Juan José Cazzulo

End products and enzyme levels of aerobic glucose fermentation in trypanosomatids

Trypanosoma cruzi (epimastigotes), Crithidia fasciculata and Leishmania mexicana (promastigotes) were grown in a brain-heart-tryptose medium supplemented with heat-inactivated fetal calf serum. T. cruzi and C. fasciculata utilized glucose completely during the log phase of growth, whereas L. mexicana used significant amounts of the carbohydrate only at the end of the log phase and at the beginning of the stationary phase. In all cases glucose consumption resulted in excretion of succinate, and much smaller amounts of acetate. C. fasciculata and L. mexicana produced very small amounts of pyruvate. C. fasciculata produced ethanol, which was taken up again and metabolysed after glucose was exhausted. Lactate and malate were not produced. The cells were disrupted by sonic disintegration, and the activities of some key enzymes of carbohydrate and amino acid catabolism were assayed in the whole homogenates. Phosphoenolpyruvate carboxykinase was present in the three organisms; L. mexicana presented the highest specific activity. The activity of this enzyme was maximal during glucose consumption, and slightly decreased after glucose was exhausted. This suggests that the role played by the enzyme is glycolytic and not gluconeogenic; the latter is the case in most higher organisms. Hexokinase and pyruvate kinase presented their highest levels in C. fasciculata and T. cruzi during glucose consumption. L. mexicana, which was in active glycolysis during the whole experimental period, presented the highest specific activities of both enzymes. Citrate synthase, on the other hand, increased in C. fasciculata and, to a lesser extent, in T. cruzi, after glucose was exhausted; the enzyme could not be detected in L. mexicana. The NAD-linked glutamate dehydrogenase increased considerably in C. fasciculata and T. cruzi after glucose was exhausted, suggesting a catabolic role for the enzyme. This increase coincided with an increase in NH3 production by both organisms after glucose consumption. The NADP-linked glutamate dehydrogenase, on the other hand, presented a maximum about the time when glucose was exhausted, and then decreased again, which suggests a catabolic role for the enzyme. Both glutamate dehydrogenases had low activities in L. mexicana; this fits in well with the low NH3 production throughout the culture of this organism. The results are in good agreement with current ideas on the mechanism of aerobic glucose fermentation by trypanosomatids, and suggest that, under the experimental conditions used, both T. cruzi and C. fasciculata used glucose perferentially over amino acids for growth.

The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is encoded by multiple polymorphic tandemly organized genes located on different chromosomes

We demonstrate that cruzipain, the major cysteine proteinase of Trypanosoma cruzi epimastigotes, is encoded by a large number of tandemly arranged genes. Restriction enzyme analysis of 20 clones containing complete repeat units of the gene, as well as sequencing of 2 of these clones, and comparison with previously published partial sequences, indicated that the sequence is conserved among the repeat units, although polymorphisms clearly exist. The repeat units contain an intergenic region of 528 bp and coding regions for pre- and pro-enzyme, a central domain and a C-terminal extension. The predicted amino acid sequences of these regions indicated a sequence identity of 30, 60, 70 and 36%, respectively, when the T. cruzi sequence was compared with the sequence of a similar cysteine proteinase from Trypanosoma brucei. Studies by pulsed field gel electrophoresis, complemented with restriction analysis, indicated that the clusters are located on 2-4 different chromosomes in several parasite isolates.

Further characterization and partial amino acid sequence of a cysteine proteinase from Trypanosoma cruzi

A cysteine proteinase from epimastigotes of Trypanosoma cruzi, Tul 2 stock, has been purified to homogeneity from cell-free extracts obtained by freezing and thawing, by a procedure involving ammonium sulfate fractionation, DEAE-Sephacel chromatography, and gel filtration on Sephadex G-200; when necessary, further purification was attained by fast protein liquid chromatography on Mono Q and Superose 6 columns. The purified enzyme was strongly inhibited by leupeptin, antipain and chymostatin (I50 values of 0.25, 0.75 and 1 microM, respectively), little inhibited by elastatinal, and unaffected by pepstatin A. The enzyme is a glycoprotein, as shown by binding to ConA-Sepharose and elution with alpha-methyl-D-mannopyranoside and alpha-methyl-D-glucopyranoside. Partial amino acid sequences were obtained from the N-terminal end (32 amino acids) of the carbamidomethylated enzyme, and from a tryptic peptide (14 amino acids) of the pyridylethylated enzyme. Both regions show considerable homology with papain and some cathepsins, such as cathepsin L, thus showing that the enzyme belongs to the cysteine proteinase family.

The Major Cysteine Proteinase of Trypanosoma cruzi: A Valid Target for Chemotherapy of Chagas Disease

Trypanosoma cruzi, the causative agent of the American Trypanosomiasis, Chagas disease, contains a major cysteine proteinase (CP), cruzipain (also known as cruzain, or GP57/51). The enzyme is a member of the papain C1 family of CPs, with a specificity intermediate between those of cathepsin L and cathepsin B. The enzyme, which is expressed at different levels by different parasite stages, is encoded by a high number of genes (up to 130 in the Tul2 strain), which code for a pre-pro-enzyme. Mature cruzipain consists of a catalytic moiety with high homology to cathepsins S and L, and a C-terminal domain, characteristic of Type I CPs of Trypanosomatids, and absent in all other C1 family CPs described so far. Irreversible inhibitors of cruzipain (peptidyl diazomethylketones, peptidyl fluoromethylketones, peptidyl vinyl sulphones) are able to block the differentiation steps in the parasites life cycle, and effectively kill the organism. Recently, a vinyl sulphone derivative (N-piperazine-Phe-hPhe-vinyl sulphone phenyl) which is an efficient inhibitor of cruzipain and kills T. cruzi by inducing an accumulation of unprocessed cruzipain in the Golgi cisternae, interfering with the secretory pathway, has been tested in vivo in a mice model (J.H. McKerrow et al.). The curative effects observed, as well as the good bioavailability of the inhibitor and its apparent lack of undesirable side effects, make it a promising lead compound for the development of new drugs for the chemotherapy of Chagas disease.

Autophagy Is Involved in Nutritional Stress Response and Differentiation in Trypanosoma cruzi

Autophagy is the major mechanism used by eukaryotic cells to degrade and recycle proteins and organelles. Bioinformatics analysis of the genome of the protozoan parasite Trypanosoma cruzi revealed the presence of all components of the Atg8 conjugation system, whereas Atg12, Atg5, and Atg10 as the major components of the Atg12 pathway could not be identified. The two TcATG4 (autophagin) homologs present in the genome were found to correctly process the two ATG8 homologs after the conserved Gly residue. Functional studies revealed that both ATG4 homologues but only one T. cruzi ATG8 homolog (TcATG8.1) complemented yeast deletion strains. During starvation of the parasite, TcAtg8.1, but not TcAtg8.2, was found by immunofluorescence to be located in autophagosome-like vesicles. This confirms its function as an Atg8/LC3 homolog and its potential to be used as an autophagosomal marker. Most importantly, autophagy is involved in differentiation between developmental stages of T. cruzi, a process that is essential for parasite maintenance and survival. These findings suggest that the autophagy pathway could represent a target for a novel chemotherapeutic strategy against Chagas disease.

Intermediate metabolism inTrypanosoma cruzi

Epimastigotes ofTrypanosoma cruzi, the causative agent of Chagas disease, catabolize proteins and amino acids with production of NH3, and glucose with production of reduced catabolites, chiefly succinate andl-alanine, even under aerobic conditions. This “aerobic fermentation of glucose” is probably due to both the presence of low levels of some cytochromes, causing a relative inefficiency of the respiratory chain for NADH reoxidation during active glucose catabolism, and the lack of NADH dehydrogenase and phosphorylation site I, resulting in the entry of reduction equivalents into the chain mostly as succinate. Phosphoenol pyruvate carboxykinase and pyruvate kinase may play an essential role in diverting glucose carbon to succinate orl-alanine, andl-malate seems to be the major metabolite for the transport of glucose carbon and reduction equivalents between glycosome and mitochondrion. The parasite contains proteinase and peptidase activities. The major lysosomal cysteine proteinase, cruzipain, has been characterized in considerable detail, and might be involved in the host/parasite relationship, in addition to its obvious role in parasite nutrition. Among the enzymes of amino acid catabolism, two glutamate dehydrogenases (one NADP- and the other NAD-linked), alanine aminotransferase, and the major enzymes of aromatic amino acid catabolism (tyrosine aminotransferase and aromatic α-hydroxy acid dehydrogenase), have been characterized and proposed to be involved in the reoxidation of glycolytic NADH.

A major cysteine proteinase is developmentally regulated in Trypanosoma cruzi

Epimastigotes of different stocks of Trypanosoma cruzi contain similar levels of proteinase activity on azocasein; amastigotes and trypomastigotes contain 10-fold lower levels of this proteolytic activity, which seems, therefore, to be developmentally regulated. The proteinase could be detected as a broad band, centered at about 60 kDa, which in some cases resolved into two close bands, in (a) SDS-polyacrylamide gels containing fibrinogen, and (b) Western blots probed with a polyclonal rabbit antiserum prepared against purified cysteine proteinase. No proteinase activity was observed at molecular weights lower than 55 kDa. The results show that the enzyme previously purified is the major cysteine proteinase present in epimastigotes of all stocks of T. cruzi tested.

Inhibition of cruzipain, the major cysteine proteinase of the protozoan parasite, Trypanosoma cruzi, by proteinase inhibitors of the cystatin superfamily.

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi epimastigotes, purified to a sequentially pure form, exists in multiple forms with pI values between 3.7 and 5.1, and an apparent molecular mass of 41 kDa. The enzyme is stable between pH 4.5–9.5. Cruzipain was found to be rapidly and tightly inhibited by various protein inhibitors of the cystatin superfamily (k ass = 1.7–79 × 106M−1s−1, K d = 1.4–72 pM). These results suggest a possible defensive role for the hosts cystatins after parasite infection, and may be of use for the design of new therapeutic drugs.

Chromosome specific markers reveal conserved linkage groups in spite of extensive chromosomal size variation in Trypanosoma cruzi

The karyotypes of three cloned stocks, CL Brener (CL), CA I/72 (CA) and Sylvio X10/7 (X10), of Trypanosoma cruzi were studied by pulsed-field gel electrophoresis followed by ethidium bromide staining and hybridization with 35 different probes, 30 of which identified single chromosomes. The chromosome-specific probes identified between 26 and 31 chromosomal bands in the three cloned stocks, corresponding to 20 unique chromosomes in CL and 19 in CA and X10. Considering the DNA content of the parasite, it was predicted that the markers recognise at least half of all T. cruzi chromosomes. A majority of identified chromosomes showed large differences in size among different strains, in some cases by up to 50%. Interestingly, CL had in general larger chromosomes than the two other studied cloned stocks. Several of the markers showed linkage and nine different linkage groups were identified, each comprising 2-4 markers. The linkage between the markers was maintained in 8 of the 9 linkage groups when a panel comprising 26 different T. cruzi strains representing major T. cruzi populations was tested. One linkage group was found to be maintained in some strains but not in others. This result shows that chromosomal rearrangements occur in the T. cruzi genome, albeit with a low frequency. Repetitive DNA, both non-coding and in one case coding, was more abundant in the cloned stock CL Brener than in CA and X10. The information presented will make it possible to select chromosomes for the construction of physical chromosomal maps required for the T. cruzi genome project.

Chromosomal localization of seven cloned antigen genes provides evidence of diploidy and further demonstration of karyotype variability in Trypanosoma cruzi

The karyotype of Trypanosoma cruzi was studied by pulsed field gel electrophoresis (PFGE) in conditions that allowed 20-25 chromosome bands to be detected. However, several of these bands were present in non-equimolar amounts, suggesting that the total chromosome number is considerably higher. The patterns obtained with the different cloned and uncloned strains were unique, suggesting that the karyotype of T. cruzi is highly variable. The chromosomal localizations of seven cloned genes were determined by Southern blotting of PFGE-separated chromosomes. Three of the clones gave rise to similar patterns and mapped on a chromosome or a family of chromosomes larger than 1.6 Mb. Two clones mapped on either single or pairs of chromosomes, which in some cases differed considerably in size between the different strains tested, suggesting that extensive chromosome rearrangements occur in T. cruzi. Another clone hybridized to several chromosomes in most strains and probably represents a family of genes. Lastly, one clone hybridized to nearly all chromosomes. Many of the clones hybridized to pairs of restriction fragments in the different strains, suggesting that they are allelic. For one of the clones it was possible to provide further evidence for the allelic nature of the fragments by establishing detailed restriction maps around them and by showing that the two fragments in a pair hybridized to chromosomes which differed slightly in size. Taken together, the results infer that the genome of T. cruzi epimastigotes is diploid.