CL Brener Clone Research Articles

Cysteine proteinases of Trypanosoma cruzi: from digestive enzymes to programmed cell death mediators

Trypanosoma cruzi, the parasite causing Chagas disease, contains a number of proteolytic enzymes. The recent completion of the genome sequence of the T. cruzi CL Brener clone suggests the presence of 70 cysteine peptidases, 40 serine peptidases (none of them from the chymotrypsin family), about 250 metallopeptidases (most leishmanolysin homologues), 25 threonine peptidases, and only two aspartyl peptidases, none of them from the pepsin family. The cysteine peptidases belong to 7 families of Clan CA, 3 families of Clan CD, and one each of Clans CE and CF In Clan CA, the C1 family is represented by cruzipains 1 and 2, biochemically well characterized, as well as cathepsin B and two other cathepsins. There are a number of homologues to calpains (family C2), probably non-functional, lacking the Ca-binding domain. Family C54 includes the Atg4 proteinases (autophagins), which seem to be involved in the autophagic process. Clan CD includes family C14, the metacaspases. We have expressed the metacaspases TcMCA3 and TcMCA5, and obtained indirect evidence of their participation in programmed cell death induced by fresh human serum in the parasite. More experiments are required to better define their role in apoptosis.

Metacaspases of Trypanosoma cruzi: Possible candidates for programmed cell death mediators

The genome of Trypanosoma cruzi, the Protozoan parasite causing the American Trypanosomiasis, Chagas disease, contains two genes, TcMCA3 and TcMCA5, with homology to those encoding metacaspases, distantly related to the caspases involved in programmed cell death (PCD) in higher eukaryotes. TcMCA3 is present in the CL Brener clone at 16 copies per haploid genome, arrayed in two tandems located in chromosomes of 0.54 and 0.98 Mbp. TcMCA5, on the other hand, is present as a single copy gene. The proteins encoded were expressed in Escherichia coli BL21 [DE3] cells, and used to generate antibodies, which allowed demonstrating that TcMCA3 is expressed in the four major developmental stages of the parasite, whereas TcMCA5 is expressed only in the epimastigote form. Moreover, recombinant TcMCA3, but not TcMCA5, was recognized by most sera from chronic Chagasic patients, showing that the protein is expressed during natural infections. All attempts to show processing and enzyme activity in the recombinant proteins have been unsuccesful so far; however, indirect evidence suggests that the metacaspases might be involved in PCD of the parasite. (1) Immunofluorescence experiments showed that both proteins change their subcellular localization during fresh human serum (FHS)-induced PCD migrating into the nucleus. (2) Epimastigotes over-expressing TcMCA5 were more sensitive to FHS-induced PCD than the controls. (3) PCD was paralelled by an increase in peptidase activity against Z-YVAD-AFC, a typical caspase substrate, and the apoptotic nuclei cells were labeled in vivo with the pan-caspase fluorescent inhibitor SR-VAD-FMK. Further experiments will be required to complete the characterization of these proteins and elucidate their role in the parasite.

The 6-phosphogluconate dehydrogenase from Trypanosoma cruzi: the absence of two inter-subunit salt bridges as a reason for enzyme instability

The third enzyme of the pentose phosphate pathway (PPP), 6-phosphogluconate dehydrogenase (6PGDH), is present in the four major stages of Trypanosoma cruzi, CL Brener clone. The enzyme was too unstable to be purified from epimastigote cell-free extracts. Two genes encoding 6PGDH were cloned and sequenced; the predicted amino acid sequences differ only in five non-essential residues. Since Southern blots suggested the presence of a single copy per haploid genome, the two genes found are probably alleles. One of these genes, encoding a protein with 78.6% identity with the Trypanosoma brucei 6PGDH, was expressed in Escherichia coli as an active recombinant enzyme, which was as unstable as the native 6PGDH. Modeling of the T. cruzi enzyme using the three-dimensional structure of the T. brucei 6PGDH as template suggested the lack of two out of five salt bridges proposed to strengthen subunit interactions in the active dimer. Restoring of these bridges by site-directed mutagenesis resulted in a more stable recombinant T. cruzi 6PGDH, which was used to determine the kinetic parameters. The K(m) value for 6-phosphogluconate (22.2+/-0.4 microM) was identical to the values reported for 6PGDHs from mammals, but the K(m) for NADP (5.9+/-0.2 microM) was significantly lower than the value reported for the human enzyme, and closer to that for the T. brucei enzyme. This suggests the possibility that inhibitors of the T. brucei 6PGDH, under development as potential drugs against African Trypanosomiasis, might also be successful for the chemotherapy of Chagas disease.

The amino terminal domain of a novel WD repeat protein from Trypanosoma cruzi contains a non-canonical mitochondrial targeting signal

WD (tryptophan/aspartic acid) repeat proteins perform a wide variety of functions in eukaryotic cells. They are characterised by the presence of a number of conserved repeat motifs that contribute to the β-propeller structures which are the common feature of this large group of proteins. We report here the properties of the first characterised member of this family in the American trypanosome, Trypanosoma cruzi (TcBPP1). In the CL Brener clone the protein is 482 amino acids long and is predicted to contain four WD repeat motifs, flanked by amino and carboxyl terminal extensions. TcBPP1 is a single copy gene present on a 1.0/1.6 Mb pair of homologous chromosomes in a locus that is syntenic with the corresponding regions of Trypanosoma brucei and Leishmania major chromosomes. Consistent with the proposed hybrid nature of the CL Brener clone, the proteins encoded by the two different alleles share only 97% identity at the amino acid level. To determine subcellular location, we examined transfected parasites for the distribution of green fluorescent protein (GFP) fused with different regions of TcBPP1. These studies demonstrated that a 115 amino acid peptide derived from the amino terminal domain of TcBPP1 is able to target GFP to the mitochondrion. Interestingly this region lacks a typical amino terminal presequence suggesting that mitochondrial import is mediated by an alternative targeting signal.

Trypanosoma cruzi: mixture of two populations can modify virulence and tissue tropism in rat

In rats, CL-Brener clone caused high mortality, severe acute myocarditis, and myositis that subsided completely in surviving animals. Accordingly, no parasite kDNA could be amplified in several organs after 4 months. The monoclonal JG strain caused null mortality, acute predominantly focal myocarditis, discrete and focal myositis, and a chronic phase with sparse inflammatory foci. Double infection with both Trypanosoma cruzi populations turned mortality very low or null. At the end of the acute phase, the heart exhibited only JG strain kDNA (LSSP-PCR), while skeletal muscles and rectum exhibited only CL-Brener kDNA. Molecular and histopathological findings were accordant. In double infection chronic phase, JG strain remains in heart and appeared in organs previously parasitized by CL-Brener clone. Understanding the virulence and histotropism shifts now described could be important to clarify the variable clinical course and epidemiological peculiarities of Chagas’ disease. Index Descriptors and Abbreviations: kDNA, kinetoplast DNA; PCR, polymerase chain reaction; LSSP-PCR, low-stringency single-specific primer-PCR

Trypanosoma cruzi: Molecular identification and characterization of new members of the Tc13 family. Description of the interaction between the Tc13 antigen from Tulahuén strain and the second extracellular loop of the β 1-adrenergic receptor

Trypanosoma cruzi Tc13 antigens belong to the trans-sialidase superfamily. Their sequences have been described only partially and, up to now, their physiological activity has not been elucidated. Here we present two new members of this family from the Tulahuén strain (Tc13 Tul) and the CL Brener clone (Tc13 CL), being the latter the first Tc13 sequence fully described. Alignment of all Tc13 sequences allowed us to define two sub-families that differ in the number of repeats and the presence or absence of the GPI addition site. Chromoblots demonstrate that Tc13 antigens are mainly located in chromosome III and its homologous. Pull down assays suggest that recombinant MBP-Tc13 Tul interacts with the second extracellular loop of the β 1-adrenergic receptor. This is the first evidence that a Tc13 antigen acts as a ligand interacting with a neurotransmitter receptor. These observations might add some light to the development of chagasic pathology. Index Descriptors and Abbreviations: β 1-AR, β 1-adrenergic receptor; β 1e2, β 1-AR second extracellular loop; GPI, glycosylphosphatidyl inositol; GSH, glutathione; GST, glutathione- S-transferase; m2 mAChR, m2 muscarinic cholinergic receptor; m2e2, m2 mAChR second extracellular loop; MBP, maltose binding protein; SjGST, Schistosoma japonicum GST; Tc13 CL, Tc13 antigen from CL Brener clone; Tc13 Tul, Tc13 antigen from Tulahuén strain

Molecular cloning and characterization of a gene encoding the 29-kDa proteasome subunit from Trypanosoma cruzi.

Antiserum raised against purified Trypanosoma cruzi proteasomes was used to isolate two cDNA clones, tcpr29 and tcpr29B, and the corresponding genomic sequence, termed tcpr29A. Both cDNAs and the gene contain a 798-bp ORF, coding for a 266-amino acid protein, with a predicted molecular mass of 29 kDa. Sequence comparisons show that the protein encoded by tcpr29 belongs to the alpha6 subfamily of proteasome subunits. Southern analysis indicated that tcpr29 subunit is encoded by a single-copy gene which maps to chromosome 20 of the CL Brener clone. Allelic variants were found in other T. cruzi isolates, suggesting heterozygosity for the gene in some and homozygosity in other strains. A spliced-leader addition site was identified 123 bp upstream from the start codon, generating a stable 1.5-kb transcript. Western analysis revealed that tcpr29A is constitutively expressed during the life cycle of the parasite.

Characterisation of a cyclophilin isoform in Trypanosoma cruzi

The immunosuppressive drug cyclosporin A (CsA) has shown antiparasitic activity against several protozoans and helminths, when complexed to proteins called cyclophilins (CyPs). In this paper, the molecular characterisation of one member of the CyP family in Trypanosoma cruzi is reported. TcCyP19 gene proved to be highly conserved compared to CyPs from other organisms and was highly homologous to a Trypanosoma brucei brucei CyPA. This gene was expressed in Escherichia coli and the purified recombinant protein exhibited a peptidyl prolyl cis- trans isomerase activity that was inhibited by CsA (IC 50=18.4±0.8 nM). The TcCyP19 gene was located on two chromosomal bands in T. cruzi CL Brener clone.

Characterisation of a cyclophilin isoform in Trypanosoma cruzi.

The immunosuppressive drug cyclosporin A (CsA) has shown antiparasitic activity against several protozoans and helminths, when complexed to proteins called cyclophilins (CyPs). In this paper, the molecular characterisation of one member of the CyP family in Trypanosoma cruzi is reported. TcCyP19 gene proved to be highly conserved compared to CyPs from other organisms and was highly homologous to a Trypanosoma brucei brucei CyPA. This gene was expressed in Escherichia coli and the purified recombinant protein exhibited a peptidyl prolyl cis-trans isomerase activity that was inhibited by CsA (IC(50) = 18.4 + /-0.8 nM). The TcCyP19 gene was located on two chromosomal bands in T. cruzi CL Brener clone.

Usefulness of microsatellite typing in population genetic studies of Trypanosoma cruzi.

Through microsatellite analysis of 53 monoclonal populations of Trypanosoma cruzi, we found a remarkable degree of genetic polymorphism with no single multilocus genotype being observed more than once. The microsatellite profile proved to be stable during 70 generations of the CL Brener clone in culture. The microsatellite profiling presented also high diagnostic sensitivity since DNA amplifications could be achieved with less than 100 fg DNA, corresponding to half parasite total DNA content. Based on these technical attributes the microsatellite assay turns out to be an important tool for direct typing T. cruzi in biological samples. By using this approach we were able to type T. cruzi in feces of artificially infected bugs and in single cells sorted by FACS. The microsatellites have shown to be excellent markers for T. cruzi phylogenetic reconstruction. We used maximum parsimony based on the minimum number of mutational steps to build an unrooted Wagner network, which confirms previous conclusions based on the analysis of the D7 domain of the LSU rDNA gene that T. cruzi is composed by two major groups. We also obtained evidence that strains belonging to rRNA group 2 are subdivided into two genetically distant clusters, and that one of these clusters is more related to rRNA group (1/2). These results suggest different origins for these strains.

Tc45, a dimorphic Trypanosoma cruzi immunogen with variable chromosomal localization, is calreticulin.

We demonstrate that Tc45, a polypeptide described as an immunogenetically restricted Trypanosoma cruzi antigen in mice, is calreticulin, a dimorphic molecule encoded by genes with variable chromosomal distribution. Previously we showed that IgG from A.SW (H2s) mice immunized with T. cruzi trypomastigotes or epimastigotes and sera from infected humans recognize Tc45, a 45 kD parasite polypeptide. Herein we describe the cloning, sequencing, and expression of the Tc45 gene. A 98% homology in the deduced amino acid sequence was found with a T. cruzi calreticulin-like molecule and 41% with Leishmania donovani and human calreticulin. In the T. cruzi CL Brener clone and in the Tulahuén strain, the gene is located in two and four chromosomes, respectively. Calreticulin was detected in several T. cruzi clones, in the Tulahuén strain, and in T. rangeli, displaying alternative 43 and 46 kD forms.

MRNA Encoding a Putative RNA Helicase of the DEAD‐Box Gene Family is Up‐Regulated in Trypomastigotes of Trypanosoma cruzi

Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands.

Infection with different Trypanosoma cruzi populations in rats: myocarditis, cardiac sympathetic denervation, and involvement of digestive organs.

We tested four isolates of Trypanosoma cruzi to assess parasite virulence and ability to cause myocarditis, cardiac sympathetic denervation, and histopathologic alterations in organs of the digestive system. The susceptibility of rats depends on the population of T. cruzi, with the ABC strain and the CL-Brener clone killing all animals, regardless of the parasitemic pattern. All tested T. cruzi populations caused acute myocarditis, but failed to induce histologic alterations in the intestine. The Cl-Brener and ABC isolates caused esophageal myositis. The myocarditis caused by the ABC, CL-Brener, and Y isolates was severe, but resolution started at the end of the acute phase. In contrast, the Col 1.7 G2 clone induced mild and sustained myocarditis. Our results also showed that T. cruzi populations able to induce severe acute myocarditis caused marked sympathetic denervation, but recovery of normal cardiac histology and innervation occurred. The sustained myocarditis induced by Col 1.7 G2 clone failed to cause sustained denervation.

Analysis of the distribution of SIRE in the nuclear genome of Trypanosoma cruzi.

The short interspersed repetitive element (SIRE) of the nuclear genome of Trypanosoma cruzi was first detected when comparing the sequences of loci that encode the TcP2beta genes. The present study was designed to assess its distribution and organization in the nuclear genome of the parasite. Southern blots of genomic DNA from different strains demonstrated that each one possesses a defined and characteristic pattern of SIRE distribution. The conservation of the SIRE sequence in T. cruzi strains allowed the development of a rapid inter-SIRE PCR reaction that yields strain-specific amplicon profiles. In the T. cruzi CL Brener clone, we found 1500 copies of the element distributed in all chromosomes. 16 genomic fragments containing SIRE (SZs) were isolated and characterized. In fragments SZ10, SZ12 and SZ31, SIRE was linked to TcRel, a novel repeated sequence that constitutes the 3' end of vp85 genes. SIRE was also linked to an unknown open reading frame in fragments SZ14 and SZ23 which might be related to the subtelomeric regions of T. cruzi chromosomes. Further sequencing of SZ fragments revealed that SIRE was also linked to protein coding genes that have not yet been described in kinetoplastids such as the one coding for PRP22 helicase and a thimet oligopeptidase. To allow the rapid-generation genetic markers associated with SIRE, we developed a SIRE-bubble PCR reaction that provided several such markers for the construction of the physical map of chromosome XVI. The results herein demonstrate that SIRE-associated sites (SAS) may be of great help in physical mapping and interpretation of T. cruzi genomic sequence data.

Mucin-like glycoprotein genes are closely linked to members of the trans-sialidase super-family at multiple sites in the Trypanosoma cruzi genome

In Trypanosoma cruzi a cell surface enzyme with trans-sialidase (TS) activity has been implicated as an important factor in establishing infection. The enzyme is encoded by genes belonging to a large super-family which on the basis of sequence has been subdivided into 4 groups. TS mediates the transfer of sialic acid residues from host glycoconjugates to acceptor molecules on the parasite surface. To study the organisation of the TS genes we isolated several distinct cosmids from a library constructed with DNA from the T. cruzi X10.6 clone. In these cosmids, the TS genes (group I) were present either as single copies or as a direct tandem repeat. A common feature of the cosmids was the presence of a related group III gene located 10–12kb downstream of the TS gene(s) and arranged in the same orientation. In several of the cosmids we also identified a mucin-like glycoprotein gene located between the group I and group III genes. The mucin-like genes are part of a large polymorphic family and contour clamped homogeneous electric field electrophoresis (CHEFE) analysis showed that they were linked to members of the TS super-family at multiple sites in the X10.6 genome. Screening of a second cosmid library made with DNA from the CL-Brener clone confirmed this multiple linkage suggesting that it is a common feature of the species. This genetic organisation may have important functional significance since the mucin-like glycoproteins are the major cell surface acceptors of sialic acid.