Regioselective Galactofuranosylation for the Synthesis of Disaccharide Patterns Found in Pathogenic Microorganisms.

Koenigs-Knorr glycosylation of acceptors with more than one free hydroxyl group by 2,3,5,6-tetrabenzoyl galactofuranosyl bromide was performed using diphenylborinic acid 2-aminoethyl ester (DPBA) as inducer of regioselectivity. High regioselectivity for the glycosylation on the equatorial hydroxyl group of the acceptor was obtained thanks to the transient formation of a borinate adduct of the corresponding 1,2-cis diol. Nevertheless formation of orthoester byproducts hampered the efficiency of the method. Interestingly electron-withdrawing groups on O-6 or on C-1 of the acceptor displaced the reaction in favor of the desired galactofuranosyl containing disaccharide. The best yield was obtained for the furanosylation of p-nitrophenyl 6-O-acetyl mannopyranoside. Precursors of other disaccharides, found in the glycocalix of some pathogens, were synthesized according to the same protocol with yields ranging from 45 to 86%. This is a good alternative for the synthesis of biologically relevant glycoconjugates.

RNA folding pathways and kinetics using 2D energy landscapes.

RNA folding pathways play an important role in various biological processes, such as (i) the hok/sok (host-killing/suppression of killing) system in E. coli to check for sufficient plasmid copy number, (ii) the conformational switch in spliced leader (SL) RNA from Leptomonas collosoma, which controls trans splicing of a portion of the '5 exon, and (iii) riboswitches--portions of the 5' untranslated region of messenger RNA that regulate genes by allostery. Since RNA folding pathways are determined by the energy landscape, we describe a novel algorithm, FFTbor2D, which computes the 2D projection of the energy landscape for a given RNA sequence. Given two metastable secondary structures A, B for a given RNA sequence, FFTbor2D computes the Boltzmann probability p(x, y) = Z(x,y)/Z that a secondary structure has base pair distance x from A and distance y from B. Using polynomial interpolationwith the fast Fourier transform,we compute p(x, y) in O(n(5)) time and O(n(2)) space, which is an improvement over an earlier method, which runs in O(n(7)) time and O(n(4)) space. FFTbor2D has potential applications in synthetic biology, where one might wish to design bistable switches having target metastable structures A, B with favorable pathway kinetics. By inverting the transition probability matrix determined from FFTbor2D output, we show that L. collosoma spliced leader RNA has larger mean first passage time from A to B on the 2D energy landscape, than 97.145% of 20,000 sequences, each having metastable structures A, B. Source code and binaries are freely available for download at http://bioinformatics.bc.edu/clotelab/FFTbor2D. The program FFTbor2D is implemented in C++, with optional OpenMP parallelization primitives.

Abstract folding space analysis based on helices.

RNA has many pivotal functions especially in the regulation of gene expression by ncRNAs. Identification of their structure is an important requirement for understanding their function. Structure prediction alone is often insufficient for this task, due to algorithmic problems, parameter inaccuracies, and biological peculiarities. Among the latter, there are base modifications, cotranscriptional folding leading to folding traps, and conformational switching as in the case of riboswitches. All these require more in-depth analysis of the folding space. The major drawback, which all methods have to cope with, is the exponential growth of the folding space. Therefore, methods are often limited in the sequence length they can analyze, or they make use of heuristics, sampling, or abstraction. Our approach adopts the abstraction strategy and remedies some problems of existing methods. We introduce a position-specific abstraction based on helices that we term helix index shapes, or hishapes for short. Utilizing a dynamic programming framework, we have implemented this abstraction in the program RNAHeliCes. Furthermore, we developed two hishape-based methods, one for energy barrier estimation, called HiPath, and one for abstract structure comparison, termed HiTed. We demonstrate the superior performance of HiPath compared to other existing methods and the competitive accuracy of HiTed. RNAHeliCes, together with HiPath and HiTed, are available for download at http://www.cyanolab.de/software/RNAHeliCes.htm.

Structural characterization of the cell division cycle in Strigomonas culicis, an endosymbiont-bearing trypanosomatid.

Strigomonas culicis (previously referred to as Blastocrithidia culicis) is a monoxenic trypanosomatid harboring a symbiotic bacterium, which maintains an obligatory relationship with the host protozoan. Investigations of the cell cycle in symbiont harboring trypanosomatids suggest that the bacterium divides in coordination with other host cell structures, particularly the nucleus. In this study we used light and electron microscopy followed by three-dimensional reconstruction to characterize the symbiont division during the cell cycle of S. culicis. We observed that during this process, the symbiotic bacterium presents different forms and is found at different positions in relationship to the host cell structures. At the G1/S phase of the protozoan cell cycle, the endosymbiont exhibits a constricted form that appears to elongate, resulting in the bacterium division, which occurs before kinetoplast and nucleus segregation. During cytokinesis, the symbionts are positioned close to each nucleus to ensure that each daughter cell will inherit a single copy of the bacterium. These observations indicated that the association of the bacterium with the protozoan nucleus coordinates the cell cycle in both organisms.

Euglena gracilis and Trypanosomatids possess common patterns in predicted mitochondrial targeting presequences.

Euglena gracilis possessing chloroplasts of secondary green algal origin and parasitic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi and Leishmania major belong to the protist phylum Euglenozoa. Euglenozoa might be among the earliest eukaryotic branches bearing ancestral traits reminiscent of the last eukaryotic common ancestor (LECA) or missing features present in other eukaryotes. LECA most likely possessed mitochondria of endosymbiotic α-proteobacterial origin. In this study, we searched for the presence of homologs of mitochondria-targeted proteins from other organisms in the currently available EST dataset of E. gracilis. The common motifs in predicted N-terminal presequences and corresponding homologs from T. brucei, T. cruzi and L. major (if found) were analyzed. Other trypanosomatid mitochondrial protein precursor (e.g., those involved in RNA editing) were also included in the analysis. Mitochondrial presequences of E. gracilis and these trypanosomatids seem to be highly variable in sequence length (5-118 aa), but apparently share statistically significant similarities. In most cases, the common (M/L)RR motif is present at the N-terminus and it is probably responsible for recognition via import apparatus of mitochondrial outer membrane. Interestingly, this motif is present inside the predicted presequence region in some cases. In most presequences, this motif is followed by a hydrophobic region rich in alanine, leucine, and valine. In conclusion, either RR motif or arginine-rich region within hydrophobic aa-s present at the N-terminus of a preprotein can be sufficient signals for mitochondrial import irrespective of presequence length in Euglenozoa.

AQPX-cluster aquaporins and aquaglyceroporins are asymmetrically distributed in trypanosomes.

Major Intrinsic Proteins (MIPs) are membrane channels that permeate water and other small solutes. Some trypanosomatid MIPs mediate the uptake of antiparasitic compounds, placing them as potential drug targets. However, a thorough study of the diversity of these channels is still missing. Here we place trypanosomatid channels in the sequence-function space of the large MIP superfamily through a sequence similarity network. This analysis exposes that trypanosomatid aquaporins integrate a distant cluster from the currently defined MIP families, here named aquaporin X (AQPX). Our phylogenetic analyses reveal that trypanosomatid MIPs distribute exclusively between aquaglyceroporin (GLP) and AQPX, being the AQPX family expanded in the Metakinetoplastina common ancestor before the origin of the parasitic order Trypanosomatida. Synteny analysis shows how African trypanosomes specifically lost AQPXs, whereas American trypanosomes specifically lost GLPs. AQPXs diverge from already described MIPs on crucial residues. Together, our results expose the diversity of trypanosomatid MIPs and will aid further functional, structural, and physiological research needed to face the potentiality of the AQPXs as gateways for trypanocidal drugs.

Detection and localization of small nuclear ribonucleoproteins (snRNPs) in trypanosomatids using anti-m3G antibodies.

This work reports for the first time the identification and immunolocalization, by confocal and conventional indirect immunofluorescence, of m3G epitopes present in ribonucleoproteins of the following trypanosomatids: Trypanosoma cruzi epimastigotes of three different strains, Blastocrithidia ssp., and Leishmania major promastigotes. The identity of these epitopes and hence the specificity of the anti-m3G monoclonal antibody were ascertained through competition reaction with 7-methylguanosine that blocks the Ig binding sites, abolishing the fluorescence in all the parasites tested and showing a specific perinuclear localization of the snRNPs, which suggests their nuclear reimport in the parasites. Using an immunoprecipitation technique, it was also possible to confirm the presence of the trimethylguanosine epitopes in trypanosomatids.

TryTransDB: A web-based resource for transport proteins in Trypanosomatidae.

TryTransDB is a web-based resource that stores transport protein data which can be retrieved using a standalone BLAST tool. We have attempted to create an integrated database that can be a one-stop shop for the researchers working with transport proteins of Trypanosomatidae family. TryTransDB (Trypanosomatidae Transport Protein Database) is a web based comprehensive resource that can fire a BLAST search against most of the transport protein sequences (protein and nucleotide) from Trypanosomatidae family organisms. This web resource further allows to compute a phylogenetic tree by performing multiple sequence alignment (MSA) using CLUSTALW suite embedded in it. Also, cross-linking to other databases helps in gathering more information for a certain transport protein in a single website.

An evaluation of lipid metabolism in the insect trypanosomatid Herpetomonas muscarum uncovers a pathway for the uptake of extracellular insect lipoproteins.

Lipid uptake and metabolism by trypanosomatid parasites from vertebrate host blood have been well established in the literature. However, there is a lack of knowledge regarding the same aspects concerning the parasites that cross the hemolymph of their invertebrate hosts. We have investigated the lipid composition and metabolism of the insect trypanosomatid Herpetomonas muscarum by 3H- palmitic acid and phosphate (32Pi) and the parasite interaction with Lipophorin (Lp) the main lipid carrying protein of insect hemolymph. Gas chromatography-mass spectrometry (GC-MS) analyses were used to identify the fatty acids and sterols composition of H.muscarum. Furthermore, we investigated the Lp binding site in the plasma membrane of parasite by Immunolocalization. We showed that H. muscarum incorporated 3H-palmitic acid and inorganic phosphate (32Pi) which were readily used as precursor molecules of lipid biosynthetic pathways. Furthermore, H. muscarum was able to take up both protein and lipid moieties of Lp which could be used as nutrient sources. Moreover, we have also demonstrated for the first time the presence of a Lp binding site in the membrane of a parasite. Such results point out the role of describing the metabolic pathways of trypanosomatids in order to provide a better understanding of parasite-host interaction peculiarities. Such studies may enhance the potential form the identification of novel chemotherapeutic targets in harmful parasites.

Polyamine-Based Thiols in Trypanosomatids: Evolution, Protein Structural Adaptations, and Biological Functions.

SIGNIFICANCE: Major pathogenic enterobacteria and protozoan parasites from the phylum Euglenozoa, such as trypanosomatids, are endowed with glutathione (GSH)-spermidine (Sp) derivatives that play important roles in signaling and metal and thiol-redox homeostasis. For some Euglenozoa lineages, the GSH-Sp conjugates represent the main redox cosubstrates around which entire new redox systems have evolved. Several proteins underwent molecular adaptations to synthesize and utilize the new polyamine-based thiols. Recent Advances: The genomes of closely related organisms have recently been sequenced, which allows mining and analysis of gene sequences that belong to these peculiar redox systems. Similarly, the three-dimensional structures of several of these proteins have been solved, which allows for comparison with their counterparts in classical redox systems that rely on GSH/glutaredoxin and thioredoxin. CRITICAL ISSUES: The evolutionary and structural aspects related to the emergence and use of GSH-Sp conjugates in Euglenozoa are reviewed focusing on unique structural specializations that proteins developed to use N1,N8-bisglutathionylspermidine (trypanothione) as redox cosubstrate. An updated overview on the biochemical and biological significance of the major enzymatic activities is also provided. FUTURE DIRECTIONS: A thiol-redox system strictly dependent on trypanothione is a feature unique to trypanosomatids. The physicochemical properties of the polyamine-GSH conjugates were a major driving force for structural adaptation of proteins that use these thiols as ligand and redox cofactor. In fact, the structural differences of indispensable components of this system can be exploited toward selective drug development. Future research should clarify whether additional cellular processes are regulated by the trypanothione system. Antioxid. Redox Signal. 28, 463-486.

Telomere biology of trypanosomatids: beginning to answer some questions.

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.

A giant protein associated with the anterior pole of a trypanosomatid cell body skeleton.

A megadalton protein was found to be a cytoskeleton component of the promastigote forms of the flagellate Phytomonas serpens. This protein migrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis as a doublet of polypeptides with a molecular mass similar to muscle beta-connectin (titin) 2500-3000 kDa. A polyclonal antibody raised against this protein reacts, by immunoblot analysis, with Phytomonas serpens and two others Phytomonas species. In addition, the Phytomonas serpens protein was immunoprecipitated after being metabolically labeled with [35S]methionine. This antibody did not cross-react with the cytoskeletal proteins of Trypanosoma cruzi, Crithidia luciliae thermophila, Crithidia fasciculata and Leptomonas samueli or with beta-connectin (titin). Indirect immunofluorescence microscopy analysis revealed a punctate fluorescence staining at the anterior region of the parasite's body skeleton. Moreover, immunogold electron microscopy of cytoskeletal preparations and of thin sections of whole cells indicates that the giant protein appears to cap the anterior end of the cell body microtubules at the level of the junctional complex. We suggest that this giant protein may serve as a linker between the cell body skeleton and the flagellum membrane.

The presence of galactofuranose and ribose units in asparagine-linked oligosaccharides of the digenetic trypanosomatid Endotrypanum schaudinni.

It was found that the digenetic trypanosomatid Endotrypanum schaudinni transferred Man7GlcNAc2 in protein N-glycosylation. Endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides were identified as Man7GlcNAc2, Man6GlcNAc2, Rib1Man6GlcNAc2 and/or Gal1Man6GlcNAc2, Man5GlcNAc containing two galactose or ribose units or one of each residues, Rib1Man5GlcNAc2 and Gal1Man5GlcNAc2. The galactoses were in the furanose configuration. Endo-beta-N-acetylglucosaminidase H-resistant glycopeptides that were retained by concanavalin A-Sepharose and eluted with alpha-methylmannoside were found to contain mannose, galactofuranose and ribose units. The presence of galactofuranoses in N-glycoproteins has been reported previously in several monogenetic trypanosomatids but only in one digenetic parasite (Trypanosoma cruzi). This and a recent publication on the structure of Blastocrithidia culicis N-linked oligosaccharides are the first reports on the presence of ribose in eukaryotic glycoconjugates.

Chemical characterisation of glycosylinositolphospholipids of Herpetomonas samuelpessoai.

The structure of two glycosylinositolphospholipids of the cell surface of the monoxenic protozoan Herpetomonas samuelpessoai have been deduced by methylation analysis, fast-atom bombardment mass spectrometry and two dimensional nuclear magnetic resonance spectroscopy. These glycolipids have features in common with the glycoinositolphospholipids of both Leishmania and Trypanosoma cruzi, resembling the former by the presence of the hybrid type core sequence Man alpha 1-->3(Man alpha 1-->6)Man alpha 1-->4GlcN alpha 1-->6 myo-inositol-1-PO4-lipid, while the 2-aminoethylphosphonate substituent on 0-6 of glucosamine and the presence of ceramide in place of glycerol lipids is more reminiscent of T. cruzi. Possible phylogenetic implications of these observations are discussed.

Purification and characterisation of a novel iso-propanol dehydrogenase from Phytomonas sp.

An alcohol dehydrogenase with two identical subunits and a subunit molecular mass of 40,000 was purified from Phytomonas sp. isolated from the lactiferous tubes of Euphorbia characias. Digitonin titration and subcellular fractionation suggest that the enzyme is present in the mitochondrion. It utilises as substrates, primary and secondary alcohols, is specific for NAD+ as coenzyme and is inhibited by HgCl(2). The pH optimum for the oxidation of ethanol is 9.5, and for the reverse reaction 8.5. The apparent Km values for iso-propanol and ethanol are 40 and 34 microM, respectively and for the reverse reaction, with acetone as substrate, 14 microM. The respective specific activities with iso-propanol and ethanol as substrate, as measured in crude extracts are 300 and 16 mU (milligram of protein)-1. In isoelectric focusing the enzyme showed three major bands with slightly differing isoelectric points that ranged from 6.4 to 6.8. The name, iso-propanol dehydrogenase is proposed for this enzyme.

Partial kinetoplast-mitochondrial gene organization and expression in the respiratory deficient plant trypanosomatid Phytomonas serpens.

In plant-dwelling trypanosomatids from the genus Phytomonas, mitochondrial functions, such as cytochrome mediated respiration, ATP production and Krebs cycle, are missing, and cell energetics is based on the glycolysis. Using Blue Native/Tricine-SDS two-dimensional gel electrophoretic analysis, we observed that mitochondrial respiratory Complexes III (cytochrome bc1) and IV (cytochrome c oxidase) were absent in Phytomonas serpens; however, Complex V (ATPase) was present. A deletion of the genes for cytochrome c oxidase subunit III (COIII) and apocytochrome b (Cyb) was identified within the 6234 bp sequenced region of the 31 kb maxicircle kinetoplast DNA. Genes, found in this region, include 12S and 9S ribosomal RNAs, subunits 7, 8 and 9 of NADH dehydrogenase (ND7, ND8 and ND9) and subunit 6 of ATPase (A6 or MURF4), as well as the genes (MURF1, MURF5 and G3) with unknown function. Most genes are actively transcribed and some mRNAs are edited. Fully edited mRNAs for A6 and G3 were abundant, while edited ND7 transcripts were rare, and only partially edited and pre-edited transcripts for ND8 were detected. The data show that the mitochondrial genome of P. serpens is functional, although its functions may be limited to expressing the ATPase and, possibly, NADH dehydrogenase complexes.

Acidocalcisomes of trypanosomatids have species-specific elemental composition.

The elemental composition and stoichiometric profile of elements present in acidocalcisomes of different genera of the Trypanosomatidae family (insect, plant, and mammalian parasites) submitted to parallel cultivation conditions were studied. X-ray microanalysis using transmission electron microscopy in conjunction with a morphometric approach was used to investigate the elemental content, number, distribution, and volumetric density of acidocalcisomes of different species. Microanalytical data showed that the different parasites possess the same elemental composition (oxygen, sodium, magnesium, phosphorus, calcium, iron, and zinc) in their acidocalcisomes. However, the relative concentrations of the elements varied among species, but not within acidocalcisomes of individual species. Iron was detected in acidocalcisomes of all species analyzed, characterizing this element as a constituent of these organelles. Taken together, the results strongly indicate a species-specific composition of acidocalcisomes in trypanosomatid parasites.

The Leptomonas seymouri spliced leader RNA promoter requires a novel transcription factor.

The spliced leader RNA gene promoter in Leptomonas seymouri requires three promoter elements for efficient and accurate transcription of the spliced leader RNA. The upstream most element appears to have a functional homolog in Leishmania species and in the African trypanosomes. The protein factor, promoter binding protein-1, interacts with the upstream element and appears to function as a basal transcription factor. Promoter binding protein-1 has three subunits; 36, 41 and 57 kDa. Using microsequencing techniques, we have obtained peptide sequence from each subunit. These data have enabled us to recently identify the Leptomonas gene that encodes the 41 kDa subunit. The 41 kDa subunit, comprised of 381 amino acids, is a founding member of a new class of transcription factors since extensive database searches revealed no homology to any known protein. This subunit, encoded by a single copy gene, has a potential nuclear localisation signal at amino acid positions 71-76. There are also multiple dileucine repeats with unknown function. Anti-41 kDa protein polyclonal antibodies are being employed to test the function of the 41 kDa subunit in PBP-1 activities.

Differential lectin recognition of glycoproteins in choanomastigote-shaped trypanosomatids: taxonomic implications.

The glycoprotein profiles of seven choanomastigote-shaped trypanosomatids (six Crithidia spp. and one Herpetomonas sp.), which have been suggested to form three distinct taxonomic groups (Crithidia, Angomonas and Strigomonas), were analyzed by Western blotting using the lectins Limax flavus (LFA), Sambucus nigra (SNA) and Maackia amurensis (MAA), which specifically recognize sialic acid residues, and concanavalin A (Con A) that recognizes mannose-like residues in glycoconjugates. All lectins showed a sugar-inhibited recognition with the parasite extracts, with the exception of LFA, which did not show any reactivity with the studied species. The SNA agglutinin presented a characteristic and specific pattern for each taxonomic group. The MAA lectin showed an identical profile for all species analyzed, while Con A grouped the choanomastigote-shaped species in two different patterns, one specific for the Angomonas group, and the other comprehending both Strigomonas and Crithidia groups. These results illustrate the heterogeneity of the genus Crithidia. The possible taxonomic redistribution of the choanomastigote-shaped trypanosomatids is also discussed.

Detection of sialoglycomolecules in five plant trypanosomatids and in an insect phytophagous isolate.

The sialoglycoprotein profiles of five plant trypanosomatids (Phytomonas spp.) and of one flagellate (Herpetomonas sp.) isolated from the salivary gland of a phytophagous insect (Phthia picta) were analyzed by Western blotting using three distinct lectins (LFA, SNA and MAA), which recognize specifically sialic acid residues in glycoconjugates. All six flagellates presented at least one polypeptide recognized by the lectins, with the exception of Phytomonas françai, which did not show any reactivity with SNA agglutinin. Phytomonas serpens and P. françai showed the most distinct pattern of sialoglycoproteins. Phytomonas mcgheei, Herpetomonas sp. and the two other Phytomonas spp., isolated from latex, displayed an identical sialomolecule profile. We discuss the possible role of the sialoglycoproteins in the physiology of these trypanosomatids.