Trypanosoma cruzi is the causative agent of Chagas disease, which affects more than 9 million people in Latin America. We have generated a draft genome sequence of the TcI strain Sylvio X10/1 and compared it to the TcVI reference strain CL Brener to identify lineage-specific features. We found virtually no differences in the core gene content of CL Brener and Sylvio X10/1 by presence/absence analysis, but 6 open reading frames from CL Brener were missing in Sylvio X10/1. Several multicopy gene families, including DGF, mucin, MASP and GP63 were found to contain substantially fewer genes in Sylvio X10/1, based on sequence read estimations. 1,861 small insertion-deletion events and 77,349 nucleotide differences, 23% of which were non-synonymous and associated with radical amino acid changes, further distinguish these two genomes. There were 336 genes indicated as under positive selection, 145 unique to T. cruzi in comparison to T. brucei and Leishmania. This study provides a framework for further comparative analyses of two major T. cruzi lineages and also highlights the need for sequencing more strains to understand fully the genomic composition of this parasite.
BACKGROUND: Giardia intestinalis is a protozoan parasite that causes diarrhea in a wide range of mammalian species. To further understand the genetic diversity between the Giardia intestinalis species, we have performed genome sequencing and analysis of a wild-type Giardia intestinalis sample from the assemblage E group, isolated from a pig. RESULTS: We identified 5012 protein coding genes, the majority of which are conserved compared to the previously sequenced genomes of the WB and GS strains in terms of microsynteny and sequence identity. Despite this, there is an unexpectedly large number of chromosomal rearrangements and several smaller structural changes that are present in all chromosomes. Novel members of the VSP, NEK Kinase and HCMP gene families were identified, which may reveal possible mechanisms for host specificity and new avenues for antigenic variation. We used comparative genomics of the three diverse Giardia intestinalis isolates P15, GS and WB to define a core proteome for this species complex and to identify lineage-specific genes. Extensive analyses of polymorphisms in the core proteome of Giardia revealed differential rates of divergence among cellular processes. CONCLUSIONS: Our results indicate that despite a well conserved core of genes there is significant genome variation between Giardia isolates, both in terms of gene content, gene polymorphisms, structural chromosomal variations and surface molecule repertoires. This study improves the annotation of the Giardia genomes and enables the identification of functionally important variation.
Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 16x coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus <0.01% in WB). The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species.
New insecticides are urgently needed because resistance to current insecticides allows resurgence of disease-transmitting mosquitoes while concerns for human toxicity from current compounds are growing. We previously reported the finding of a free cysteine (Cys) residue at the entrance of the active site of acetylcholinesterase (AChE) in some insects but not in mammals, birds, and fish. These insects have two AChE genes (AP and AO), and only AP-AChE carries the Cys residue. Most of these insects are disease vectors such as the African malaria mosquito (Anopheles gambiae sensu stricto) or crop pests such as aphids. Recently we reported a Cys-targeting small molecule that irreversibly inhibited all AChE activity extracted from aphids while an identical exposure caused no effect on the human AChE. Full inhibition of AChE in aphids indicates that AP-AChE contributes most of the enzymatic activity and suggests that the Cys residue might serve as a target for developing better aphicides. It is therefore worth investigating whether the Cys-targeting strategy is applicable to mosquitocides. Herein, we report that, under conditions that spare the human AChE, a methanethiosulfonate-containing molecule at 6 microM irreversibly inhibited 95% of the AChE activity extracted from An. gambiae s. str. and >80% of the activity from the yellow fever mosquito (Aedes aegypti L.) or the northern house mosquito (Culex pipiens L.) that is a vector of St. Louis encephalitis. This type of inhibition is fast ( approximately 30 min) and due to conjugation of the inhibitor to the active-site Cys of mosquito AP-AChE, according to our observed reactivation of the methanethiosulfonate-inhibited AChE by 2-mercaptoethanol. We also note that our sulfhydryl agents partially and irreversibly inhibited the human AChE after prolonged exposure (>4 hr). This slow inhibition is due to partial enzyme denaturation by the inhibitor and/or micelles of the inhibitor, according to our studies using atomic force microscopy, circular dichroism spectroscopy, X-ray crystallography, time-resolved fluorescence spectroscopy, and liquid chromatography triple quadrupole mass spectrometry. These results support our view that the mosquito-specific Cys is a viable target for developing new mosquitocides to control disease vectors and to alleviate resistance problems with reduced toxicity toward non-target species.
Whole-genome sequencing of the protozoan pathogen Trypanosoma cruzi revealed that the diploid genome contains a predicted 22,570 proteins encoded by genes, of which 12,570 represent allelic pairs. Over 50% of the genome consists of repeated sequences, such as retrotransposons and genes for large families of surface molecules, which include trans-sialidases, mucins, gp63s, and a large novel family (>1300 copies) of mucin-associated surface protein (MASP) genes. Analyses of the T. cruzi, T. brucei, and Leishmania major (Tritryp) genomes imply differences from other eukaryotes in DNA repair and initiation of replication and reflect their unusual mitochondrial DNA. Although the Tritryp lack several classes of signaling molecules, their kinomes contain a large and diverse set of protein kinases and phosphatases; their size and diversity imply previously unknown interactions and regulatory processes, which may be targets for intervention.
A comparison of gene content and genome architecture of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, three related pathogens with different life cycles and disease pathology, revealed a conserved core proteome of about 6200 genes in large syntenic polycistronic gene clusters. Many species-specific genes, especially large surface antigen families, occur at nonsyntenic chromosome-internal and subtelomeric regions. Retroelements, structural RNAs, and gene family expansion are often associated with syntenic discontinuities that-along with gene divergence, acquisition and loss, and rearrangement within the syntenic regions-have shaped the genomes of each parasite. Contrary to recent reports, our analyses reveal no evidence that these species are descended from an ancestor that contained a photosynthetic endosymbiont.
Title: Crystallization and X-ray diffraction data analysis of leukotriene A4 hydrolase from Saccharomyces cerevisiae Andersson B, Kull F, Haeggstrom JZ, Thunnissen MM Ref: Acta Crystallographica D Biol Crystallogr, 59:1093, 2003 : PubMed
The Saccharomyces cerevisiae leukotriene A4 (LTA4) hydrolase (scLTA4 hydrolase) has been crystallized in order to study the two activities of LTA4 hydrolase in an evolutionary perspective. Single well diffracting crystals are obtained after switching from the hanging-drop method to liquid-liquid diffusion in capillaries using PEG 8000 as precipitant. These crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.8, b = 98.1, c = 99.2 A. Intensity data to 2.3 A resolution were collected from a native scLTA4 hydrolase crystal using synchrotron radiation. A molecular-replacement solution was obtained using the human LTA4 hydrolase structure and the program BEAST.
Leukotriene (LT) A4 hydrolase/aminopeptidase is a bifunctional zinc enzyme that catalyzes the final step in the biosynthesis of LTB4, a potent chemoattractant and immune modulating lipid mediator. Here, we report a high-resolution crystal structure of LTA4 hydrolase in complex with captopril, a classical inhibitor of the zinc peptidase angiotensin-converting enzyme. Captopril makes few interactions with the protein, but its free thiol group is bound to the zinc, apparently accounting for most of its inhibitory action on LTA4 hydrolase. In addition, we have determined the structures of LTA4 hydrolase in complex with two selective tight-binding inhibitors, a thioamine and a hydroxamic acid. Their common benzyloxyphenyl tail, designed to mimic the carbon backbone of LTA4, binds into a narrow hydrophobic cavity in the protein. The free hydroxyl group of the hydroxamic acid makes a suboptimal, monodentate complex with the zinc, and strategies for improved inhibitor design can be deduced from the structure. Taken together, the three crystal structures provide the molecular basis for the divergent pharmacological profiles of LTA4 hydrolase inhibitors. Moreover, they help define the binding pocket for the fatty acid-derived epoxide LTA4 as well as the subsites for a tripeptide substrate, which in turn have important implications for the molecular mechanisms of enzyme catalyses.
A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.
The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.
