Magda E. Alvarado

Identification and expression of the protein ubiquitination system in Giardia intestinalis

Giardia intestinalis is a single-cell eukaryotic microorganism, regarded as one of the earliest divergent eukaryotes and thus an attractive model to study the evolution of regulatory systems. Giardia has two different forms throughout its life cycle, cyst and trophozoite, and changes from one to the other in response to environmental signals. The two differentiation processes involve a differential gene expression as well as a quick and specific protein turnover that may be mediated by the ubiquitin/proteasome system. The aim of this work was to search for unreported components of the ubiquitination system and to experimentally demonstrate their expression in the parasite and during the two differentiation processes. We found activity of protein ubiquitination in G. intestinalis trophozoites and analyzed the transcription of the ubiquitin gene, as well as that of the activating (E1), conjugating (E2), and ligase (E3) ubiquitin enzymes during encystation and excystation. A constant ubiquitin expression persisted during the parasite’s differentiation processes, whereas variation in transcription was observed in the other genes under study.

Transcription of meiotic-like-pathway genes in Giardia intestinalis

The reproductive mechanism of Giardia intestinalis, considered one of the earliest divergent eukaryotes, has not been fully defined yet. Some evidence supports the hypothesis that Giardia is an exclusively asexual organism with a clonal population structure. However, the high genetic variability, the variation in ploidy during its life cycle, the low heterozygosity and the existence of genes involved in the meiotic-like recombination pathway in the parasites genome cast doubt on exclusively asexual nature of Giardia. In this work, semiquantitative RT-PCR analysis was used to assess the transcription pattern of three meiosis-like-specific genes involved in homologues recombination: dmc1, hop1 and spo11. The mRNAs were amplified during the parasites differentiation processes, encystation and excystation, and expression was found at each stage of its life cycle. A semiquantitative assessment also suggests that expression of some of the genes is regulated during encystation process.

Calmodulin expression during Giardia intestinalis differentiation and identification of calmodulin-binding proteins during the trophozoite stage

Calmodulin (CaM) is the primary sensor for calcium in the cell. It modulates various functions by activating CaM-binding proteins (CaMBPs). This study examined the calcium/CaM-dependent system in the ancient eukaryote Giardia intestinalis. A specific antibody against the parasite’s CaM was developed; this protein’s expression and location during different stages of the parasite’s life cycle were analyzed. The results showed that it is a housekeeping protein which is possibly involved in the parasite’s motility. No CaMBP has been identified in G. intestinalis to date. Pull-down assays were used for isolating proteins which specifically bind to CaM in a calcium-dependent way. Three of them were identified through mass spectrometry; they were GASP180, α-tubulin, and pyruvate phosphate dikinase (PPDK).The first two are cytoskeleton proteins, and the last one is an essential enzyme for glycolysis. The presence of binding sites was analyzed through bioinformatics in each protein sequence. This is the first report of a CaMBP in this organism; it is considered to be a very interesting differentiation model, indicating that CaM is involved at least in two vital processes: G. intestinalis motility and energetic metabolism

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.

Quick and efficient purification of Giardia intestinalis cysts from fecal samples

Giardia intestinalis is an intestinal parasite that has sparked considerable interest because of the public health problem it creates and because it is regarded as one of the earliest divergent eukaryotes. The present report describes a new method for quick, clean, and effective isolation of G. intestinalis cysts from fecal samples. The isolated cysts have the quality required for biochemical studies of the excystation process.

In silico analysis of the EF-hand proteins in the genome of Giardia intestinalis assembly A

Giardia intestinalis is a parasite that inhabits the small intestine of humans and other mammals, causing a disease that can manifest itself with acute diarrhea. This parasite is an early divergent eukaryote with a compact genome and a life cycle composed of two distinct cell types: the trophozoite, the replicative form, and the cyst, the infectious form. Signal transduction pathways implicated in differentiation processes of G. intestinalis are largely unknown. Calcium, considered an essential messenger in cell signaling, has been shown to regulate a myriad of key cell processes including metabolism, motility, and exocytosis, among other important functions, through calcium-binding proteins (CaBPs). The most important and largest family of CaBPs is the EF-hand protein family. To investigate the nature of calcium signaling pathways present in this protozoan, an in silico analysis of the genome to identify genes encoding EF-hand proteins was undertaken. Twenty-eight sequences containing EF-hand domains were found; most of which have only a pair of domains, and half of the sequences were divergent or unique to Giardia. In addition, the transcription pattern for eight genes encoding EF-hand proteins was assessed during encystation. It was found that all the genes were differentially transcribed suggesting a different function in this process. The in silico results suggest that in G. intestinalis, calcium is involved in the regulation of protein phosphorylation through kinases and phosphatases.

Experimental and bioinformatic characterization of CaBP2933 an EF-Hand protein of Giardia intestinalis

Giardia intestinalis is a parasite that inhabits the small intestine of humans. This parasite is a divergent eukaryote with a compact genome. The calcium ion is an essential messenger in cell signaling. Calciums role as a messenger is mediated through calcium-binding proteins (CaBPs) that decode the message. The most important family of CaBPs is the EF-Hand protein family. In this study we have explored the role of EF-Hand protein CaBP2933. We analyzed its location, confirmed its ability to bind calcium and identified some of its interacting proteins. Take together our results suggest that CaBP2933 is involved in vesicular trafficking during encystation, via an interaction with kinesin-3 motor protein.

Calcium-binding proteins that are type B″ regulatory subunits of phosphatase 2A in Giardia intestinalis

Giardia intestinalis is a protozoan parasite that colonizes the upper part of the small intestine of its mammalian hosts. The trophozoite, which is the replicative stage, has a complex cytoskeleton that allows it to move and adhere to intestinal cells. It has been proposed that protein phosphatase 2A (PP2A) participates in the regulation of changes to the parasite cytoskeleton during its life cycle. However, how PP2A is involved in this regulation remains unclear since its substrates and regulators have not been characterized. In this work, we report the bioinformatic and experimental analysis of two potential regulatory B″ subunits of PP2A in Giardia, both of which are calcium-binding proteins. In this work, in silico and experimental evidence of the binding of both proteins to calcium is presented; the proteins are shown to interact with the catalytic PP2A subunit in the trophozoite stage, and they exhibit different subcellular localization patterns. Because PP2A is a heterotrimer, homology analysis of the different subunits of PP2A indicates that fewer holoenzyme combinations can be formed in this parasite than in other organisms. Our results suggest that the localization of PP2A may be associated with calcium-dependent signaling through its B″ type regulatory subunits.