Identification of PDZ5, a candidate universal minicircle sequence binding protein of Trypanosoma cruzi

The dodecamer universal minicircle sequence is a conserved sequence present in minicircles of trypanosomatid kinetoplast DNA studied so far. This sequence is recognised by a protein named universal minicircle sequence binding protein, described for Crithidia fasciculata, involved in minicircle DNA replication. We have identified a Trypanosoma cruzi gene homologue of the Crithidia fasciculata universal minicircle sequence binding protein. Similar to the Crithidia fasciculata universal minicircle sequence binding protein, the Trypanosoma cruzi protein, named PDZ5, contains five zinc finger motifs. Pulsed field gel electrophoresis indicated that the pdz5 gene is located in the chromosomal band XX of the Trypanosoma cruzi genome. The predicted amino acid sequence of PDZ5 shows a high degree of similarity with several trypanosomatid zinc finger proteins. Specific antibody raised against Crithidia fasciculata universal minicircle sequence binding protein recognises both the recombinant and endogenous PDZ5. The complete pdz5 coding sequence cloned in bacteria expresses a recombinant PDZ5 protein that binds specifically to the universal minicircle sequence dodecamer. These data strongly suggest that PDZ5 represents a Trypanosoma cruzi universal minicircle sequence binding protein.     

Mini-exon Gene Sequences Define Six Groups within the Genus Crithidia

ABSTRACT. To develop molecular markers for lower trypanosmatids, we have examined the mini-exon gene repeats of 17 isolates that were classified as Crithidia by traditional methods. Representative repeats were amplified by polymerase chain reaction and the amplification products were cloned and used as hybridization probes against genomic DNA. Six hybridization groups of Crithidia were defined on the basis of the DNA blotting experiments. The three endosymbiont-bearing species ( C. deanei, C. desouzai and C. oncopelti ) and C. acanthocephali each belonged to single-member hybridization groups, while the C. fasciculata group contained additional named and undesignated species. The Crithidia lucilae thermophila probe hybridized to multiple undesignated isolates. The DNA sequence of the cloned products revealed that the specificity of the hybridization probes was due to substantial differences in the intron and the non-transcribed spacer regions. These data indicate substantial heterogeneity within the mini-exon gene locus of the taxon Crithidia .     

The Crithidia fasciculata CRK gene encodes a novel cdc2-related protein containing large inserts between highly conserved domains.

A gene (CRK) encoding a cdc2-related protein has been identified in the trypanosomatid Crithidia fasciculata. CRK has a high degree of sequence identity with the human cdc2 gene and contains the sixteen amino acid PSTAIR motif, characteristic of p34cdc2 protein-serine/threonine kinases, with four amino acid substitutions in the motif. In addition, two inserts of more than sixty amino acids have been found between conserved domains of this putative protein-serine/threonine kinase. CRK is a single copy gene and is expressed on a 3.8 kb mRNA. Anti-CRK antibodies detect a 53kDa protein in extracts of C.fasciculata in agreement with the size predicted from the nucleotide sequence of the cloned gene. These antibodies also recognize proteins of 48 and 60 kDa in extracts of the trypanosomatid Leishmania tarentolae. Antibodies against the human PSTAIR peptide detect the p34cdc2 protein in human nuclear extracts but fail to detect a 34 kDa protein in C.fasciculata extracts. These results suggest that novel higher molecular weight forms of the cdc2 protein family may be involved in cell cycle control in trypanosomes.     

Molecular cloning and characterization of two new isoforms of the protein kinase A catalytic subunit from the human parasite Leishmania

Leishmania are protozoan parasites that cause extensive morbidity and mortality in humans. Genes for two new isoforms of the protein kinase A catalytic subunit (PKAC) in Leishmania, Lmpkac2a and Lmpkac2b, were cloned and characterized. The predicted open reading frames for these isoforms are 93.4% identical over 338 amino acids (aa). The conserved PK catalytic cores (subdomains I-XI) are identical, while the carboxy-terminal extensions differ by only two aa. However, LmPKAC2 shares only 62% identity over the 255 aa catalytic core region with the previously described LmPKAC1 (c-lpk2). Unlike LmPKAC1, the location of the FXXF motif at the carboxy-terminus is conserved in both LmPKAC2 isoforms; however, the aa sequence, LXXF, in isoform-2a is unusual. The leishmanial isoforms can be distinguished by their NH(2)-terminal extensions, which show minimal similarity at the primary sequence level. Structural analysis of the three enzymes based on the crystal structure of mammalian PKAs predicts that both LmPKAC2 isoforms, unlike LmPKAC1, have identical alpha-helix structures in the NH(2)-terminal extension. Lmpkac2 genes are located on chromosome 35 just downstream from the leishmanial prp8 gene. This genomic organization is conserved in two species of Leishmania and Crithidia fasciculata and allowed for the partial analysis of Cfpkac2a. Phylogenetic analysis groups the two LmPKAC2 isoforms together and separately from LmPKAC1, which is more similar to the Euglena gracilis PKAC, EPK2. These findings provide the basis for additional studies on the role of the PKA family in parasite differentiation and virulence.     

The majority of minicircle DNA in Crithidia fasciculata strain CF-C1 is of a single class with nearly homogeneous DNA sequence

DNA minicircles found within the kinetoplast of the trypanosomatid Crithidia fasciculata, like those of most other kinetoplastid species, are heterogeneous in sequence. The pattern of minicircle DNA fragments generated by cleavage of kinetoplast DNA with various restriction enzymes has been used to demonstrate this heterogeneity. Here we describe a strain of Crithidia fasciculata in which more than 90% of the DNA minicircles exhibit a common pattern of restriction enzyme cleavage sites. A map of cleavage sites within this major minicircle DNA class is presented for seven restriction enzymes with hexanucleotide recognition sequences. Sequence homogeneity at an even finer level is reflected in minicircle DNA digestion patterns generated by restriction enzymes with tetranucleotide recognition sites. Partial DNA sequence analysis of multiple clones from the major minicircle class shows nearly complete homogeneity at the nucleotide level. The existence of a near homogeneous complement of DNA minicircles in Crithidia should facilitate the study of their replication in this organism.     

Trypanosomatidae codon usage and GC distribution.

A study of Trypanosomatidae GC distribution and codon usage is presented. The codon usage patterns in coincidence with the phylogenetical data are similar in Crithidia and Leishmania, whereas they are more divergent in Trypanosoma brucei and T. cruzi. The analysis of the GC mutational pressure in these organisms reveals that T. brucei, and to a lesser extent T. cruzi, have evolved towards a more balanced use of all bases, whereas Leishmania and Crithidia retain features of a primeval genetic apparatus. Tables with the approximated GC mutational pressure in homologous genes, and codon usage in Trypanosomatidae are presented.     

A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.

Replication of the kinetoplast DNA minicircle light strand initiates at a highly conserved 12-nucleotide sequence, termed the universal minicircle sequence. A Crithidia fasciculata single-stranded DNA-binding protein interacts specifically with the guanine-rich heavy strand of this origin-associated sequence (Y. Tzfati, H. Abeliovich, I. Kapeller, and J. Shlomai, Proc. Natl. Acad. Sci. USA 89:6891-6895, 1992). Using the universal minicircle sequence heavy-strand probe to screen a C. fasciculata cDNA expression library, we have isolated two overlapping cDNA clones encoding the trypanosomatid universal minicircle sequence-binding protein. The complete cDNA sequence defines an open reading frame encoding a 116-amino-acid polypeptide chain consisting of five repetitions of a CCHC zinc finger motif. A significant similarity is found between this universal minicircle sequence-binding protein and two other single-stranded DNA-binding proteins identified in humans and in Leishmania major. All three proteins bind specifically to single-stranded guanine-rich DNA ligands. Partial amino acid sequence of the endogenous protein, purified to homogeneity from C. fasciculata, was identical to that deduced from the cDNA nucleotide sequence. DNA-binding characteristics of the cDNA-encoded fusion protein expressed in bacteria were identical to those of the endogenous C. fasciculata protein. Hybridization analyses reveal that the gene encoding the minicircle origin-binding protein is nuclear and may occur in the C. fasciculata chromosome as a cluster of several structural genes.     

Analyses of surface membrane carbohydrates in parasitic flagellates of the order kinetoplastida using lectins.

Crithidia fasciculata, Leishmania donovani, Leishmania major, Leishmania mexicana amazonensis, Leishmania tropica, Leishmania tarentolae, Trypanosoma sp. from Formosan bats (Tb), Trypanosoma lewisi, Trypanosoma musculi, and different strains of Trypanosoma cruzi (Tc) were cultivated at 27 degrees C in a liquid culture medium. Flagellates harvested from log phase culture were analyzed for their lectin agglutinating characteristics with concanavalin A (Con A), Peanut agglutinin, Ricinus communis agglutinin 120, soybean agglutinin (SBA), Ulex europeus agglutinin (UEA) and wheat germ agglutinin (WGA). Results indicated that all these flagellates might have D-galactose and methyl- alpha-D-manopyranoside on their surface. The presence of L-Fucose, which complexes specifically with UEA, could not be demonstrated on the surface of these flagellates. Results from quantitative comparison of surface molecules of Tb and the Tulahuen strain of Tc suggested that Tb may have more WGA-binding molecules while Tc may have more ConA-binding molecules. Pretreatment of the flagellates with 0.05% trypsin at 37 degrees C for 30 minutes caused some reduction of agglutination titers. Cell agglutination with lectins was completely inhibited or reversed in the presence of the specific lectin-binding monosaccharides.     

Cytochrome c reductase purified from Crithidia fasciculata contains an atypical cytochrome c1.

Cytochrome c reductase purified from the trypanosomatid Crithidia fasciculata retained antimycin A sensitivity and catalyzed the reduction of horse heart ferricytochrome c in the presence of reduced coenzyme Q10. The complex contained heme b and heme c1 in a ratio of 2:1. Nine major protein bands ranging in size from 55.3 to approximately 12.8 kDa were resolved by SDS-polyacrylamide gel electrophoresis. A 31.6-kDa protein was identified as cytochrome c1 by the presence of a covalently attached heme. A red shift in the alpha-absorbance band of the cytochrome c1 absolute absorbance spectrum, difference absorbance spectrum, and pyridine ferrohemochrome absorbance spectrum suggested that the heme prosthetic group of C. fasciculata cytochrome c1 is bound to the apoprotein through only one thioether bond. A fragment of the cytochrome c1 gene was amplified from C. fasciculata, Trypanosoma brucei, Leishmania tarentolae, and Bodo caudatus. The deduced heme binding site sequence of each of these kinetoplastid species, Phe-Ala-Pro-Cys-His, contains a phenylalanine rather that a cysteine at the first position so that only one thioether bond can be formed between heme and apoprotein. This phenylalanine substitution and the presence of a conserved proline in the sequence may represent compensatory changes that are necessary for optimal interaction of the cytochromes c1 with the atypical cytochromes c of these species.     

Leishmania major elongation factor 1B complex has trypanothione S-transferase and peroxidase activity

In the Trypanosomatidae, trypanothione has subsumed many of the roles of glutathione in defense against chemical and oxidant stress. Crithidia fasciculata lacks glutathione S-transferase, but contains an unusual trypanothione S-transferase activity that is associated with eukaryotic translation elongation factor 1B (eEF1B). Here we describe the cloning, expression, and reconstitution of the purified alpha, beta, and gamma subunits of eEF1B from Leishmania major. Individual subunits lacked trypanothione S-transferase activity. Only eEF1B, formed by reconstitution or co-expression of the three subunits, was able to conjugate a variety of electrophilic substrates to trypanothione or glutathionylspermidine, but not glutathione. In contrast to the C. fasciculata eEF1B, the L. major enzyme also displayed peroxidase activity against a variety of organic hydroperoxides. The enzyme showed no activity with hydrogen peroxide and greatest activity with linoleic acid hydroperoxide (1 unit mg(-1)). Kinetic studies suggest a ternary complex mechanism, with Km values of 140 mum for trypanothione and 7.4 mm for cumene hydroperoxide and kcat=25 s(-1). Immunofluorescence studies indicate that the enzyme may be localized to the surface of the endoplasmic reticulum. These results suggest that, in addition to its role in protein synthesis, the Leishmania eEF1B may help protect the parasite from lipid peroxidation.     

Five trypanosomatid species of insects distinguished by isoenzymes.

Blastocrithidia culicis, Crithidia deanei, Crithidia fasciculata, Herpetomonas samuelpessoai, Leptomonas seymouri and Leishmania tarentolae grown in cultures were compared by electrophoretic mobility for isoenzymes in 6 enzymes. All species were found distinct in these characteristics. Endosymbiotic C. deanei, which was identical to the aposymbiotic C. deanei in 5 enzymes, had an extra band in aspartate aminotransferase. No differences in isoenzymes were found between members of one species maintained in 2 different culture media.     

A 189-bp fragment of Crithidia fasciculata maxicircle DNA confers autonomous replication in Saccharomyces cerevisiae

A 189-bp fragment capable of promoting high-frequency transformation in Saccharomyces cerevisiae has been isolated from the maxicircle of the insect trypanosomatid Crithidia fasciculata. Chimeric plasmids containing this autonomously replicating sequence (ars) are maintained as extrachromosomal elements in S. cerevisiae. The nucleotide sequence of the maxicircle fragment, termed ars189, has been determined, and its position has been mapped in the maxicircle. The ars189 fragment has an A + T content of 79.4% and shows a large asymmetry in the distribution of adenine and thymine residues between the two strands. In one strand (the T strand) thymine accounts for 118 out of 189 nucleotides while adenine accounts for only 32 nucleotides. The ars189 DNA does not hybridize with minicircles, and its sequence appears to be unique in the C. fasciculata maxicircle genome. This sequence also shows extensive homology to a sequence within a 2.6-kb ars fragment of the Leishmania tarentolae maxicircle. In addition, ars189 contains two copies of a yeast consensus ars sequence (A/T)TTTATPuTTT(T/A).     

Evolution of codon usage and base contents in kinetoplastid protozoans

In this study we analyze and compare the trends in codon usage in five representative species of kinetoplastid protozoans (Crithidia fasciculata, Leishmania donovani, L. major, Trypanosoma cruzi and T. brucei), with the purpose of investigating the processes underlying these trends. A principal component analysis shows that the G+C content at the third codon position represents the main source of codon-usage variation, both within species (among genes) and among species. The non- Trypanosoma species exhibit narrow distributions in codon usage, while both Trypanosoma species present large within-species heterogeneity. The three non-Trypanosoma species have very similar codon-usage preferences. These codon preferences are also shared by the highly expressed genes of T. cruzi and to a lesser degree by those of T. brucei. This leads to the conclusion that the codon preferences shared by these species are the ancestral ones in the kinetoplastids. On the other hand, the study of noncoding sequences shows that Trypanosoma species exhibit mutational biases toward A + T richness, while the non- Trypanosoma species present mutational pressure in the opposite direction. These data taken together allow us to infer the origin of the different codon-usage distributions observed in the five species studied. In C. fasciculata and Leishmania, both mutational biases and (translational) selection pull toward G + C richness, resulting in a narrow distribution. In Trypanosoma species the mutational pressure toward A + T richness produced a shift in their genomes that differentially affected coding and noncoding sequences. The effect of these pressures on the third codon position of genes seems to have been inversely proportional to the level of gene expression.      

A comparison of the amino acid sequences of Crithidia fasciculata and Trypanosoma rhodesiense cytochromes c

Apocytochrome c was isolated from procyclic trypomastigotes of Trypanosoma rhodesiense EATRO 1895 and purified on Amberlite IRC-50 ion-exchange resin. Tryptic peptides were generated from the purified apoprotein and a partial amino acid sequence was determined. A comparison of the amino acid sequence of Crithidia fasciculata with the partial amino acid sequence of T. rhodesiense reveals significant homology.     

Hexose and Glycerol Absorption by Some Trypanosomatidae

SYNOPSIS. Culture forms of 9 species of Trypanosomatidae were studied. Of these, Crithidia fasciculata, Leishmania brasiliensis, L. donovani, L. tropica, Trypanosoma conorhini, T. cruzi and T. rangeli consumed significantly less glycerol than glucose, both when the 2 substrates were offered singly or simultaneously. On the other hand, T. gambiense and T. rhodesiense consumed as much glycerol as glucose when the 2 substrates were given separately. When both substrates were offered simultaneously, more glycerol than glucose was consumed, but the sum of glucose + glycerol carbon taken up approximated closely that taken from glucose alone. This held for species consuming only little glycerol, indicating that in all cases some mutual inhibition took place. Atttempts to adapt T. cruzi to glycerol consumption were unsuccessful. When T. gambiense and T. rhosiense were cultivated in the presence of glycerol, their normrapid glycerol consumption remained also unchanged but, curiothey consumed more glucose than before "adaptation." Iodoacetami and NaF strongly inhibited glucose and glycerol absorption of 7 gambiense ; phloridzin, deoxygalactose and deoxyglucose gave nonificant inhibition. KCN slightly stimulated glucose absorption, weakly inhibited glycerol absorption. Omission of sodium from medium was without effect, while lack of CO₂ markedly inhiglucose and glycerol uptake. Galactose was not a satisfactory sub strate for T. gambiense and T. rhodesiense , nor were deoxygalactos and deoxyglucose. These 3 hexoses were consumed in small amonly and did not allow maintenance of motility.     

Various elements of the structure of mini-ring kinetoplast DNA of Crithidia fasciculata

The nucleotide sequence of 1.4 kbp SmaI-fragment of minicircle DNA from kinetoplasts of Crithidia fasciculata has been determined and some sequence elements characterized. The sequence contains several oligo(dT)blocks located on the same strand in phase with a period of DNA helix turn, thus representing a "bent helix". Both sides of the bent helix region are flanked by sequences capable of forming a cloverleaf structure. There are also two direct 150 bp repeats located 180 degrees apart on the circular map of the molecule. Each repeat contains the sites of H-strand and L-strand replication origin. The specific stem-loop secondary structure may be folded by the nucleotide sequence within the origins region. The alignment of the sequence determined with two other C. fasciculata minicircle sequences spanning over the bent helix and the adjacent regions has indicated the presence of several conserved sequence blocks, one of them representing the sequence of the bend. The divergence of three sequences occurred mainly by small insertions-deletions. Several open reading frames were found, the largest of which being capable of coding for the approximately 200 amino acids polypeptide.