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Introduction: Post-translational modifications of proteins provide cellular physiology with a broad range of adaptability to the external environment flexibly and rapidly. In the case of the protozoan parasite Giardia lamblia , the study of these modifications has gained relevance in recent years, mainly focusing on methylation and deacetylation of proteins. This study investigates the significance of acetylation in this protozoan parasite. Methods: This study explores the role of acetylation in G. lamblia through a combination of immunofluorescence assays, manipulation of acetyltransferase enzymes, and the use of garcinol, an acetylation inhibitor, during the growth phase. Results: The acetylation of histone marks H3K9 and H3K27 occurs during growth and is followed by deacetylation during encystation. Transfections modifying acetyltransferase activity induced a latent cellular state, underscoring the importance of protein acetylation for parasite survival. Garcinol treatment during growth caused significant morphological changes, including plasma membrane blebbing and apoptotic-like bodies. Immunofluorescence revealed these bodies contained α-tubulin/acetylated α-tubulin and reactive oxygen species. Flow cytometry and Annexin V staining indicated early apoptosis within 24 hours of treatment. Additionally, garcinol led to the deacetylation of H3K9 and H3K27, with redistribution of tubulin and acetylated tubulin from microtubules to the cytosol. Significantly, garcinol prevented parasite recrudescence after treatment withdrawal. Discussion: These results demonstrate that acetylation is essential for trophozoite survival and highlight the natural histone acetyltransferase inhibitor garcinol as a potential candidate for drug development against giardiasis, considering its giardicidal activity. [ABSTRACT FROM AUTHOR]

Introduction: Giardia lamblia is a flagellated protozoan parasite causing giardiasis, a common intestinal infection characterized by diarrhea, abdominal cramps, and nausea. Treatments employed to combat this parasitic infection have remained unchanged for the past 40 years, leading to the emergence of resistant strains and prompting the search for new therapeutic agents. Methods: This study investigated the cytotoxic effects of ivermectin (IVM) on G. lamblia trophozoites. We conducted dose-response experiments to assess IVM-induced cytotoxicity. We utilized various biochemical and ultrastructural analyses to explore the underlying mechanisms of cell death, including reactive oxygen species (ROS) production, DNA fragmentation, cell cycle arrest, and apoptosis markers. Results: Our findings demonstrate that IVM induces dose-dependent cytotoxicity and triggers cell death pathways. We found that IVM treatment generates elevated levels of reactive oxygen species (ROS), DNA fragmentation, and arrests of trophozoites in the cell cycle's S phase. Additionally, ultrastructural analysis reveals morphological alterations consistent with apoptosis, such as cytoplasmic vacuolization, chromatin condensation, and tubulin distribution. Discussion: The insights gained from this study may contribute to developing new therapeutic strategies against giardiasis, addressing the challenge posed by drug-resistant strains. [ABSTRACT FROM AUTHOR]

The genetically related assemblages of the intestinal protozoa parasite Giardia lamblia are morphologically indistinguishable and are often derived from specific hosts. The Giardia assemblages are separated by large genetic distances, which might account for their relevant biological and pathogenic differences. In this work, we analyzed the RNAs cargo released into exosomal-like vesicles (ElVs) by the assemblages A and B, which differentially infect humans, and the assemblage E, which infects hoofed animals. The RNA sequencing analysis revealed that the ElVs of each assemblage contained distinct small RNA (sRNA) biotypes, suggesting a preference for specific packaging in each assemblage. These sRNAs were classified into three categories, ribosomal-small RNAs (rsRNAs), messenger-small RNAs (msRNAs), and transfer-small RNAs (tsRNAs), which may play a regulatory role in parasite communication and contribute to host-specificity and pathogenesis. Uptake experiments showed, for the first time, that ElVs were successfully internalized by the parasite trophozoites. Furthermore, we observed that the sRNAs contained inside these ElVs were first located below the plasma membrane but then distributed along the cytoplasm. Overall, the study provides new insights into the molecular mechanisms underlying the host-specificity and pathogenesis of G. lamblia and highlights the potential role of sRNAs in parasite communication and regulation. [ABSTRACT FROM AUTHOR]

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In eukaryotes, histone lysine methylation is associated with either active or repressed chromatin states, depending on the status of methylation. Even when the amino-terminus of Giardia lamblia histones diverges from other organisms, these regions contain lysine residues that are potential targets for methylation. When we examined the role of the histone methyltransferase 1 (HMT1) in the regulation of the encystation process by giardial histone methyltransferase 1 (GlHMT1) overexpression or downregulation, we observed an increase or a decrease in cyst production, respectively, compared to wild-type trophozoites. A time-lapse analysis of encystation showed that overexpression of GlHMT1 induced an earlier and faster process than in wild-type cells together with an upregulation of mRNA expression of cyst wall proteins. Subcellular localization studies indicated that GlHMT1-hemaglutinin was mainly associated with the nuclear and perinuclear region in both growing and encysting parasites, in agreement with bioinformatics analyses showing that GlHMT-1 possesses nuclear localization signals in addition to the classical SU(var)3-9, Enhancer-of-Zeste, Trithorax (SET), and post-SET domains. Altogether, these findings suggest that the function of HMT1 is critical for the success and timing of the encystation process, and reinforce the idea that epigenetic marks are critical for cyst formation in G. lamblia. [ABSTRACT FROM AUTHOR]

Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation. Author Summary: Giardiasis is a major cause of non-viral/non-bacterial diarrheal disease worldwide and has been included within the WHO Neglected Disease Initiative since 2004. Infection begins with the ingestion of Giardia lamblia in cyst form, which, after exposure to gastric acid in the host stomach and proteases in the duodenum, gives rise to trophozoites. The inverse process is called encystation and begins when the trophozoites migrate to the lower part of the small intestine where they receive signals that trigger synthesis of the components of the cyst wall. The cyst form enables the parasite to survive in the environment, infect a new host and evade the immune response. In this work, we explored the role of protein S-palmitoylation, a unique reversible post-translational modification, during Giardia encystation, because de novo generation of endomembrane compartments, protein sorting and vesicle fusion occur in this process. Our findings may contribute to the design of therapeutic agents against this important human pathogen. [ABSTRACT FROM AUTHOR]

Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation. [ABSTRACT FROM AUTHOR]

Post-translational modifications are able to regulate protein function and cellular processes in a rapid and reversible way. SUMOylation, the post-translational modification of proteins by the addition of SUMO, is a highly conserved process that seems to be present in modern cells. However, the mechanism of protein SUMOylation in earlier divergent eukaryotes, such as Giardia lamblia, is only starting to become apparent. In this work, we report the presence of a single SUMO gene encoding to SUMO protein in Giardia. Monoclonal antibodies against recombinant Giardia SUMO protein revealed the cytoplasmic localization of native SUMO in wild-type trophozoites. Moreover, the over-expression of SUMO protein showed a mainly cytoplasmic localization, though also neighboring the plasma membrane, flagella, and around and even inside the nuclei. Western blot assays revealed a number of SUMOylated proteins in a range between 20 and 120 kDa. The genes corresponding to putative enzymes involved in the SUMOylation pathway were also explored. Our results as a whole suggest that SUMOylation is a process conserved in the eukaryotic lineage, and that its study is significant for understanding the biology of this interesting parasite and the role of post-translational modification in its evolution. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The manner in which membrane-associated proteins interact with the membrane defines their subcellular fate and function. This interaction relies on the characteristics of the proteins, their journey after synthesis, and their interaction with other proteins or enzymes. Understanding these properties may help to define the function of a protein and also the role of an organelle. In the case of microorganisms like protozoa parasites, it may help to understand singular features that will eventually lead to the design of parasite-specific drugs. The protozoa parasite Giardia lamblia is an example of a widespread parasite that has been infecting humans and animals from ancestral times, adjusting itself to the changes of the environment inside and outside the host. Several membrane-associated proteins have been posted in the genome database GiardiaDB, although only a few of them have been characterized. This review discusses the data regarding membrane-associated proteins in relationship with lipids and specific organelles and their implication in the discovery of anti-giardial therapies. [ABSTRACT FROM AUTHOR]

The protozoan parasite Giardia lamblia uses arginine deiminase (ADI) to produce energy from free L-arginine under anaerobic conditions. In this work, we demonstrate that, in addition to its known role as a metabolic enzyme, it also functions as a peptidylarginine deiminase, converting protein-bound arginine into citrulline. G. lamblia ADI specifically binds to and citrullinates the arginine in the conserved CRGKA tail of variant-specific surface proteins (VSPs), affecting both antigenic switching and antibody-mediated cell death. During encystation, ADI translocates from the cytoplasm to the nuclei and appears to play a regulatory role in the expression of encystation-specific genes. ADI is also sumoylated, which might modulate its activity. Our findings reveal a dual role played by ADI and define novel regulatory pathways used by Giardia for survival. [ABSTRACT FROM AUTHOR]