectively. Giardia duodenalis was also found in five specimens (2.5%) firstly: assemblage B in a white-cheeked gibbon and beaver, and assemblage F in a Chinese leopard and two Siberian tigers, respectively. Thirteen genotypes of E. bieneusi (seven previously reported genotypes and six new genotypes) were detected in 32 specimens (15.8%), of which most were reported for the first time. A phylogenetic analysis of E. bieneusi showed that five genotypes (three known and two new) clustered in group 1; three known genotypes clustered in group 2; one known genotype clustered in group 4; and the remaining four genotypes clustered in a new group. In conclusion, zoonotic Cryptosporidium spp., G. duodenalis, and E. bieneusi are maintained in wildlife and transmitted between them. Zoonotic disease outbreaks of these infectious agents possibly originate in wildlife reservoirs.

[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.

Harnessing the power of new genetic tools to illuminate Giardia biology and pathogenesis.

Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially tetraploid) Giardia trophozoites have presented a formidable challenge to the development of molecular genetic tools to interrogate gene function. High sequence divergence and the high percentage of hypothetical proteins lacking homology to proteins in other eukaryotes have limited our understanding of Giardia protein function, slowing drug target validation and development. For more than 25 years, Giardia A and B assemblages have been readily amenable to transfection with plasmids or linear DNA templates. Here, we highlight the utility and power of genetic approaches developed to assess protein function in Giardia, with particular emphasis on the more recent clustered regularly interspaced palindromic repeats/Cas9-based methods for knockdowns and knockouts. Robust and reliable molecular genetic approaches are fundamental toward the interrogation of Giardia protein function and evaluation of druggable targets. New genetic approaches tailored for the double diploid Giardia are imperative for understanding Giardia's unique biology and pathogenesis.

Comparative genomics of Giardia duodenalis sub-assemblage AI beaver (Be-2) and human (WB-C6) strains show remarkable homozygosity, sequence similarity, and conservation of VSP genes.

Giardia duodenalis, a major cause of waterborne infection, infects a wide range of mammalian hosts and is subdivided into eight genetically well-defined assemblages named A through H. However, fragmented genomes and a lack of comparative analysis within and between the assemblages render unclear the molecular mechanisms controlling host specificity and differential disease outcomes. To address this, we generated a near-complete de novo genome of AI assemblage using the Oxford Nanopore platform by sequencing the Be-2 genome. We generated 148,144 long-reads with quality scores of > 7. The final genome assembly consists of only nine contigs with an N50 of 3,045,186 bp. This assembly agrees closely with the assembly of another strain in the AI assemblage (WB-C6). However, a critical difference is that a region previously placed in the five-prime region of Chr5 belongs to Chr4 of Be-2. We find a high degree of conservation in the ploidy, homozygosity, and the presence of cysteine-rich variant-specific surface proteins (VSPs) within the AI assemblage. Our assembly provides a nearly complete genome of a member of the AI assemblage of G. duodenalis, aiding population genomic studies capable of elucidating Giardia transmission, host range, and pathogenicity.

Molecular analysis of household transmission of Giardia lamblia in a region of high endemicity in Peru.

BACKGROUND: Giardia lamblia is ubiquitous in multiple communities of nonindustrialized nations. Genotypes A1, A2, and B (Nash groups 1, 2, and 3, respectively) are found in humans, whereas genotypes C and D are typically found in dogs. However, genotypes A and B have occasionally been identified in dogs. METHODS: Fecal Giardia isolates from 22 families and their dogs, living in Pampas de San Juan, were collected over 7 weeks in 2002 and 6 weeks in 2003. Samples were genotyped, followed by sequencing and haplotyping of many of these isolates by using loci on chromosomes 3 and 5. RESULTS: Human infections were all caused by isolates of genotypes A2 and B. Human coinfections with genotypes A2 and B were common, and the reassortment pattern of different subtypes of A2 isolates supports prior observations that suggested recombination among genotype A2 isolates. All dogs had genotypes C and/or D, with one exception of a dog with a mixed B/D genotype infection. CONCLUSIONS: In a region of high endemicity where infected dogs and humans constantly commingle, different genotypes of Giardia are almost always found in dogs and humans, suggesting that zoonotic transmission is very uncommon.

Genotypes of Giardia duodenalis in Household Dogs and Cats from Poland.

BACKGROUND: Giardia duodenalis is a widespread protozoan parasite affecting humans and many species of animals, including dogs and cats. Due to its zoonotic potential, it is important to know the frequency of this parasite in companion animals. The aim of this study was to determine current epidemiological status of G. duodenalis in household dogs and cats. METHODS: In this study, 293 fecal samples from pet dogs and cats were collected from January 2017 to July 2019 and tested for G. duodenalis by PCR (using ß-giardin gene). The animals were divided into groups depending on their age, breed and fecal consistency. RESULTS: The examination allowed for detection of G. duodenalis in 6.0% of canine and 3.9% of feline fecal samples. The highest frequency was revealed in young (under one-year old) dogs. Sequencing confirmed the presence of assemblages C and D in dogs and A and F in cats. CONCLUSION: The study showed current frequency of G. duodenalis in dogs and cats and also revealed the occurrence of host-specific assemblages as well as zoonotic assemblage A.

Identification of Actin Filament-Associated Proteins in Giardia lamblia.

The deep-branching protozoan parasite Giardia lamblia is the causative agent of the intestinal disease giardiasis. Consistent with its proposed evolutionary position, many pathways are minimalistic or divergent, including its actin cytoskeleton. Giardia is the only eukaryote known to lack all canonical actin-binding proteins. Previously, our lab identified a number of noncanonical Giardia lamblia actin (GlActin) interactors; however, these proteins appeared to interact only with monomeric or globular actin (G-actin) rather than with filamentous actin (F-actin). To identify F-actin interactors, we used a chemical cross-linker to preserve native interactions followed by an anti-GlActin antibody, protein A affinity chromatography, and liquid chromatography coupled to mass spectrometry. We found 46 putative actin interactors enriched under the conditions favoring F-actin. Data are available via ProteomeXchange with identifier PXD026067. None of the proteins identified contain known actin-interacting motifs, and many lacked conserved domains. Each potential interactor was then tagged with the fluorescent protein mNeonGreen and visualized in live cells. We categorized the proteins based on their primary localization; localizations included ventral disc, marginal plate, nuclei, flagella, plasma membrane, and internal membranes. One protein from each of the six categories was colocalized with GlActin using immunofluorescence microscopy. We also co-immunoprecipitated one protein from each category and confirmed three of the six potential interactions. Most of the localization patterns are consistent with previously demonstrated GlActin functions, but the ventral disc represents a new category of actin interactor localization. These results suggest a role for GlActin in ventral disc function, which has previously been controversial. IMPORTANCE Giardia lamblia is an intestinal parasite that colonizes the small intestine and causes diarrhea, which can lead to dehydration and malnutrition. Giardia actin (GlActin) has a conserved role in Giardia cells, despite being a highly divergent protein with none of the conserved regulators found in model organisms. Here, we identify and localize 46 interactors of polymerized actin. These putative interactors localize to a number of places in the cell, underlining GlActin's importance in multiple cellular processes. Surprisingly, eight of these proteins localize to the ventral disc, Giardia's host attachment organelle. Since host attachment is required for infection, proteins involved in this process are an appealing target for new drugs. While treatments for Giardia exist, drug resistance is becoming more common, resulting in a need for new treatments. Giardia and human systems are highly dissimilar, thus drugs specifically tailored to Giardia proteins would be less likely to have side effects.

Giardia intestinalis in Thailand: identification of genotypes.

This study was undertaken to determine the genetic diversities of Giardia intestinalis isolated in Thailand. G. intestinalis cysts were collected from stool samples of 61 subjects residing in Bangkok or in rural communities of Thailand with and without gastrointestinal symptoms. All the cyst samples gave positive tpi amplicons (100% sensitivity), either of the 148- or the 81-bp tpi segments. Cyst assemblage identification of the 148- and 81-bp tpi gene segments by polymerase chain reaction showed that 8% of the cysts were assemblage A, 41% assemblage A and B combined, and 51% assemblage B. The prevalence of assemblage A was significantly lower than that of assemblage B and the mixed types. Restriction fragment length polymorphism (RFLP) of the 384-bp beta-giardin gene segment revealed that 12% and 88% of the assemblage A cysts were AI and AII respectively. RFLP, based on the 432-bp gdh gene segment, showed 45.5% of the assemblage B cysts to be BIII and 54.5% to be BIV. The AI sub-assemblage was less prevalent than the others. All subjects with AI and 50% of the subjects with BIII sub-assemblage cysts were symptomatic; 80% of symptomatic Bangkok residents were adults/elderly while 85% of the rural cases were children.

What's eating you? An update on Giardia, the microbiome and the immune response.

Giardia intestinalis has been observed in human stools since the invention of the microscope. However, it was not recognized as a pathogen until experimental infections in humans in the 1950s resulted in diarrheal illness [1]. We now know that this protozoan is capable of inducing a malabsorptive diarrhea and that the parasite is a major contributor to stunting in young children [2]. However, the majority of infections with this parasite are not accompanied by overt diarrhea and several studies indicate that it actually has a protective effect against moderate-severe diarrhea [3]. There is therefore significant interest in the mechanisms responsible for the wide variation observed in the clinical outcomes of infection with Giardia. This review will highlight recent work on the interactions among the parasite, the host microbiome and the immune response as contributing to this variation.

Gene polymorphism in transforming growth factor-beta codon 10 is associated with susceptibility to Giardiasis.

Secretory immunoglobulin A (S-IgA) antibodies have a central role in anti-Giardial defence. It has been demonstrated that transforming growth factor-beta1 (TGF-beta1) stimulates B lymphocytes to produce and secrete S-IgA. We sought to determine the association between TGF-beta1 polymorphism (T+869C) with susceptibility to Giardiasis. The TGF-beta1 genotypes and levels of salivary (S-IgA) were analysed in individuals with Giardiasis (97 symptomatic and 57 asymptomatic) and controls (n = 92). Individuals with symptomatic Giardiasis had the lowest levels of S-IgA compared to individuals in asymptomatic Giardiasis and control groups (97%, 73% and 43%, <1 g L(-1), respectively, P = 0.002). The frequency of allele C and CC genotypes of TGF-beta1 polymorphism was significantly higher among symptomatic patients than asymptomatic and control groups. Logistic regression analysis demonstrated that the individuals homozygous for allele C of TGF-beta1 had a significantly higher risk for symptomatic Giardiasis with odds ratio of 2.76 (95% CI: 3.88, 1.71, P = 0.007). Among the participants with TT genotype per cent of individuals with S-IgA level of more than 1 g L(-1) was almost twice the percentage in CC genotype individuals (14% versus 7% respectively P = 0.01). Our data suggest that CC genotype of TGF-beta1 polymorphism at codon 10 is associated with occurrence of Giardiasis.

Encystation commitment in Giardia duodenalis: a long and winding road.

Cholesterol and bile salts are relevant modulators of Giardia encystation. Although several molecules within signaling cascades have been identified, and changes in their expression observed during giardial encystation, their underlying interactions leading to expression of cyst wall markers (CWPs and precursors of the GalNAc homopolymer) are not well defined. Recent experimental data and the completion of the Giardia Genome Project Database (GiardiaDB) allow us now to consider the role of bile salts as "natural stimuli" and the potential involvement of a Raf/MEK/ERK pathway mediating cholesterol-regulated expression of cyst-specific genes. These new findings may provide promising targets for diagnostics, drug design and prophylactic intervention against giardiasis.

Molecular epidemiology of Giardia and Cryptosporidium infections - What's new?

New information generated since 2016 from the application of molecular tools to infections with Giardia and Cryptosporidium is critically summarised. In the context of molecular epidemiology, nomenclature, taxonomy, in vitro culture, detection, zoonoses, population genetics and pathogenicity, are covered. Whole genome sequencing has had the greatest impact in the last three years. Future advances will provide a much better understanding of the zoonotic potential of both parasites, their diversity and how this is linked to pathogenesis in different hosts.

Delayed development of the protective IL-17A response following a Giardia muris infection in neonatal mice.

Giardia is an intestinal protozoan parasite that has the ability to infect a wide range of hosts, which can result in the clinical condition 'giardiasis'. Over the years, experimental research has shown the crucial involvement of IL-17A to steer the protective immune response against Giardia. The development of the protective response, as reflected by a significant drop in cyst secretion, typically takes around 3 to 4 weeks. However, early-life infections often have a more chronic character lasting for several weeks or months. Therefore, the aim of the current study was to investigate the dynamics of a Giardia muris infection and the subsequent host immune response in neonatal mice infected 4 days after birth. The outcome of the study showed that a G. muris infection in pre-weaned mice failed to trigger a protective IL-17A response, which could explain the prolonged course of infection in comparison to older mice. Only after weaning, a protective intestinal immune response started to develop, characterized by an upregulation of IL-17A and Mbl2 and the secretion of parasite-specific IgA.

Structure-based identification of a potential non-catalytic binding site for rational drug design in the fructose 1,6-biphosphate aldolase from Giardia lamblia.

Giardia lamblia is the causal agent of giardiasis, one of the most prevalent parasitosis in the world. Even though effective pharmacotherapies against this parasite are available, the disadvantages associated with its use call for the development of new antigiardial compounds. Based on the Giardia dependence on glycolysis as a main energy source, glycolytic enzymes appear to be attractive targets with antiparasitic potential. Among these, fructose 1,6-biphosphate aldolase (GlFBPA) has been highlighted as a promising target for drug design. Current efforts are based on the design of competitive inhibitors of GlFBPA; however, in the kinetic context of metabolic pathways, competitive inhibitors seem to have low potential as therapeutic agents. In this work, we performed an experimental and in silico structure-based approach to propose a non-catalytic binding site which could be used as a hot spot for antigardial drug design. The druggability of the selected binding site was experimentally tested; the alteration of the selected region by site directed mutagenesis disturbs the catalytic properties and the stability of the enzyme. A computational automated search of binding sites supported the potential of this region as functionally relevant. A preliminary docking study was performed, in order to explore the feasibility and type of molecules to be able to accommodate in the proposed binding region. Altogether, the results validate the proposed region as a specific molecular binding site with pharmacological potential.

A modified PCR-RFLP method to determine genetic diversity of Giardia lamblia human isolates based on triosephosphate isomerase (TPI) gene.

An infection of digestive system, Giardiasis, caused by tiny parasites called Giardia lamblia (also known Giardia intestinalis or Giardia duodenalis). Giardia sp. is the most common intestinal parasite of humans and other animals throughout the world. Isolates of G. lamblia are classified into eight assemblages based on isoenzyme and DNA analyses. Assemblages A and B infect humans and a broad range of other hosts. The purpose of this study was to genotype human isolates of G. lamblia by PCR-RFLP in Karaj city. 60 positive fecal samples of G. lamblia were collected. DNA extraction and amplification of TPI gene were successfully conducted by nested-PCR. Subsequently, all samples were positive. Sequencing on 5 samples was conducted to determine genetic differences. The presence of 2 genotypes of G. lamblia (A and B) was revealed by the alignment of the TPI sequences obtained with reference sequences. The results of RFLP technique show that 35 of 60 (58.3%) isolates belonged to assemblage A, and 17 of 60 (28.3%) belonged to assemblage B but 1(1.7%) sample was not determined. Whereas, 7 (11.6%) specimens were detected as mixed infections. The latter RFLP was carried out to identify subtypes.The final results were 100% (35/35) AII, 82.3% (14/17) BIII, and 17.7% (3/17) BIV. This study suggests that the modified RFLP method is favorably time saving and easily achievable and highly economical. Hence, the sub-assemblage AII might be dominant in Karaj city.

Giardia secretome highlights secreted tenascins as a key component of pathogenesis.

Background: Giardia is a protozoan parasite of public health relevance that causes gastroenteritis in a wide range of hosts. Two genetically distinct lineages (assemblages A and B) are responsible for the human disease. Although it is clear that differences in virulence occur, the pathogenesis and virulence of Giardia remain poorly understood. Results: The genome of Giardia is believed to contain open reading frames that could encode as many as 6000 proteins. By successfully applying quantitative proteomic analyses to the whole parasite and to the supernatants derived from parasite culture of assemblages A and B, we confirm expression of ∼1600 proteins from each assemblage, the vast majority of which are common to both lineages. To look for signature enrichment of secreted proteins, we considered the ratio of proteins in the supernatant compared with the pellet, which defined a small group of enriched proteins, putatively secreted at a steady state by cultured growing trophozoites of both assemblages. This secretome is enriched with proteins annotated to have N-terminal signal peptide. The most abundant secreted proteins include known virulence factors such as cathepsin B cysteine proteases and members of a Giardia superfamily of cysteine-rich proteins that comprise variant surface proteins, high-cysteine membrane proteins, and a new class of virulence factors, the Giardia tenascins. We demonstrate that physiological function of human enteric epithelial cells is disrupted by such soluble factors even in the absence of the trophozoites. Conclusions: We are able to propose a straightforward model of Giardia pathogenesis incorporating key roles for the major Giardia-derived soluble mediators.