A new gene inventory of the ubiquitin and ubiquitin-like conjugation pathways in Giardia intestinalis.

BACKGROUND: Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE: To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS: We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS: We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the ß-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS: Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.

In vitro interaction between Plasmodium falciparum myosin B (PfMyoB) and myosin A tail interacting protein (MTIP).

Apicomplexan parasites, including Plasmodium falciparum, are obligate intracellular organisms that utilize a strategy termed "gliding" to move and invade host cells, causing disease. Gliding is carried out by a protein complex known as the glideosome, which includes an actin-myosin motor. To date, six myosins have been identified in P. falciparum (PfMyoA, B, C, D, E, and F), but only the role of PfMyoA, the myosin of the glideosome that is involved in the process of red blood cell and mosquito cell invasion, has been established. Based on previous observations, we speculated that PfMyoA and PfMyoB may have similar or redundant functions. To test this hypothesis, we searched for in vitro interactions between PfMyoB and MTIP (myosin A tail interacting protein), the myosin light chain of PfMyoA. A set of differentially tagged PfMyoA, PfMyoB, and MTIP recombinant proteins was employed to specifically and simultaneously detect each myosin in competition assays and inhibition assays using specific peptides. MTIP potentially acts as the light chain of PfMyoB.

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.

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.

Interactions between Giardia duodenalis Sm proteins and their association with spliceosomal snRNAs.

Giardia duodenalis is a parasite that colonises the intestines of humans and other vertebrates, causing diarrhoea and poor nutrient absorption. G. duodenalis is sometimes considered an early diverging eukaryote, and its genome exhibits simplified molecular machinery for many cellular processes, which makes it an interesting model to study. The spliceosome, one of the most complex molecular machines in the eukaryotic cell, is responsible for intron excision and exon splicing. Just over a decade ago, it was believed that the G. duodenalis genome did not contain introns or undergo splicing. Research now shows that this speculation was incorrect and that uncommon mechanisms, such as trans-splicing from different genes, occur. In silico studies of the parasite suggest the possibility of a simplified spliceosome and spliceosomal small nuclear RNA (snRNA) candidates; however, none of these components have been identified in vivo. Here, we developed a strategy to study the in vivo expression, interactions and localisation of these spliceosome components in G. duodenalis. Haemagglutinin (HA)-tagged SmB and SmD3 proteins, which form part of the spliceosome core, were overexpressed in the parasite. Immunoprecipitation with anti-HA revealed that the SmD3 protein is associated with the proteins SmB, SmD1, SmD2, SmE and SmF in vivo. In addition, the U1, U2 and U4 snRNA candidates reported previously were found in the protein complex, suggesting that these molecules are spliceosomal snRNAs of G. duodenalis and they contained a 2,2,7-trimethylguanosine modification at their 5' end. Our data indicate that the actively expressed spliceosome in G. duodenalis is similar to that of highly evolved protists and higher animals.

Myosin B of Plasmodium falciparum (PfMyoB): in silico prediction of its three-dimensional structure and its possible interaction with MTIP.

The mobility and invasion strategy of Plasmodium falciparum is governed by a protein complex known as the glideosome, which contains an actin-myosin motor. It has been shown that myosin A of the parasite (PfMyoA) is the myosin of the glideosome, and the interaction of PfMyoA with myosin tail domain interacting protein (MTIP) determines its correct location and its ability to function in the complex. Because PfMyoA and myosin B of P. falciparum (PfMyoB) share high sequence identity, are both small proteins without a tail domain, belong to the class XIV myosins, and are expressed in late schizonts and merozoites, we suspect that these myosins may have similar or redundant functions. Therefore, this work examined the structural similarity between PfMyoA and PfMyoB and performed a molecular docking between PfMyoB and MTIP. Three-dimensional (3D) models obtained for PfMyoA and PfMyoB achieved high scores in the structural validation programs used, and their superimposition revealed high structural similarity, supporting the hypothesis of possible similar functions for these two proteins. The 3D interaction models obtained and energy values found suggested that interaction between PfMyoB and MTIP is possible. Given the apparent abundance of PfMyoA relative to PfMyoB in the parasite, we believe that the interaction between PfMyoB and MTIP would only be detectable in specific cellular environments because under normal circumstances, it would be masked by the interaction between PfMyoA and MTIP.

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.

Activity of the Giardia intestinalis proteasome during encystation and its connection with the expression of the cyst wall protein 1 (CWP1).

Giardia is a parasite whose life cycle is composed of two stages: replicative trophozoites, responsible for the symptoms of the disease, and infective cysts, resistant to adverse environments outside of hosts. Proteasomes are multicatalytic peptidase complexes responsible for the specific degradation of proteins in eukaryotic cells. This study assessed the proteasome activity in the trophozoite and during encystation. Strong activation of the proteasome was observed during the differentiation of trophozoites into cysts, reaching its maximum level 24 h after the stimulus. We also found that the Giardia proteasome presents unusual characteristics related to higher eukaryotic proteasomes, making it an eventual therapeutic target. Here we tested the effects on the synthesis of a cyst wall protein by chemical inactivation of the proteasome and by overexpression or partial inhibition of the deubiquitinating protein RPN11 in transfected cells. Moreover, an analysis of the intracellular localization of RPN11 (an integral part of the proteasome regulatory particle) revealed major changes associated with the differentiation of trophozoites into cysts. This evidence further supports the important role of the proteasome in Giardia encystation.

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 parasite's 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 parasite's 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.

Genetic diversity of Giardia intestinalis populations in Colombia.

INTRODUCTION: Giardia intestinalis is a protozoan parasite that causes a gastrointestinal infection known as giardiosis, which is transmitted primarily through fecal-oral contamination. Genetic studies of axenically cultivated Giardia isolates have identified two major genetic groups distributed throughout the world. In the present study 24 native strains of the parasite were analyzed by the RAPD technique (Random Amplified Polymorphic DNA). OBJECTIVE: To determine the level of polymorphism and the complexity of Giardia intestinalis circulating strains in specific areas of Colombia. MATERIALS AND METHODS: The RAPD method was used, as it allows for a quick, simple and reliable analysis that requires no prior knowledge of the genetics of the parasite. A RAPD analysis was conducted on native isolates collected in Colombia between 1997 and 2001, established in continuous cultures. Several primers were tested separately, in order to enhance the capacity for discrimination of the method. RESULTS: Of the 24 strains that were included in the study, 22 were arranged in independent clusters. The strains that were from the same geographic area and collected at about the same time, generally displayed highly similar but distinguishable RAPD patterns. Clones isolated from a strain were analyzed as well, and it was possible to differentiate them molecularly. CONCLUSION: The studied strains showed to belong to genotype A . The results suggest that the Colombian strains studied consist of a heterogeneous mixture of closely related populations.

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. Calcium's 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.

Do genes from the cholesterol synthesis pathway exist and express in Giardia intestinalis?

Giardia intestinalis undergo biochemical and morphological changes to survive under extreme environmental conditions. One of these changes is encystation. The trophozoites colonizing the upper part of the small intestine differentiate into a cyst, the infective form of the parasite. In in vitro cultures, the formation of cysts is induced by the depletion of cholesterol. It was reported that Giardia cannot synthesize cholesterol de novo. However, through bioinformatic studies, we found the genes that codify for the enzymes in the cholesterol biosynthesis pathway. We were able to verify the existence and define the transcription of four genes in the trophozoite and in parasites subjected to the encystation and excystation processes.

[Genotyping of the Plasmodium falciparum msp1 (block 2) and dhfr (codon 108) genes in field samples collected in four endemic Colombian localities]. / Genotipificación de los genes msp1 (bloque 2) y dhfr (codón108) de Plasmodium falciparum en muestras de campo recolectadas en cuatro localidades endémicas de Colombia.

INTRODUCTION: Plasmodium falciparum is a highly polymorphic parasite, which allows it to evade the host's immune response, spread drug resistance and favours transmission. OBJECTIVES: To analyse the genetic diversity of P. falciparum populations in samples from four endemic localities in Colombia. MATERIALS AND METHODS: 123 blood samples were collected on filter paper from patients with non-complicated P. falciparum malaria during 2002 to 2004. The samples were genotyped using polymerase chain reaction with specific primers for the polymorphic region of block 2 of the msp1 gene and the 108 codon of the dhfr gene. RESULTS: In msp1 block 2, 95.9% (118/123; 95% CI: 90.8-98.7) of the samples harboured MAD20; 6.5% K1 (8/123; 95% CI: 2.8-12.4) and 2.4% RO33 (3/123; 95% CI: 0.5-6.9). For the dhfrgene the mutant allele N 108 was found in all the samples amplified, T 108 in 3.2% and the wild type S108 in 34.1%. Taking together all the results from both genes, 61.8% (76/123; 95% CI: 52.6-70.4) of the samples were simple infections and 38.2% (47/123; 95% CI: 29.6-47.4) were mixed infections. MAD20/N108-S108 (30.1%) was the most frequent combination among the latter. CONCLUSIONS: Simple infections, i.e, a single allelic type in each one of the genes studied, prevailed among the circulating parasite populations. In this study the genetic composition of P. falciparum parasite populations was very homogeneous.

Partial inhibition of two genes that encode spliceosomal proteins in Giardia intestinalis.

INTRODUCTION: Giardia intestinalis is an early divergent organism that was recently shown to have introns. The machinery responsible for the removal of introns in higher eukaryotes is the spliceosome, which consists of five ribonucleoproteins. Each of these ribonucleoproteins has a small nuclear RNA, a set of seven Sm proteins (B, D1, D2, D3, E, F and G) and several specific proteins. Some genes that encode spliceosome proteins have been bioinformatically identified in the parasite genome. Although it is assumed that the spliceosome is responsible for splicing in this parasite, biochemical characterization is lacking. Objective. To inhibit two G. intestinalis spliceosome protein genes in order to determine whether this inhibition affects parasite growth or encystation. Materials and methods. Antisense sequences of the genes encoding the spliceosomal parasite proteins SmB and SmD3 were cloned into a specific G. intestinalis vector. G. intestinalis individuals were subsequently transfected with the recombinant vectors and those parasites that incorporated the vector were selected. A decrease in mRNA levels by real-time PCR was confirmed and the growth and encystation in wild and transfected parasites was assessed. Results. A decrease of 40% and 70% of SmB and SmD3 mRNA levels, respectively, was observed. Growth and encystation in these parasites were not affected. Conclusion. Decrease of SmB and SmD3 mRNA levels does not affect the parasite, indicating that the spliceosome remains functional or that splicing is not essential for parasite viability.

Production of recombinant proteins from Plasmodium falciparum in Escherichia coli.

INTRODUCTION: The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum. However, the proteins of P. falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies.  OBJECTIVE: To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8).  MATERIALS AND METHODS: The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice.  RESULTS: The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli, compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies.  CONCLUSION: The use of genetically modified strains of E. coli was essential to achieve high expression levels of the four evaluated P. falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P. falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.

[Identification of the gene sequence of telomerase catalytic subunit in Plasmodium falciparum]. / Identificación de la secuencia del gen de la subunidad catalítica de la telomerasa en Plasmodium falciparum.

INTRODUCTION: The enzyme telomerase regulates telomere length by synthesis of telomeric repeats to compensate for telomeric loss in each DNA replication cycle. Therefore, telomerase is a potential target to block growth of cells with high replication rates. In Plasmodium falciparum, telomerase activity has been documented, but little information on its structure and role. METHODS: Herein, alignment of multiple sequences was undertaken comparing telomerase catalytic subunit sequences as found in existing databases. A consensus sequence was compared with the sequences in the P. falciparum genome project and as a result, a candidate sequence for a portion of the telomerase gene was recovered. Primer sets were designed for DNA and RNA amplifications. RESULTS: DNA fragments corresponding to telomerase conserved domains were amplified by using reverse transcription and PCR of cDNA. With a combination of bioinformatics and sequencing methods, the sequence of telomerase catalytic subunit gene (TERT) in P. falciparum was discovered, and its presence and transcription demonstrated.

A new gene inventory of the ubiquitin and ubiquitin-like conjugation pathways in Giardia intestinalis

BACKGROUND Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the β-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.

Genetic diversity of Plasmodium falciparum field samples from an isolated Colombian village.

Colombian field isolates of Plasmodium falciparum were analyzed for genetic diversity. Fifty-three samples were collected as thick smears from patients living in Panguí, an isolated area with low migration. While the samples were being collected, Panguí was experiencing an epidemic outbreak of malaria. The samples were typified using nested polymerase chain reaction (PCR) amplification of block 2 of the merozoite surface protein 1 (MSP1) gene and nested PCR with mutation-specific primers for position 108 of the dihydrofolate reductase enzyme gene. The results for the circulating population of parasites in Panguí show low diversity--four allelic forms--using MSP1 as a marker, a fact that contrasts with data reported for certain Asian and African zones. A high percentage of mixed infections was observed, as was high complexity of the infection. No differential distributions were found for any allelic type.

Transcription of metabolic enzyme genes during the excystation of Giardia lamblia.

The present study evaluates the expression of genes of Giardia lamblia, one of the most simple and most early diverging eukaryotes, that encode the metabolic enzymes pyruvate: ferredoxin oxidoreductase (PFOR), acetyl-CoA synthetase (ACS), alcohol dehydrogenase E (ADHE) and glutamate dehydrogenase (GDH) and the cyst wall protein (CWP1) gene in trophozoites, cysts and during the excystation process. Primers were designed to amplify mRNA fragments through quantitative reverse-transcriptase-polymerase-chain-reaction. In trophozoites, all transcripts of the enzymes studied were present. In cysts, three of the transcripts were detected: CWP1, GDH and ACS; but the relative levels of the mRNA of GDH and ACS were very different between trophozoites and cysts. During excystation, PFOR and ADHE transcripts appeared after the first induction phase, and the mRNAs of ACS and GDH increased throughout the process.

[Preparation of DNA libraries of Plasmodium falciparum for searching calmodulin binding protein genes, GOGAT and Pfmyo A]. / Preparación de genotecas de ADNc de Plasmodium falciparum para la búsqueda de los genes de proteínas de unión a calmodulina, GOGAT y Pfmyo A.

A cDNA library of Plasmodium falciparum (Colombian strain FCB2) asexual stage was constructed in the lambda ZipLox vector. The lambda ZipLox library and a lambda ZAPII (Dd2 strain) were screened for genes coding for proteins that bind with or are related to calmodulin (CaM). Screening was accomplished with Hot start PCR assays and hybridization with radiolabeled probes. Actin I, CaM, glutamate synthase (GOGAT) and the three myosin clones--Pfmyo A, Pfmyo B and Pfmyo C--were identified. The clones coding for actin I, CaM and GOGAT were retrieved from the lambda ZipLox library, and the GOGAT and Pfmyo A clones from the lambda ZAP II library. The GOGAT clone contained an insert of 2,413 base pairs corresponding to 24.8% of the reported sequence. The Pfmyo A insert was 2,457 base pairs long, and represented the complete mRNA coding for this gene. Finally, the first report of a complete cDNA clone containing the P. falciparum myosin A is presented.