SUMOylation and deimination of proteins: two epigenetic modifications involved in Giardia encystation.

SUMOylation, a posttranslational modification of proteins, has been recently described as vital in eukaryotic cells. In a previous work, we analyzed the role of SUMO protein and the genes encoding the putative enzymes of the SUMOylation pathway in the parasite Giardia lamblia. Although we observed several SUMOylated proteins, only the enzyme Arginine Deiminase (ADI) was confirmed as a SUMOylated substrate. ADI is involved in the survival of the parasite and, besides its role in ATP production, it also catalyzes the modification of arginine residues to citrulline in the cytoplasmic tail of surface proteins. During encystation, however, ADI translocates to the nuclei and downregulates the expression of the Cyst Wall Protein 2 (CWP2). In this work, we made site-specific mutation of the ADI SUMOylation site (Lys101) and observed that transgenic trophozoites did not translocate to the nuclei at the first steps of encystation but shuttled in the nuclei late during this process through classic nuclear localization signals. Inside the nuclei, ADI acts as a peptidyl arginine deiminase, being probably involved in the downregulation of CWPs expression and cyst wall formation. Our results strongly indicate that ADI plays a regulatory role during encystation in which posttranslational modifications of proteins are key players.

Diphyllobothrium sp. in Canis familiaris from the subtropical area of Argentina (Puerto Iguazú, Misiones).

This paper reports the first finding of Diphyllobothrium sp. eggs in Canis familiaris (domestic dog) from Puerto Iguazú, a subtropical city of Misiones province, Argentina. In 2013, two positive cases of Diphyllobothrium sp. eggs were detected during an annual parasitological survey of dogs. Dog feces were collected in vials containing 10% formalin and processed using Telemann's sedimentation and Sheather's flotation techniques. The two cases were detected in rural areas of the municipality. Since Misiones is not a part of the endemic area of diphyllobothriasis and given the fact that it is located in the three-border area of Argentina, Brazil and Paraguay, we consider this finding of great importance to public health. We stress the need for updating the current knowledge about the life cycle of these parasites considering the range of intermediate and definitive hosts, their zoonotic potential, and the epidemiological situation in non-endemic areas.

Receptor-mediated endocytosis and trafficking between endosomal-lysosomal vacuoles in Giardia lamblia.

The early branching Giardia lamblia has highly polarized vacuoles, located underneath the plasma membrane, which have at least some of the characteristics of endosomes and of lysosomes. These peripheral vacuoles (PVs) are necessary for nutrient uptake and the maintenance of plasma membrane composition, but whether they carry out sorting and segregation of receptors and ligands is a matter of debate. Here, we showed that the internalization of low-density lipoprotein (LDL) to the PVs is highly dynamic in trophozoites with a rate similar to the internalization of the low-density lipoprotein receptor-related protein 1. Moreover, by analyzing receptor-mediated and fluid-phase endocytosis in living cells, we showed that after endocytosis LDL but not dextran moved laterally between the PVs. We speculate on PV functional heterogeneity and maturation in this parasite.

Giardia lamblia low-density lipoprotein receptor-related protein is involved in selective lipoprotein endocytosis and parasite replication.

As Giardia lamblia is unable to synthesize cholesterol de novo, this steroid might be obtained from the host's intestinal milieu by endocytosis of lipoproteins. In this work, we identified a putative Giardia lamblia low-density lipoprotein receptor-related proteins (GlLRP), a type I membrane protein, which shares the substrate N-terminal binding domain and a FXNPXY-type endocytic motif with human LRPs. Expression of tagged GlLRP showed that it was localized predominantly in the endoplasmic reticulum, lysosomal-like peripheral vacuoles and plasma membrane. However, the FXNPXY-deleted GlLRP was retained at the plasma membrane suggesting that it is abnormally transported and processed. The low-density lipoprotein and chylomicrons interacted with GlLRP, with this interaction being necessary for lipoprotein internalization and cell proliferation. Finally, we show that GlLRP binds directly to the medium subunit of Giardia adaptor protein 2, indicating that receptor-mediated internalization occurs through an adaptin mechanism.

Investigating how clathrin adaptor complex AP-2 participates in Giardia lamblia encystation.

The protozoan parasite Giardia lamblia acquires cholesterol from the environment since it is unable to synthesise cholesterol de novo and this is vital for trophozoite growth. Conversely, the lack of cholesterol was described as an essential event to trigger encystation, the differentiation of trophozoites to mature cysts. During the G. lamblia cell cycle, cholesterol is acquired as a free molecule as well as through receptor-mediated endocytosis (RME) of lipoproteins. In this work, we describe the involvement of RME in the cell differentiation process of G. lamblia. We found that a reduction in the expression of the medium subunit (Glµ2) of the giardial adaptin protein GlAP2 impaired RME, triggering the process of encystation in growing cells. Contrary to expectations, decreasing Glµ2 expression produced a cohort of trophozoites that yielded significantly less mature cysts when cells were induced to encyst. Analysis of the subcellular localization of Glµ2 and the cyst wall protein 1 (CWP1) during encystation was later performed, to dissect the process. Our results showed, on one hand, that blocking RME by inhibiting Glµ2 expression, and probably cholesterol entry, is sufficient to induce cell differentiation but not to complete the process of encystation. On the other hand, we observed that GlAP2 is necessary to accomplish the final steps of encystation by sorting CWP1 to the plasma membrane for cyst wall formation. The understanding of the mechanisms involved in cyst formation should provide novel insights into the control of giardiasis, an endemic worldwide neglected disease.

Immunodominant proteins α-1 giardin and ß-giardin are expressed in both assemblages A and B of Giardia lamblia.

BACKGROUND: To date, eight assemblages of Giardia lamblia have been described, but only assemblages A and B are known to infect humans. Despite the fact that the genomic, biological, and clinical differences found between these two assemblages has raised the possibility that they may be considered different species, there is relatively limited information on their phenotypic differences. In the present study, we developed monoclonal antibodies against alpha-1 and beta giardin, two immunodominant proteins produced during G. lamblia infection, and studied their expression and localization in WB (assemblage A) and GS trophozoites (assemblage B). RESULTS: The polyclonal antibodies generated against WB trophozoites, particularly those recognizing intracellular proteins as well as the proteins present at the plasma membrane (variable-specific surface proteins), showed cross-reactivity with intracellular proteins in GS trophozoites. The use of monoclonal antibodies against beta giardin indicated ventral disc localization, particularly at the periphery in WB trophozoites. Interestingly, although beta giardin was also restricted to the ventral disc in GS trophozoites, the pattern of localization clearly differed in this assemblage. On the other hand, monoclonal antibodies against alpha-1 giardin showed plasma membrane localization in both assemblages with the bare area of GS trophozoites also being distinguished. Moreover, the same localization at the plasma membrane was observed in Portland-1 (Assemblage A) and in P15 (Assemblage E) trophozoites. CONCLUSIONS: We found differences in localization of the beta giardin protein between assemblages A and B, but the same pattern of localization of alpha-1 giardin in strains from Assemblages A, B and E. These findings reinforce the need for more studies based on phenotypic characteristics in order to disclose how far one assemblage is from the other.

Adaptor protein 2 regulates receptor-mediated endocytosis and cyst formation in Giardia lamblia.

The parasite Giardia lamblia possesses PVs (peripheral vacuoles) that function as both endosomes and lysosomes and are implicated in the adaptation, differentiation and survival of the parasite in different environments. The mechanisms by which Giardia traffics essential proteins to these organelles and regulates their secretion have important implications in the control of parasite dissemination. In the present study, we describe the participation of the heterotetrameric clathrin-adaptor protein gAP2 (Giardia adaptor protein 2) complex in lysosomal protein trafficking. A specific monoclonal antibody against the medium subunit (gmu2) of gAP2 showed localization of this complex to the PVs, cytoplasm and plasma membrane in the growing trophozoites. gAP2 also co-localized with clathrin in the PVs, suggesting its involvement in endocytosis. Uptake experiments using standard molecules for the study of endocytosis revealed that gAP2 specifically participated in the endocytosis of LDL (low-density lipoprotein). Targeted down-regulation of the gene encoding gmu2 in growing and encysting trophozoites resulted in a large decrease in the amount of cell growth and cyst wall formation, suggesting a distinct mechanism in which gAP2 is directly involved in both endocytosis and vesicular trafficking.

First report of parasitism by Hexametra boddaertii (Nematoda: Ascaridae) in Oxyrhopus guibei (Serpentes: Colubridae).

The current study summarizes the postmortem examination of a specimen of Oxyrhopus guibei (Serpentes, Colubridae) collected in Iguazu National Park (Argentina), and found deceased a week following arrival to the serpentarium of the National Institute of Tropical Medicine (Argentina). Although the snake appeared to be in good health, a necropsy performed following its death identified the presence of a large number of roundworms in the coelomic cavity, with indications of peritonitis and serosal adherence. Additional observations from the necropsy revealed small calcifications in the mesothelium of the coelomic cavity; solid and expressive content in the gallbladder; massive gastrointestinal obstruction due to nematodes; and lung edema and congestion. Histopathological analyses of lung sections also showed proliferative heterophilic and histiocytic pneumonia. Parasites isolated from both the intestine and coelomic cavity were identified as Hexametra boddaertii by a combination of light and scanning electron microscopic examination. Results from this necropsy identify O. guibei as a new host for H. boddaertii, and is the first report of a natural infection by Hexametra in Argentina. Since Hexametra parasites may contribute to several pathological conditions in humans, and with the recent availability of O. guibei specimens through the illegal pet trade, it is necessary to consider the possibility of zoonotic helminth transmission of Hexametra from snake to human.

Lysosomal protein trafficking in Giardia lamblia: common and distinct features.

Giardia is a flagellated protozoan parasite that has to face different microenvironments during its life cycle in order to survive. All cells exchange materials with the extracellular medium through the reciprocal processes of endocytosis and secretion. Unlike more evolved cells, Giardia lacks a defined endosomal/lysosomal system, but instead possesses peripheral vacuoles that play roles in endocytosis, degradation, recycling, and secretion of proteins during growth and differentiation of the parasite. This review focuses on recent reports defining the role of different molecules involved in protein trafficking to the peripheral vacuoles, and discusses possible mechanisms of receptor recycling. Since Giardia is an early-branching protist, the study of this parasite may lead to a clearer understanding of the minimal machinery required for protein transport in eukaryotic cells.

Vacuolar protein sorting receptor in Giardia lamblia.

In Giardia, lysosome-like peripheral vacuoles (PVs) need to specifically coordinate their endosomal and lysosomal functions to be able to successfully perform endocytosis, protein degradation and protein delivery, but how cargo, ligands and molecular components generate specific routes to the PVs remains poorly understood. Recently, we found that delivering membrane Cathepsin C and the soluble acid phosphatase (AcPh) to the PVs is adaptin (AP1)-dependent. However, the receptor that links AcPh and AP1 was never described. We have studied protein-binding to AcPh by using H6-tagged AcPh, and found that a membrane protein interacted with AcPh. This protein, named GlVps (for Giardia lamblia Vacuolar protein sorting), mainly localized to the ER-nuclear envelope and in some PVs, probably functioning as the sorting receptor for AcPh. The tyrosine-binding motif found in the C-terminal cytoplasmic tail domain of GlVps was essential for its exit from the endoplasmic reticulum and transport to the vacuoles, with this motif being necessary for the interaction with the medium subunit of AP1. Thus, the mechanism by which soluble proteins, such as AcPh, reach the peripheral vacuoles in Giardia appears to be very similar to the mechanism of lysosomal protein-sorting in more evolved eukaryotic cells.

Long double-stranded RNA produces specific gene downregulation in Giardia lamblia.

In many eukaryotes, the introduction of double-stranded RNA (dsRNA) into cells triggers the degradation of mRNAs through a post-transcriptional gene-silencing mechanism called RNA interference or RNAi. In the present study, we found that endogenous long-dsRNA was substantially more effective at producing interference than endogenous, or exogenous, short-dsRNA expression in Giardia lamblia . The effects of this interference were not evident in the highly expressed protein tubulin or the stage-specific cyst wall protein 2. However, long-dsRNA caused potent and specific interference in the medium subunits of adaptins, the RNA-dependent RNA polymerase, and the exogenous green fluorescence protein. Our results suggest that the ability of dsRNA antisense to inhibit the expression of these specific types of proteins is indicative of a gene-specific mechanism.