Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase

Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.     

Giardipain-1, a protease secreted by Giardia duodenalis trophozoites,causes junctional,barrier and apoptotic damage in epithelial cell monolayers

The adhesion of Giardia duodenalis trophozoites to intestinal epithelial cells allows the onset and maintenance of giardiasis. During these interactions, epithelial cells can be committed to apoptosis by enzymes secreted by the parasites, including cysteine proteases that are increasingly identified as virulence factors in parasitic protozoa. In this work, a monoclonal antibody (mAb1G3) raised against G. duodenalis surface components was found to react with a 25?kDa protein expressed in the cell surface and flagella of G. duodenalis trophozoites. When trophozoites expressing this protein were cultured with IEC-6 intestinal epithelial cell monolayers, a dynamic release of this protein was observed with mAbIG3. Proteomic analysis identified the protein as a mature cathepsin B-like (gCatB) enzyme, whose proteolytic activity, detected in zymograms, was eliminated by CatB inhibitor E-64. This protein was named giardipain-1 due to its functional papain-like features and was purified by affinity chromatography using mAbIG3. Upon exposure to the purified, mature and secreted forms of giardipain-1, IEC-6 epithelial cell monolayers displayed membrane blebbing and phosphatidylserine exposure on the outer cell surface, indicating an apoptotic process. In Madin Darby Canine Kidney (MDCK) cell monolayers, giardipain-1 leads to the appearance of pore-like regions and of gaps along cell–cell junctions, to decreased transepithelial electrical resistance (TER), caspase-3 activation and poly-ADP-ribose polymerase (PARP) fragmentation. At early times during exposure, giardipain-1 co-localized at cell–cell junctions, associated with occludin and induced the delocalization and degradation of tight junction proteins occludin and claudin-1. The damage caused to epithelial monolayers by giardipain-1 was blocked by pre-incubation with the CatB B Inhibitor E-64. Furthermore, silencing the giardipain-1 gene in trophozoites lowered the proteolytic activity of giardipain-1 and reduced the damage in IEC-6 monolayers. The damage observed appears to be specific to giardipain activity since almost no damage was observed when IEC-6 monolayers were incubated with papain, a non-related cysteine protease. Hence this study suggests that giardipain-1 triggers, in epithelial cells, degradation of cell–cell junctional components and apoptotic damage, supporting the notion of giardiapain-1 as a virulence factor of Giardia.     

Intestinal epithelial cells related lncRNA and mRNA expression profiles in dextran sulphate sodium-induced colitis

Intestinal epithelial barrier damage caused by intestinal epithelial cells (IECs) dysfunction plays a crucial role in the pathogenesis and development of inflammatory bowel disease (IBD). Recently, some studies have suggested the emerging role of long non-coding RNAs (lncRNAs) in IBD. The aim of this study was to reveal lncRNAs and mRNA expression profiles in IECs from a mouse model of colitis and to expand our understanding in the intestinal epithelial barrier regulation. IECs from the colons of wild-type mice and dextran sulphate sodium (DSS)-induced mice were isolated for high-throughput RNA-sequencing. A total of 254 up-regulated and 1013 down-regulated mRNAs and 542 up-regulated and 766 down-regulated lncRNAs were detected in the DSS group compared with the Control group. Four mRNAs and six lncRNAs were validated by real-time quantitative PCR. Function analysis showed that dysregulated mRNAs participated in TLR7 signalling pathway, IL-1 receptor activity, BMP receptor binding and IL-17 signalling pathway. Furthermore, the possibility of indirect interactions between differentially expressed mRNAs and lncRNAs was illustrated by the competing endogenous RNA (ceRNA) network. LncRNA ENSMUST00000128026 was predicted to bind to mmu-miR-6899-3p, regulating Dnmbp expression. LncRNA NONMMUT143162.1 was predicted to competitively bind to mmu-miR-6899-3p, regulating Tnip3 expression. Finally, the protein-protein interaction (PPI) network analysis was constructed with 311 nodes and 563 edges. And the highest connectivity degrees were Mmp9, Fpr2 and Ccl3. These results provide novel insights into the functions of lncRNAs and mRNAs involved in the regulation of the intestinal epithelial barrier.     

Giardia intestinalis: study of infestation procedures of young mice before weaning

Despite the prevalence of giardiasis little is known about the host-parasite specificity. With Giardia intestinalis-suckling mouse model, the success of infestation was depending on the parasite form, cyst or trophozoite, and, for the same parasite form, on the studied strain. Furthermore, the intestinal modification during the wean were unfavourable to Giardia intestinalis colonization.     

Recent insights into the mucosal reactions associated with Giardia lamblia infections

Giardia lamblia is an intestinal protozoan parasite infecting humans and various other mammalian hosts. The most important clinical signs of giardiasis are diarrhoea and malabsorption. Giardia lamblia is able to undergo continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). While intestinal antibodies, and more specifically anti-VSP IgA antibodies, were proven to be involved in modulating antigenic variation of the parasite the participation of the local antibody response in control of the parasite infection is still controversial. Conversely, previous studies based on experimental infections in mice showed that cellular immune mechanisms are essential for elimination of the parasite from its intestinal habitat. Furthermore, recent data indicated that inflammatory mast cells have a potential to directly, or indirectly, interfere in duodenal growth of G. lamblia trophozoites. However, this finding was challenged by other reports, which did not find a correlation between intestinal inflammation and resistance to infection. Since intestinal infiltration of inflammatory cells and/or CD8+T-cells were demonstrated to coincide with villus-shortening and crypt hyperplasia immunological reactions were considered to be a potential factor of pathogenesis in giardiasis. The contribution of physiological factors to pathogenesis was essentially assessed in vitro by co-cultivation of G. lamblia trophozoites with epithelial cell lines. By using this in vitro model, molecular (through surface lectins) and mechanical (through ventral disk) adhesion of trophozoites to the epithelium was shown to be crucial for increased epithelial permeability. This phenomenon as well as other Giardia-induced intestinal abnormalities such as loss of intestinal brush border surface area, villus flattening, inhibition of disaccharidase activities, and eventually also overgrowth of the enteric bacterial flora seem to be involved in the pathophysiology of giardiasis. However, it remains to be elucidated whether at least part of these pathological effects are causatively linked to the clinical manifestation of the disease.     

Giardia disrupts the arrangement of tight, adherens and desmosomal junction proteins of intestinal cells

Giardia duodenalis is a parasitic protozoan that causes diarrhea and other symptoms which together constitute a disease known as giardiasis. Although the disease has been well defined, the mechanisms involving the establishment of the infection have not yet been fully elucidated. In this study, we show that after 24h of interaction between parasites and intestinal Caco-2 cells, there was an alteration of the paracellular permeability, as observed by an approximate 42% of reduction in the transepithelial electrical resistance and permeation to ruthenium red, which was concomitant with ultrastructural changes. Nevertheless, epithelium viability was not affected. We also demonstrate that there was no change in expression of junctional proteins (tight and adherens) but that the distribution of these proteins in Caco-2 cells after parasite adhesion was significantly altered, as observed via laser scanning confocal microscopy 3D reconstruction. The present work shows that adhesion of Giardia duodenalis trophozoites to intestinal cells in vitro induces disturbances of the tight, adherens and desmosomal junctions.     

Lactobacillus casei as a probiotic in malnourished Giardia lamblia-infected mice: a biochemical and histopathological study

The study describes the in vivo activity of Lactobacillus casei in malnourished Giardia lamblia-infected BALB/c mice. By experimentation, it was found that daily administration of the probiotic 7 days before inoculation with Giardia trophozoites in malnourished mice efficiently reduced both the severity and duration of giardiasis. More specifically, excretion of Giardia cysts and trophozoites counts were reduced, while faecal lactobacilli counts increased significantly in probiotic-fed malnourished mice, compared with control mice. Interestingly, it was also observed that oral feeding of the probiotic to malnourished mice abrogated all the anthropometric and biochemical anomalies. Histologically, morphological and cellular alteration of microvillus membrane integrity revealed that probiotic administration ameliorated the mucosal damage in malnourished, probiotic-inoculated, Giardia-infected mice compared with the severe microvillus atrophy, ?dematous and vacuolated epithelial cells, and ileitis in malnourished Giardia-infected mice. The results clearly show the antigiardial effect of the probiotic in vivo by modulating the gut cells to inhibit the colonization and multiplication of Giardia trophozoites, thus reducing the severity and duration of murine giardiasis.     

Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced by the parasite Giardia intestinalis

Giardia intestinalis is a flagellated protozoan which causes enteric disease worldwide. Giardia trophozoites infect epithelial cells of the proximal small intestine and can cause acute or chronic diarrhea. The mechanism of epithelial injury in giardiasis remains unknown. A number of enteric pathogens, including protozoan parasites, are able to induce enterocyte apoptosis. The aim of this work was to assess whether G. intestinalis strain WB clone C6 is able to induce apoptosis in the human intestinal epithelial cell line HCT-8, and to investigate the role of caspases in this process. Results demonstrated that the parasite induces cell apoptosis, as confirmed by DNA fragmentation analysis, detection of active caspase-3 and degradation of the caspase-3 substrate PARP [poly(ADP-ribose) polymerase]. Furthermore, G. intestinalis infection induces activation of both the intrinsic and the extrinsic apoptotic pathways, down-regulation of the antiapoptotic protein Bcl-2 and up-regulation of the proapoptotic Bax, suggesting a possible role for caspase-dependent apoptosis in the pathogenesis of giardiasis.     

Modeling Long-Term Host Cell-Giardia lamblia Interactions in an In Vitro Co-Culture System

Globally, there are greater than 700,000 deaths per year associated with diarrheal disease. The flagellated intestinal parasite, Giardia lamblia, is one of the most common intestinal pathogens in both humans and animals throughout the world. While attached to the gastrointestinal epithelium, Giardia induces epithelial cell apoptosis, disrupts tight junctions, and increases intestinal permeability. The underlying cellular and molecular mechanisms of giardiasis, including the role lamina propria immune cells, such as macrophages, play in parasite control or clearance are poorly understood. Thus far, one of the major obstacles in ascertaining the mechanisms of Giardia pathology is the lack of a functionally relevant model for the long-term study of the parasite in vitro. Here we report on the development of an in vitro co-culture model which maintains the basolateral-apical architecture of the small intestine and allows for long-term survival of the parasite. Using transwell inserts, Caco-2 intestinal epithelial cells and IC-21 macrophages are co-cultured in the presence of Giardia trophozoites. Using the developed model, we show that Giardia trophozoites survive over 21 days and proliferate in a combination media of Caco-2 cell and Giardia medium. Giardia induces apoptosis of epithelial cells through caspase-3 activation and macrophages do not abrogate this response. Additionally, macrophages induce Caco-2 cells to secrete the pro-inflammatory cytokines, GRO and IL-8, a response abolished by Giardia indicating parasite induced suppression of the host immune response. The co-culture model provides additional complexity and information when compared to a single-cell model. This model will be a valuable tool for answering long-standing questions on host-parasite biology that may lead to discovery of new therapeutic interventions.     

Inhibition of Giardia intestinalis by extracellular factors from Lactobacilli: an in vitro study.

The aim of the present work was to evaluate the effect of spent culture supernatants of different strains of lactobacilli on giardia trophozoites. The growth of Giardia intestinalis strain WB, as well as the attachment to the human intestinal epithelial cell line Caco-2, was evaluated by using proliferation and adhesion assays with radiolabeled parasites. In addition, scanning electron microscopy and flow cytometric analysis were performed. The effect of spent culture supernatants from lactobacilli was strain dependent. Lactobacillus johnsonii La1 significantly inhibited the proliferation of G. intestinalis trophozoites. Although the effect was strongly pH dependent, it was not simply due to lactic acid. According to flow cytometric analysis, trophozoites were arrested in G(1) phase but neither significant necrosis nor apoptosis could be detected. Bacterial cells or their spent culture supernatants were unable to modify trophozoite attachment to Caco-2 cells. However, trophozoites treated with spent culture supernatants had little, if any, proliferative capacity. These results suggest that La1 produces some substance(s) able to inhibit proliferation of Giardia trophozoites. Partial characterization of the factors involved in the antigiardiasic action showed that they have a low molecular mass and are inactivated by heating. On this basis, it seems worthwhile to explore how colonization of the proximal small bowel with these lactic acid bacteria could interfere with giardiasis in vivo.     

Synchronisation of Giardia lamblia: identification of cell cycle stage-specific genes and a differentiation restriction point

The intestinal parasite Giardia lamblia undergoes cell differentiations that entail entry into and departure from the replicative cell cycle. The pathophysiology of giardiasis depends directly upon the ability of the trophozoite form to replicate in the host upper small intestine. Thus, cell proliferation is tightly linked to disease. However, studies of cell cycle regulation in Giardia have been hampered by the inability to synchronise cultures. Here we report that Giardia isolates of the major human genotypes A and B can be synchronised using aphidicolin, a mycotoxin that reversibly inhibits replicative DNA polymerases in eukaryotic cells. Aphidicolin arrests Giardia trophozoites in the early DNA synthesis (S) phase of the cell cycle. We identified a set of cell cycle orthologues in the Giardia genome using bioinformatic analyses and showed that synchronised parasites express these genes in a cell cycle stage-specific manner. The synchronisation method also showed that during encystation, exit from the ordinary cell cycle occurs preferentially in G(2) and defines a restriction point for differentiation. Synchronisation opens up possibilities for further molecular and cell biological studies of chromosome replication, mitosis and segregation of the complex cytoskeleton in Giardia.     

An indirect haemagglutination test to detect serum antibodies toGiardia lamblia

We used an indirect haemagglutination test withGiardia lamblia trophozoites as the antigen to detectanti-Giardia lamblia antibodies in serum, the soluble tritonatedGiardia lamblia antigen being used for detecting anti-giardial antibodies in sera of 60 human subjects. Titers in some of these subjects were 1 : 80-1 : 2560. whereas titers in some subjects were negative button to 1 : 20. The results indicated thatGiardia lamblia, an intestinal parasite, induced a systemic antibody response and the indirect haemagglu tination test foranti-Giardia lamblia antibodies is a simple specific and reproducible system which may be useful in epidemiologic and immunologic studies of giardiasis. The specificity of the anti-bodies was demonstrated by the ability of liveGiardia lamblia trophozoites, but not Entamoeba histolytica to absorb the antibody activity.