Induction of resistance to azole drugs in Trypanosoma cruzi

Trypanosoma cruzi is the protozoan parasite that causes Chagas' disease, a frequently fatal illness affecting the heart and gastrointestinal systems. An estimated 16 million to 18 million people in Latin America and 50,000 to 100,000 people in the United States are infected with this pathogen. Treatment options for T. cruzi infections are suboptimal due to the toxicities and limited effectiveness of the available drugs. Azole antimicrobial agents have been discovered to have antitrypanosomal activity by inhibition of ergosterol synthesis. The triazole itraconazole was recently shown to produce a parasitologic cure rate of 53% in chronically infected patients (W. Apt et al., Am. J. Trop. Med. Hyg. 59:133-138, 1998), a result which may lead to more use of this family of drugs for the treatment of T. cruzi infections. In the experiments reported on here, resistance to azoles was induced in vitro by serial passage of mammalian-stage parasites in the presence of fluconazole for 4 months. These parasites were cross resistant to the other azoles, ketoconazole, miconazole, and itraconazole. They remained susceptible to benznidazole and amphotericin B. The azole-resistant phenotype was stable for more than 2 months of in vitro serial passage without fluconazole. In addition, the parasites resisted treatment in mice receiving ketoconazole. The rapid development of azole resistance in T. cruzi in vitro suggests that resistance to azole drugs has the potential to occur in patients and may pose an impediment to the progress being made in the treatment of T. cruzi infection.     

Ca2+ content and expression of an acidocalcisomal calcium pump are elevated in intracellular forms of Trypanosoma cruzi

The survival of a eukaryotic protozoan as an obligate parasite in the interior of a eukaryotic host cell implies its adaptation to an environment with a very different ionic composition from that of its extracellular habitat. This is particularly important in the case of Ca2+, the intracellular concentration of which is 3 orders of magnitude lower than the extracellular value. Ca2+ entry across the plasma membrane is a widely recognized mechanism for Ca2+ signaling, needed for a number of intracellular processes, and obviously, it would be restricted in the case of intracellular parasites. Here we show that Trypanosoma cruzi amastigotes possess a higher Ca2+ content than the extracellular stages of the parasite. This correlates with the higher expression of a calcium pump, the gene for which was cloned and sequenced. The deduced protein product (Tca1) of this gene has a calculated molecular mass of 121,141 Da and exhibits 34 to 38% identity with vacuolar Ca2+-ATPases of Saccharomyces cerevisiae and Dictyostelium discoideum, respectively. The tca1 gene suppresses the Ca2+ hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca2+ accumulation. Indirect immunofluorescence and immunoelectron microscopy analysis indicate that Tca1 colocalizes with the vacuolar H+-ATPase to the plasma membrane and to intracellular vacuoles of T. cruzi. These vacuoles were shown to have the same size and distribution as the calcium-containing vacuoles identified by the potassium pyroantimoniate-osmium technique and as the electron-dense vacuoles observed in whole unfixed parasites by transmission electron microscopy and identified in a previous work (D. A. Scott, R. Docampo, J. A. Dvorak, S. Shi, and R. D. Leapman, J. Biol. Chem. 272:28020-28029, 1997) as being acidic and possessing a high calcium content (i.e., acidocalcisomes). Together, these results suggest that acidocalcisomes are distinct from other previously recognized organelles present in these parasites and underscore the ability of intracellular parasites to adapt to the hostile environment of their hosts.     

A 1.4 A crystal structure for the hypoxanthine phosphoribosyltransferase of Trypanosoma cruzi

The hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi, etiologic agent of Chagas' disease, was cocrystallized with the inosine analogue Formycin B (FmB) and the structure determined to 1.4 A resolution. This is the highest resolution structure yet reported for a phosphoribosyltransferase (PRT), and the asymmetric unit of the crystal contains a dimer of closely associated, nearly identical subunits. A conserved nonproline cis peptide in one active-site loop exposes the main-chain nitrogen to the enzyme active site, while the adjacent lysine side chain interacts with the other subunit of the dimer, thereby providing a possible mechanism for communication between the subunits and their active sites. The three-dimensional coordinates for the invariant Ser103-Tyr104 dipeptide are reported here for the first time. These are the only highly conserved residues in a second active-site loop, termed the long flexible loop, which is predicted to close over the active site of HPRTs to protect a labile transition state [Eads et al. (1994) Cell 78, 325-334]. This structure represents a major step forward in efforts to design/discover potent selective inhibitors of the HPRT of T. cruzi.     

Intraspecific diversity in the response of Trypanosoma cruzi to environmental stress

Epimastigotes of 5 Trypanosoma cruzi stocks were cultivated in liver infusion tryptose (LIT) medium at 23-35 C or cocultivated with vertebrate cells at 35 C. A temperature decrease from 26 to 23 C resulted in a stable 60% increase in population doubling time. In zymodeme I and II stocks, a temperature increase to 35 C resulted in a transient approximately 25% increase in doubling time during the first month followed by a approximately 30% decrease after 2 mo. A zymodeme III stock did not grow at 35 C. Flow cytometric analyses showed that the total DNA/cell, guanine + cytosine (G-C), and adenine + thymidine content of 2 zymodeme II stocks increased by 3-11% when cultivated in LIT at 35 C, whereas the DNA values of 2 zymodeme I stocks did not change. The increased DNA levels, due predominantly to an increased kinetoplast G-C content, returned to normal levels when the culture temperature was reduced to 26 C. The effects of cocultivation with vertebrate cells at 35 C were identical to cultivation in LIT at 35 C except that the DNA increase in a zymodeme II stock was not stable. Total DNA/cell, nuclear, and kinetoplast DNA decreased by 8-13% upon prolonged cocultivation. No change in total protein, antigen profiles, complement sensitivity, or heat shock protein gene expression was observed as a consequence of culturing the parasites above 26 C.     

Inhibiting effects of spermidine derivatives on Trypanosoma cruzi trypanothione reductase

Trypanothione reductase is a vital component of the antioxidant defenses of trypanosomes. This enzyme reduces trypanothione, a spermidine-glutathione conjugate. The inhibitory effects of several spermidine derivatives on the reduction of trypanothione by Trypanosoma cruzi trypanothione reductase were assessed. Spermidine derivatives containing hydrophobic aromatic substituents were found to be competitive inhibitors of trypanothione reductase. N4-acylated spermidine derivatives were less effective inhibitors than the corresponding N4-alkylated derivatives. The most effective compounds studied were N1, N8-bis(2-naphthylmethyl)spermidine and N4-(2-naphthylmethyl)spermidine, with Ki values of 9.5 and 108 microM, respectively.     

Effect of thapsigargin on calcium homeostasis in Trypanosoma cruzi trypomastigotes and epimastigotes

By using the fluorescent calcium indicator fura-2, it was found that the concentration of free Ca2+ in the cytoplasm of Trypanosoma cruzi trypomastigotes incubated in the presence or absence of external calcium was maintained at very low levels (10-20 nM). When trypomastigotes were incubated in the presence of succinate and ATP and permeabilized with digitonin, they lowered the medium calcium concentration to a submicromolar level. In the presence of 1 microM FCCP the initial rate of Ca2+ sequestration by these permeabilized cells was very slow. When succinate alone was present, the initial rate of Ca2+ accumulation was slower than with ATP plus succinate, and the calcium set point was about 0.6 microM. The succinate dependence and FCCP sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. High concentrations of the tumor promoter thapsigargin slightly increased cytosolic Ca2+ in the presence of extracellular Ca2+ but had no effect on the FCCP- and oligomycin/antimycin A-insensitive Ca2+ pool. In addition, when used at those concentrations (4-20 microM), thapsigargin was shown to release Ca2+ from the mitochondria and to decrease the inner mitochondrial membrane potential of trypomastigotes and epimastigotes as measured using safranine O. Despite the presence of inositol phosphates as determined by [3H]inositol incorporation, no IP3-sensitive Ca2+ release could be detected in trypomastigotes.     

A pteridine reductase gene ptr1 contiguous to a P-glycoprotein confers resistance to antifolates in Trypanosoma cruzi

We have isolated the pteridine reductase-1 gene (ptr1), from Trypanosoma cruzi (Y strain), located contiguous to the Trypanosoma cruzi P-glycoprotein-2 (tcpgp2). The gene encodes a member of the family of short-chain dehydrogenases, enzymes that are involved in several oxidoreduction reactions. One member of the family, pteridine reductase-1 (PTR1) has been previously described in Leishmania as being involved in antifolate resistance. The ptr1 gene from T. cruzi presents an 828 bp open reading frame, coding for a 276 amino acid protein with a predicted molecular mass of 30 kDa. The deduced amino acid sequence exhibited a remarkable homology with the ptr1 genes of Leishmania major and Leishmania tarentolae. Southern blot analysis using a specific probe indicated that T. cruzi PTR1 is encoded by a single copy gene located in two chromosomes of about 0.9 and 1.2 Mb. Western blot analysis using a polyclonal antiserum against recombinant PTR1 revealed that the protein is only expressed in the epimastigote forms of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. Purified recombinant PTR1 exhibits a NADPH-dependent pteridine reductase activity comparable with those described in Leishmania. Gene transfection experiments using the pTEX expression vector show that, under the conditions tested, T. cruzi PTR1 is involved in resistance to the methotrexate, aminopterin and trimethoprim antifolates.     

The activities of four anticancer alkyllysophospholipids against Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei

The in-vitro activities of four anticancer alkyllysophospholipids, ET-18-OCH3 (edelfosine), hexadecylphosphocholine (miltefosine), ilmofosine and SRI 62-834, were determined with ED50 values for Leishmania donovani and Trypanosoma cruzi amastigotes between 0.2 and 5.0 microM and for Trypanosoma brucei trypomastigotes between 7.0 and 50.8 microM. In BALB/c mice miltefosine and ilmofosine were the most active compounds with ED50 values of 2.9 and 14.5 mg/kg x 5 doses against L. donovani and a suppressive effect on T. cruzi infections at 30 mg/kg x 5 doses.     

Trypanosoma rangeli sialidase: cloning, expression and similarity to T. cruzi trans-sialidase

Sialidases are hydrolytic enzymes present from virus to higher eukaryotes, catalyzing the removal of sialic acid from glycoconjugates. Some protozoa Trypanosomatidae secrete high levels of sialidase into the medium. We have now purified the secreted sialidase from Trypanosoma rangeli. Its N-terminal sequence reveals 100% identity with the corresponding region of the trans-sialidase from T. cruzi. Trans-sialidase, although homologous to viral and bacterial sialidases, displays a novel sialyltransferase activity and is involved in host cell invasion. Several homologous transsialidase-like genes were cloned from genomic DNA of T. rangeli, and grouped in three subfamilies. Active sialidase-encoding genes were found in one of them. The recombinant sialidase shows similar properties to those of the native enzyme, including undetectable trans-sialidase activity. Nevertheless, it has an overall identity of 68.9% with the catalytic domain of T. cruzi trans-sialidase, increasing to 86.7% admitting conservative substitutions. Only three other eukaryotic sialidases have been previously cloned, none of them showing significant homology to trans-sialidase. The isolation of a highly similar sialidase is relevant to further identify the molecular determinants allowing trans-sialidase activity. As a first approach, chimeric constructs between sialidase and trans-sialidase were generated, one of them rendering a sialidase with three times lower Km than the natural enzyme.     

Growth inhibitory effect of juvenile hormone analogues on epimastigotes of Trypanosoma cruzi

Several compounds, structurally related to the insect growth regulator Fenoxycarb, exhibited interesting inhibition action to control proliferation of Trypanosoma cruzi, the parasite responsible for Chagas' disease. Some of these drugs were shown to be potent growth inhibitors of this parasite. All of these drugs had previously presented juvenoid activity on several non-related bug species such as Tenebrio molitor, Galleria mellonella, Dysdercus cingulatos, and Pyrrhocoris apterus.     

Inhibitory effects of human alpha 2-macroglobulin on Trypanosoma cruzi epimastigote proteinases

The inactivation of Trypanosoma cruzi proteinases by human alpha 2-macroglobulin (alpha 2-M), a major plasma proteinase inhibitor was studied. Evidences regarding the interaction between alpha 2-M and proteolytic enzymes contained in crude cell-free extracts of T. cruzi were derived from electrophoretic and enzymatic assays. The former showed conformational and structural changes occurring in alpha 2-M, as judged by the appearance of transformed 'fast' form on native PAGE; generation of bands of approximately 90 kDa on reduced SDS-PAGE and formation of covalent complexes enzyme-inhibitor on SDS-PAGE. On the other hand, the total proteolytic activity on azocasein dropped significantly in the presence of alpha 2-M, although partial activity was still maintained. The proteinases detected as a double band of 44 and 53 kDa on gelatin SDS-PAGE were also inhibited by alpha 2-M. Results suggest that the study of specific interactions between alpha 2-M and T. cruzi-proteinases, probably with cruzipain, could be biologically important in the fate of T. cruzi-infection and Chagas' disease.     

Comparison of chagasic and non-chagasic myocardiopathies by ELISA and immunoblotting with antigens of Trypanosoma cruzi and Trypanosoma rangeli

Trypanosoma cruzi associated myocardiopathy, or Chagas disease, continues to be a serious problem in Venezuela, for which there is neither a vaccine nor a cure. In order to learn more about the humoral immune response to trypanosomal antigens, and to try to identify dominant antigens, we used ELISA and immunoblotting to study the reactivity of sera from patients with chagasic and non-chagasic myocardiopathies, against surface and secreted proteins from T. cruzi and T. rangeli. Both species are found in the same insect vector, but only T. cruzi is thought to be pathogenic in vertebrates. The ELISA results fell into three patterns: (1) high reactivity values with both T. cruzi and T. rangeli surface and secreted proteins; (2) high values to T. cruzi but low values with T. rangeli; and (3) high values to T. rangeli and low values with T. cruzi. This finding that some chagasic sera react more strongly against T. rangeli than against T. cruzi is intriguing, and warrants further investigation. When chagasic sera were tested on Western blots of total extracts of T. cruzi and T. rangeli, the pattern of reactive bands was similar against both parasites, but no two sera showed an identical pattern. Furthermore, there was no correlation between a particular immunoblotting pattern and either the antibody titer, or the severity of the disease. Several T. cruzi and T. rangeli antigens were recognized by sera from healthy controls as well as from patients with other tropical diseases endemic in Venezuela. Overall, our results suggest that the humoral immune response to trypanosomal antigens is complex, and no single antigen may be the determining factor in the pathogenesis of chagasic myocardiopathy.     

Effects of fluorinated inositols on the phosphoinositide metabolism and the proliferation of Trypanosoma cruzi

Inositol has been cited as being essential for the growth of micro-organisms and animals. Its action rests mostly in the formation of a set of inositol-containing lipids, including phosphatidylinositol and its phosphorylated derivatives. To evaluate the functional responses coupled to the phosphoinositide metabolism in Trypanosoma cruzi we used myo-inositol and its six fluorinated analogues. Their uptake into epimastigotes was characterised using tritium-labeled myo-inositol and monodeoxyfluoro-myo-inositols. The analogues were tested for their ability to inhibit [3H]-myo-inositol incorporation into phosphoinositides and the proliferation of epimastigotes and amastigotes. The results showed differences between T. cruzi and mammalian systems in the responses to the fluorinated analogues. We found that the 3-, 5- and 6-fluoro analogues did not enter the cells but had an inhibitory effect on the incorporation of the radioactive inositol into lipids and on the amastigotes' and epimastigotes' replication. The most effective inhibitor, 1-D-6-deoxy-6-myo-inositol, had no effect on mammalian cell division.     

Comparability and reliability of ELISA, immunofluorescence, and indirect hemagglutination assays for Trypanosoma cruzi and Trypanosoma rangeli

Serum samples and filter paper spot samples from 48 adults were assayed in three laboratories for Trypanosoma cruzi and Trypanosoma rangeli by ELISA, immunofluorescence (IF), and indirect hemagglutination (HA) in a controlled, blind experiment. IF (serum samples) attained higher levels of comparability necessary for validity than did ELISA (serum samples) in these laboratories. ELISA was inferior to IF and HA (spot samples) in testing for T. cruzi. ELISA was not reliable (intrasubject r = .51) and was not in agreement between two laboratories (kappa = .09). The experiment indicates that ELISA is not the best choice of assay for T. cruzi in epidemiologic surveys. Assays for T. rangeli by ELISA and IF lacked comparability to the extent that those two techniques could not be considered useful for T. rangeli.     

Attachment of Trypanosoma cruzi epimastigotes to hydrophobic substrates and use of this property to separate stages and promote metacyclogenesis

Sphingosine and its analogs, which inhibit protein kinase C (PKC), are known to be potent inducers of apoptosis in tumor cells. However, we were concerned that sphingosine might also interfere with anti-tumor cells of the immune system. Therefore, we evaluated the effect of sphingosine on activation of human monocytes by interleukin-2 (IL-2) for killing of leukemic cells. Monocytes, purified by elutriation and adherence, were activated with IL-2 or interferon-gamma (IFN-gamma) in the presence or absence of sphingosine or another inhibitor for 18 h. Then the monocytes were washed and the culture medium was replaced with fresh medium to remove the sphingosine. HL- 60 and K562 leukemic cells were added to the monocyte cultures. Over the next 48 h, the cytotoxic activity of the monocytes towards the leukemic cells was assessed by means of an 111-indium-releasing assay. IL-2-activated monocytes lysed 48 +/- 3% of HL-60 cells and 44 +/- 3% of K562 cells. Sphingosine, dihydrosphingosine, N,N-dimethylsphingosine, and the PKC inhibitor H7 inhibited the activation of monocytes by IL-2, blocking cytotoxic activity against the leukemic cells by approximately 75%. These inhibitors were not toxic to monocytes at the concentrations used. In a PKC assay, sphingosine and H7 inhibited PKC activity in IL-2-treated monocytes. Thus, sphingosines, by inhibiting PKC activity, inhibited activation of monocytes by IL-2, which inhibited the killing of leukemic cells.     

Low probability of transmission of Trypanosoma cruzi to humans by domiciliary Triatoma sordida in Bolivia

The role of Triatoma sordida in the domestic transmission of Trypanosoma cruzi was assessed in 7 rural localities in Velasco Province, Department of Santa Cruz, Bolivia. Tri. sordida, the only triatomine species identified in these localities, was found inside 58.0% of houses but not in large numbers (3.1 bugs per infested house on average). A total of 220 faecal samples from domiciliary bugs was examined microscopically and by the polymerase chain reaction for the presence of trypanosomes: 21.4% were infected. Analysis of blood meals of domiciliary Tri. sordida showed that humans were the commonest host (70.4%), followed by chickens and dogs. Four of 418 persons tested were seropositive for Tryp. cruzi. Only 2 of a second group of 62 persons living in dwellings infested by Tri. sordida were seropositive. Tryp. cruzi infection was demonstrated in dogs and domestic rats. Three other species of small mammals were found to be infected with trypanosomes. In our study area, domestic Tri. sordida are mainly incriminated in the transmission of Tryp. cruzi to synanthropic animals, whereas transmission to humans is very rare. The presence in houses of small populations of Tri. sordida infected with Tryp. cruzi is therefore currently insufficient for this insect to constitute a major epidemiological risk factor.