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Abstract 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.

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]

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]

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. [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]

In the relationships between host and parasites, there is a cross-talk that involves diverse mechanisms developed by two different genetic systems during years of evolution. On the one hand, immunocompetent hosts have developed effective innate and acquired immune responses that are used to restrict or avoid parasitism. On the other hand, parasites evade the immune response, expressing different antigens on their surface or by using other specific mechanisms, such as nutrient depletion. In this review, we analyze the survival mechanisms used by the protozoan parasite Giardia lamblia during infection. In particular, we examine the multiple roles played by the enzyme arginine deiminase during colonization of the gut, also involving the parasite's mechanism of antigenic variation. Potential drug targets for the treatment of giardiasis are also discussed.