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.     

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.     

Lipid metabolism in Giardia: a post-genomic perspective

Giardia lamblia, a protozoan parasite, infects a wide variety of vertebrates, including humans. Studies indicate that this anaerobic protist possesses a limited ability to synthesize lipid molecules de novo and depends on supplies from its environment for growth and differentiation. It has been suggested that most lipids and fatty acids are taken up by endocytic and non-endocytic pathways and are used by Giardia for energy production and membrane/organelle biosynthesis. The purpose of this article is to provide an update on recent progress in the field of lipid research of this parasite and the validation of lipid metabolic pathways through recent genomic information. Based on current cellular, biochemical and genomic data, a comprehensive pathway has been proposed to facilitate our understanding of lipid and fatty acid metabolism/syntheses in this waterborne pathogen. We envision that the current review will be helpful in identifying targets from the pathways that could be used to design novel therapies to control giardiasis and related diseases.     

Analysis of phosphorylated proteins and inhibition of kinase activity during Giardia intestinalis excystation

The parasite Giardia intestinalis undergoes a differentiation process that allows it to infect its mammal host. That process is excystation. We examined the importance of protein phosphorylation during the passage from cyst to trophozoite. Cysts obtained from patients with giardiasis were excysted in vitro and the soluble cytoplasmic proteins were analyzed during the three phases of the process, using a specific staining for phosphoproteins. We found two phosphorylated proteins and identified them with MALDI-TOF as 14-3-3 and Hsp70. Modifications were detected in both proteins, which could indicate a role in differentiation of the parasite. In addition, the inhibition of serine–threonine kinases during excystation specifically affected the cytokinesis of the excyzoite, thus inhibiting the completion of trophozoite formation.     

Characterization of SNAREs determines the absence of a typical Golgi apparatus in the ancient eukaryote Giardia lamblia

Giardia is a eukaryotic protozoal parasite with unusual characteristics, such as the absence of a morphologically evident Golgi apparatus. Although both constitutive and regulated pathways for protein secretion are evident in Giardia, little is known about the mechanisms involved in vesicular docking and fusion. In higher eukaryotes, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of the vesicle-associated membrane protein and syntaxin families play essential roles in these processes. In this work we identified and characterized genes for 17 SNAREs in Giardia to define the minimal set of subcellular organelles present during growth and encystation, in particular the presence or not of a Golgi apparatus. Expression and localization of all Giardia SNAREs demonstrate their presence in distinct subcellular compartments, which may represent the extent of the endomembrane system in eukaryotes. Remarkably, Giardia SNAREs, homologous to Golgi SNAREs from other organisms, do not allow the detection of a typical Golgi apparatus in either proliferating or differentiating trophozoites. However, some features of the Golgi, such as the packaging and sorting function, seem to be performed by the endoplasmic reticulum and/or the nuclear envelope. Moreover, depletion of individual genes demonstrated that several SNAREs are essential for viability, whereas others are dispensable. Thus, Giardia requires a smaller number of SNAREs compared with other eukaryotes to accomplish all of the vesicle trafficking events that are critical for the growth and differentiation of this important human pathogen.     

The molecular mechanisms of giardia encystation

The protozoan parasite Giardia lamblia is transmitted as an environmentally resistant cyst. The encystation process is attracting attention not only from the viewpoint of disease transmission, but also as a model for differentiation. Here, Hugo Luján, Michael Mowatt and Theodore Nash discuss molecular events underlying this process, including the induction of expression and transport of cyst wall proteins and the induction of Golgi-like activity. They also propose that the signal for encystation derives from cholesterol deprivation in the lower small intestine.     

How nuclei of Giardia pass through cell differentiation: semi-open mitosis followed by nuclear interconnection

Differentiation into infectious cysts (encystation) and multiplication of pathogenic trophozoites after hatching from the cyst (excystation) are fundamental processes in the life cycle of the human intestinal parasite Giardia intestinalis. During encystation, a bi-nucleated trophozoite transforms to a dormant tetra-nucleated cyst enveloped by a protective cyst wall. Nuclear division during encystation is not followed by cytokinesis. In contrast to the well-studied mechanism of cyst wall formation, information on nuclei behavior is incomplete and basic cytological data are lacking. Here we present evidence that (1) the nuclei divide by semi-open mitosis during early encystment; (2) the daughter nuclei coming from different parent nuclei are always arranged in pairs; (3) in both pairs, the nuclei are interconnected via bridges formed by fusion of their nuclear envelopes; (4) each interconnected nuclear pair is associated with one basal body tetrad of the undivided diplomonad mastigont; and (5) the interconnection between nuclei persists through the cyst stage being a characteristic feature of encysted Giardia. Based on the presented results, a model of nuclei behavior during Giardia differentiation is proposed.     

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.     

Protein trafficking in Giardia lamblia

Giardia, a protozoan parasite of humans and other vertebrates, is a common cause of intestinal disease worldwide. Besides its medical importance, Giardia is considered an excellent system to study the evolution of fundamental cellular processes because it belongs to the earliest branches of the eukaryotic lineage of descent. Giardia trophozoites lack organelles typical of higher eukaryotes such mitochondria, peroxisomes and compartments involved in intracellular protein trafficking and secretion, such as the Golgi apparatus and secretory granules. Nevertheless, the minimal machinery for protein transport and sorting is present in this parasite. When Giardia undergoes encystation, the biogenesis of secretory organelles necessary to transport cyst wall constituents to the cell surface takes place. Recent studies in both vegetative and encysting trophozoites have provided interesting information regarding the secretory pathway of this important human pathogen.     

In vitro encystation of Giardia lamblia: analysis with two-dimensional electrophoresis of differentially expressed proteins

The reconstruction of Giardia lamblia life cycle in vitro is an excellent tool for the study of the parasite's molecular biology. The present work describes techniques developed that better define parasite differentiation. An encystation protocol is presented along with a method for isolation and purification of the produced cysts. The cyst morphology at the light microscopy level is identical to that of in vivo cysts. A two-dimension protein map obtained by high-resolution electrophoresis indicated that most of the parasite's proteins are acid. Based on this result, the two dimension gel electrophoresis used a pH 4-7 gradient in the first, isoelectric focusing dimension. Differences in protein expression during the stages of encystation were clearly discerned, as well as images of the parasite obtained by light and by transmission electron microscopy that describe the morphological and the ultrastructural changes that occur as the cysts are produced in vitro.     

Mechanisms of Giardia lamblia differentiation into cysts.

Microbiologists have long been intrigued by the ability of parasitic organisms to adapt to changes in the environment. Since most parasites occupy several niches during their journey between vectors and hosts, they have developed adaptive responses which allow them to survive under adverse conditions. Therefore, the life cycles of protozoan and helminthic parasites are excellent models with which to study numerous mechanisms involved in cell differentiation, such as the regulation of gene expression, signal transduction pathways, and organelle biogenesis. Unfortunately, many of these studies are very difficult because the conditions needed to elicit developmental changes in parasites remain undetermined in most cases. Recently, several interesting findings were reported on the process of differentiation of Giardia lamblia trophozoites into cysts. G. lamblia is a flagellated protozoan that inhabits the upper small intestine of its vertebrate host and is a major cause of enteric disease worldwide. It belongs to the earliest identified lineage among eukaryotes and therefore offers a unique insight into the progression from primitive to more complex eukaryotic cells. The discovery of a specific stimulus that induces trophozoites to differentiate into cysts, the identification and characterization of encystation-specific molecules, the elucidation of novel biochemical pathways, and the development of useful reagents and techniques have made this parasite an excellent model with which to study differentiation in eukaryotic cells. In this review, we summarize the most recent fundings on several aspects of Giardia differentiation and discuss the significance of these findings within the context of current knowledge in the field.     

Nomenclature and genetic groupings of Giardia infecting mammals

Giardia is a ubiquitous and well-known enteric parasite affecting humans and a range of domestic and wild mammals. It is one of the most common parasites of domestic dogs and dairy cattle and a frequently recognized waterborne pathogen. Giardiasis is considered to be a re-emerging infection because of its association with outbreaks of diarrhoea in child-care centres. Although only a single species has been recognized as causing disease in humans and most other mammals, molecular characterization of morphologically identical isolates from humans and numerous other species of mammals has confirmed the heterogeneity of this parasite and provided a basis for a clearer understanding of the taxonomy and zoonotic potential of Giardia.     

Giardia duodenalis: dendritic cell defects in IL-6 deficient mice contribute to susceptibility to intestinal infection

Interleukin (IL)-6 is important in numerous infections. IL-6 can promote T cell survival and differentiation toward Th17 cells, as well as B cell proliferation and differentiation to plasma cells. Giardia duodenalis is a protozoan parasite that replicates in the lumen of the small intestine in humans and many other mammals resulting in diarrhea, cramps and developmental delays in children. IL-6 is required for control of this infection, but it is unclear what its role is or which cells are required to produce this cytokine to generate efficient immunity. We have analyzed infections in a series of chimeric mice in which specific cell types lacked the ability to produce IL-6 in order to determine which sources of IL-6 played an important role in controlling this infection. Analysis of bone marrow chimeras indicate that radiation-sensitive, bone-marrow derived cells must produce IL-6. T cell chimeras show that T cell production of IL-6 is not required. Finally, by transferring dendritic cells from wild-type mice into IL-6 deficient recipients, we show that dendritic cell defects are responsible for the inability of IL-6 deficient mice to respond to Giardia challenge.     

Prevalence of Cryptosporidium spp. and Giardia spp. in five marine mammal species

Cryptosporidium spp. and Giardia spp. are protozoan parasites that are often associated with severe diarrheal disease in a variety of mammals. Although these parasites have been extensively studied in terrestrial ecosystems, little is known about either parasite in the marine environment. Therefore, the objective of this study was to determine the prevalence of both Cryptosporidium spp. and Giardia spp. in 5 marine mammal species. Fecal samples were collected from 39 bowhead whales (Balaena mysticetus), 49 North Atlantic right whales (Eubalaena glacialis), 31 ringed seals (Phoca hispida), 22 bearded seals (Erignathus barbatus), and 18 beluga whales (Delphinapterus leucas) between 1998 and 2003. Using an immunofluorescent assay, parasites were detected in the feces of bowhead whales, right whales, and ringed seals, while neither parasite was detected in samples from bearded seals or beluga whales. Overall, prevalences were highest in ringed seals (Cryptosporidium spp., 22.6%; Giardia spp., 64.5%) and right whales (Cryptosporidium spp., 24.5%; Giardia spp., 71.4%) and lowest in bowhead whales (Cryptosporidium spp., 5.1%; Giardia spp., 33.3%). To our knowledge, this is the first report of Cryptosporidium spp. and Giardia spp. in either whale species and of Cryptosporidium spp. in the ringed seal.     

Evidence from bioinformatics, expression and inhibition studies of phosphoinositide-3 kinase signalling in Giardia intestinalis

Background  

Giardia intestinalis is a parasitic protozoan and major cause of diarrhoeal disease. Disease transmission is dependent on the ability of the parasite to differentiate back and forth between an intestine-colonising trophozoite and an environmentally-resistant infective cyst. Our current understanding of the intracellular signalling mechanisms that regulate parasite replication and differentiation is limited, yet such information could suggest new methods of disease control. Phosphoinositide-3 kinase (PI3K) signalling pathways have a central involvement in many vital eukaryotic processes, such as regulation of cell growth, intracellular membrane trafficking and cell motility. Here we present evidence for the existence of functional PI3K intracellular signalling pathways in G. intestinalis.     

Oral immunization of BALB/c mice by intragastric delivery of Streptococcus gordonii-expressing Giardia cyst wall protein 2 decreases cyst shedding in challenged mice

Giardia lamblia (Giardia duodenalis or Giardia intestinalis) is a protozoan parasite of vertebrates with broad host specificity. Specific antibodies directed against cyst antigens can interfere with the cyst wall-building process. In this study, we engineered Streptococcus gordonii to express a 26 kDa fragment of cyst wall protein 2 (CWP2), containing a relevant B cell epitope, on the cell surface. This is the first report of S. gordonii expressing a protein of parasite origin. As S. gordonii was intended for intestinal delivery of CWP2, it was determined that this oral commensal bacterium is able to persist in the murine intestine for 30 days. Immunization with recombinant streptococci expressing the 26 kDa fragment resulted in higher antibody levels. Specific anti-CWP2 IgA antibodies were detected in fecal samples and anti-CWP2 IgG antibodies were detected in serum demonstrating the efficacy of S. gordonii for intragastric antigen delivery. In a pilot challenge experiment, immunized mice demonstrated a significant 70% reduction in cyst output.     

Phospholipid remodeling/generation in Giardia: the role of the Lands cycle.

Recent results suggest that Giardia is able to carry out deacylation/reacylation reactions (the Lands cycle) to generate new phospholipids, effectively bypassing the de novo synthesis of the entire phospholipid molecule. The successful operation of this deacylation/reacylation cycle is important for Giardia because this protozoan parasite possesses limited lipid synthesis ability. This article discusses how Giardia might use the Lands cycle to alter phospholipids acquired from the host during its colonization in the human small intestine.