Crystal Structure of a Monomeric Thiolase-Like Protein Type 1 (TLP1) from Mycobacterium smegmatis

An analysis of the Mycobacterium smegmatis genome suggests that it codes for several thiolases and thiolase-like proteins. Thiolases are an important family of enzymes that are involved in fatty acid metabolism. They occur as either dimers or tetramers. Thiolases catalyze the Claisen condensation of two acetyl-Coenzyme A molecules in the synthetic direction and the thiolytic cleavage of 3-ketoacyl-Coenzyme A molecules in the degradative direction. Some of the M. smegmatis genes have been annotated as thiolases of the poorly characterized SCP2-thiolase subfamily. The mammalian SCP2-thiolase consists of an N-terminal thiolase domain followed by an additional C-terminal domain called sterol carrier protein-2 or SCP2. The M. smegmatis protein selected in the present study, referred to here as the thiolase-like protein type 1 (MsTLP1), has been biochemically and structurally characterized. Unlike classical thiolases, MsTLP1 is a monomer in solution. Its structure has been determined at 2.7 Å resolution by the single wavelength anomalous dispersion method. The structure of the protomer confirms that the N-terminal domain has the thiolase fold. An extra C-terminal domain is indeed observed. Interestingly, it consists of six β-strands forming an anti-parallel β-barrel which is completely different from the expected SCP2-fold. Detailed sequence and structural comparisons with thiolases show that the residues known to be essential for catalysis are not conserved in MsTLP1. Consistent with this observation, activity measurements show that MsTLP1 does not catalyze the thiolase reaction. This is the first structural report of a monomeric thiolase-like protein from any organism. These studies show that MsTLP1 belongs to a new group of thiolase related proteins of unknown function.     

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC₅₀ values in the range concentration of 0.25–0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC₅₀ value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC₅₀ = 34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX® Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.     

Trypanosoma cruzi: A kinetoplast-associated protein of the photolyase/cryptochrome family

A photolyase-like protein gene found in the Trypanosoma cruzi genome database was cloned and expressed in Escherichia coli resulting in the formation of inclusion bodies. Antibodies against this protein were used to determine expression of the protein in the different forms of the parasite. It was visualized in the epimastigote form but not in amastigote or trypomastigote forms obtained from culture in Vero cells. In epimastigotes, this protein is located at the level of the mitochondrion associated to both sides of the kinetoplast. Sequence analyses indicated that this protein, as well as other photolyases from Leishmania spp. and Trypanosoma brucei are related to single-stranded photolyases or cryptochromes DASH.     

Synthesis and characterization of a pyridine-2-thiol N-oxide gold(I) complex with potent antiproliferative effect against Trypanosoma cruzi and Leishmania sp. insight into its mechanism of action

In the search for new therapeutic tools against parasitic diseases caused by the Kinetoplastids Leishmania spp. and Trypanosoma cruzi, a novel gold(I) triphenylphosphine complex with the bioactive coligand pyridine-2-thiol N-oxide (mpo) was synthesized and characterized by using analytical and conductometric measurements, electrospray ionization-mass spectrometry (ESI) and electronic, FTIR and ¹H and ³¹P NMR spectroscopies. A dinuclear structure is suggested for the complex. At a 1 microM concentration the complex induced in vitro after 30 min a potent leishmanicidal effect (LD₅₀) against promastigotes of Leishmania (L.) mexicana while on Leishmania (V.) braziliensis with the same concentration only a leishmanistatic effect (IC₇₅) was observed 48 h after treatment. Similar differential susceptibilities were also found when testing the ligand mpo, but at a higher dose (5 μM). In addition, the compound showed growth inhibitory effect on Dm28c T. cruzi epimastigotes in culture (IC₅₀ 0.09 μM), being even more active than the anti-trypanosomal reference drug Nifurtimox (IC₅₀ 6 μM). DNA interaction studies showed that this biomolecule does not constitute a main target for the mpo complex currently tested. Instead, the significant potentiation of the antiproliferative effect against both Leishmania species and T. cruzi could be associated to the inhibition of NADH fumarate reductase, a kinetoplastid parasite-specific enzyme absent in the host. Furthermore, due to its low unspecific cytotoxicity on mammalian cells (J774 macrophages), the new gold complex showed a selective anti-parasite activity. It constitutes a promising new potent chemotherapeutic alternative to be evaluated in vivo in experimental models of leishmaniasis and Chagas´ disease.     

Molecular epidemiology of domestic and sylvatic Trypanosoma cruzi infection in rural northwestern Argentina

Genetic diversity of Trypanosoma cruzi populations and parasite transmission dynamics have been well documented throughout the Americas, but few studies have been conducted in the Gran Chaco ecoregion, one of the most highly endemic areas for Chagas disease, caused by T. cruzi. In this study, we assessed the distribution of T. cruzi lineages (identified by PCR strategies) in Triatoma infestans, domestic dogs, cats, humans and sylvatic mammals from two neighbouring rural areas with different histories of transmission and vector control in northern Argentina. Lineage II predominated amongst the 99 isolates characterised and lineage I amongst the six isolates obtained from sylvatic mammals. T. cruzi lineage IIe predominated in domestic habitats; it was found in 87% of 54 isolates from Tr. infestans, in 82% of 33 isolates from dogs, and in the four cats found infected. Domestic and sylvatic cycles overlapped in the study area in the late 1980s, when intense domestic transmission occurred, and still overlap marginally. The introduction of T. cruzi from sylvatic into domestic habitats is likely to occur very rarely in the current epidemiological context. The household distribution of T. cruzi lineages showed that Tr. infestans, dogs and cats from a given house compound shared the same parasite lineage in most cases. Based on molecular evidence, this result lends further support to the importance of dogs and cats as domestic reservoir hosts of T. cruzi. We believe that in Argentina, this is the first time that lineage IIc has been isolated from naturally infected domestic dogs and Tr. infestans.     

Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence

Virulence of Trypanosoma cruzi depends on a variety of genetic and biochemical factors. It has been proposed that components of the parasites’ antioxidant system may play a key part in this process by pre-adapting the pathogen to the oxidative environment encountered during host cell invasion. Using several isolates (10 strains) belonging to the two major phylogenetic lineages (T. cruzi-I and T. cruzi-II), we investigated whether there was an association between virulence (ranging from highly aggressive to attenuated isolates at the parasitemia and histopathological level) and the antioxidant enzyme content. Antibodies raised against trypanothione synthetase (TcTS), ascorbate peroxidase (TcAPX), mitochondrial and cytosolic tryparedoxin peroxidases (TcMPX and TcCPX) and trypanothione reductase (TcTR) were used to evaluate the antioxidant enzyme levels in epimastigote and metacyclic trypomastigote forms in the T. cruzi strains. Levels of TcCPX, TcMPX and TcTS were shown to increase during differentiation from the non-infective epimastigote to the infective metacyclic trypomastigote stage in all parasite strains examined. Peroxiredoxins were found to be present at higher levels in the metacyclic infective forms of the virulent isolates compared with the attenuated strains. Additionally, an increased resistance of epimastigotes from virulent T. cruzi populations to hydrogen peroxide and peroxynitrite challenge was observed. In mouse infection models, a direct correlation was found between protein levels of TcCPX, TcMPX and TcTS, and the parasitemia elicited by the different isolates studied (Pearson’s coefficient: 0.617, 0.771, 0.499; respectively, P < 0.01). No correlation with parasitemia was found for TcAPX and TcTR proteins in any of the strains analyzed. Our data support that enzymes of the parasite antioxidant armamentarium at the onset of infection represent new virulence factors involved in the establishment of disease.     

The peptidases of Trypanosoma cruzi: Digestive enzymes, virulence factors, and mediators of autophagy and programmed cell death

Trypanosoma cruzi, the agent of the American Trypanosomiasis, Chagas disease, contains cysteine, serine, threonine, aspartyl and metallo peptidases. The most abundant among these enzymes is cruzipain, a cysteine proteinase expressed as a mixture of isoforms, some of them membrane-bound. The enzyme is an immunodominant antigen in human chronic Chagas disease and seems to be important in the host/parasite relationship. Inhibitors of cruzipain kill the parasite and cure infected mice, thus validating the enzyme as a very promising target for the development of new drugs against the disease. In addition, a 30 kDa cathepsin B-like enzyme, two metacaspases and two autophagins have been described. Serine peptidases described in the parasite include oligopeptidase B, a member of the prolyl oligopeptidase family involved in Ca²⁺-signaling during mammalian cell invasion; a prolyl endopeptidase (Tc80), against which inhibitors are being developed, and a lysosomal serine carboxypeptidase. Metallopeptidases homologous to the gp63 of Leishmania spp. are present, as well as two metallocarboxypeptidases belonging to the M32 family, previously found only in prokaryotes. The proteasome has properties similar to those of other eukaryotes, and its inhibition by lactacystin blocks some differentiation steps in the life cycle of the parasite. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Trypanosomatid essential metabolic pathway: New approaches about heme fate in Trypanosoma cruzi

Trypanosoma cruzi, the causal agent of Chagas disease, has a complex life cycle and depends on hosts for its nutritional needs. Our group has investigated heme (Fe-protoporphyrin IX) internalization and the effects on parasite growth, following the fate of this porphyrin in the parasite. Here, we show that epimastigotes cultivated with heme yielded the compounds α-meso-hydroxyheme, verdoheme and biliverdin (as determined by HPLC), suggesting an active heme degradation pathway in this parasite. Furthermore, through immunoprecipitation and immunoblotting assays of epimastigote extracts, we observed recognition by an antibody against mammalian HO-1. We also detected the localization of the HO-1-like protein in the parasite using immunocytochemistry, with antibody staining primarily in the cytoplasm. Although HO has not been described in the parasite’s genome, our results offer new insights into heme metabolism in T. cruzi, revealing potential future therapeutic targets.     

Trypanosoma cruzi: Synergistic cytotoxicity of multiple amphipathic anti-microbial peptides to T. cruzi and potential bacterial hosts

The parasite Trypanasoma cruzi is responsible for Chagas disease and its triatomine vector, Rhodnius prolixus, has a symbiotic relationship with the soil bacterium, Rhodococcus rhodnii.R. rhodnii that was previously genetically engineered to produce the anti-microbial peptide, cecropin A was co-infected with T. cruzi into R. prolixus resulting in clearance of the infectious T. cruzi in 65% of the vectors. Similar anti-microbial peptides have been isolated elsewhere and were studied for differential toxicity against T. cruzi and R. rhodnii. Of the six anti-microbial peptides tested, apidaecin, magainin II, melittin, and cecropin A were deemed potential candidates for the Chagas paratransgenic system as they were capable of killing T.cruzi at concentrations that exhibit little or no toxic effects on R. rhodnii. Subsequent treatments of T. cruzi with these peptides in pair-wise combinations resulted in synergistic killing, indicating that improvement of the 65% parasite clearance seen in previous experiments may be possible utilizing combinations of different anti-microbial peptides.     

Trypanosoma brucei: Reduction of GPI-phospholipase C protein during differentiation is dependent on replication of newly transformed cells

The protozoan parasite Trypanosoma brucei lives in the bloodstream of vertebrates or in a tsetse fly. Expression of a GPI-phospholipase C polypeptide (GPI-PLCp) in the parasite is restricted to the bloodstream form. Events controlling the amount of GPI-PLCp expressed during differentiation are not completely understood. Our metabolic “pulse-chase” analysis reveals that GPI-PLCp is stable in bloodstream form. However, during differentiation of bloodstream to insect stage (procyclic) T. brucei, translation GPI-PLC mRNA ceases within 8 h of initiating transformation. GPI-PLCp is not lost precipitously from newly transformed procyclic trypanosomes. Nascent procyclics contain 400-fold more GPI-PLCp than established insect stage T. brucei. Reduction of GPI-PLCp in early-stage procyclics is linked to parasite replication. Sixteen cell divisions are required to reduce the amount of GPI-PLCp in newly differentiated procyclics to levels present in the established procyclic. GPI-PLCp is retained in strains of T. brucei that fail to replicate after differentiation of the bloodstream to the procyclic form. Thus, at least two factors control levels of GPI-PLCp during differentiation of bloodstream T. brucei; (i) repression of GPI-PLC mRNA translation, and (ii) sustained replication of newly transformed procyclic T. brucei. These studies illustrate the importance of repeated cell divisions in controlling the steady-state amount of GPI-PLCp during differentiation of the African trypanosome.     

Interactions of antimicrobial peptides with Leishmania and trypanosomes and their functional role in host parasitism

Antimicrobial peptides (AMPs) are multifunctional components of the innate systems of both insect and mammalian hosts of the pathogenic trypanosomatids Leishmania and Trypanosoma species. Structurally diverse AMPs from a wide range of organisms have in vitro activity against these parasites acting mainly to disrupt surface-membranes. In some cases AMPs also localize intracellularly to affect calcium levels, mitochondrial function and induce autophagy, necrosis and apoptosis. In this review we discuss the work done in the area of AMP interactions with trypanosomatid protozoa, propose potential targets of AMP activity at the cellular level and discuss how AMPs might influence parasite growth and differentiation in their hosts to determine the outcome of natural infection.     

Bioluminescent imaging of Trypanosoma cruzi infection

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a major public health problem in Central and South America. The pathogenesis of Chagas disease is complex and the natural course of infection is not completely understood. The recent development of bioluminescence imaging technology has facilitated studies of a number of infectious and non-infectious diseases. We developed luminescent T. cruzi to facilitate similar studies of Chagas disease pathogenesis. Luminescent T. cruzi trypomastigotes and amastigotes were imaged in infections of rat myoblast cultures, which demonstrated a clear correlation of photon emission signal strength to the number of parasites used. This was also observed in mice infected with different numbers of luminescent parasites, where a stringent correlation of photon emission to parasite number was observed early at the site of inoculation, followed by dissemination of parasites to different sites over the course of a 25-day infection. Whole animal imaging from ventral, dorsal and lateral perspectives provided clear evidence of parasite dissemination. The tissue distribution of T. cruzi was further determined by imaging heart, spleen, skeletal muscle, lungs, kidneys, liver and intestines ex vivo. These results illustrate the natural dissemination of T. cruzi during infection and unveil a new tool for studying a number of aspects of Chagas disease, including rapid in vitro screening of potential therapeutical agents, roles of parasite and host factors in the outcome of infection, and analysis of differential tissue tropism in various parasite-host strain combinations.     

Proteomic analysis of the Trypanosoma cruzi ribosomal proteins

Trypanosoma cruzi is a parasite responsible for Chagas disease. The identification of new targets for chemotherapy is a major challenge for the control of this disease. Several lines of evidences suggest that the translational system in trypanosomatids show important differences compared to other eukaryotes. However, there little is known information about this. We have performed a detailed data mining search for ribosomal protein genes in T. cruzi genome data base combined with mass spectrometry analysis of purified T. cruzi ribosomes. Our results show that T. cruzi ribosomal proteins have ∼50% sequence identity to yeast ones. Nevertheless, some parasite proteins are longer due to the presence of several N- or C-terminal extensions, which are exclusive of trypanosomatids. In particular, L19 and S21 show C-terminal extensions of 168 and 164 amino acids, respectively. In addition, we detected two 60S subunit proteins that had not been previously detected in the T. cruzi total proteome; namely, L22 and L42.     

Characterization of a novel Obg-like ATPase in the protozoan Trypanosoma cruzi

We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185 bp long and encodes a 44.3 kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes.     

Detection of matrix metallopeptidase-9-like proteins in Trypanosoma cruzi

In this study, the cell-associated and extracellular peptidases of Trypanosoma cruzi grown in modified Roitman’s complex (MRC) medium were analyzed by measuring peptidase activity in gelatin-containing zymograms. Our results showed that the cell-associated peptidases as well as peptidases extracellularly released by T. cruzi displayed two distinct proteolytic classes: cysteine and metallopeptidase activities. The major cysteine peptidase, cruzipain, synthesized by T. cruzi cells was detected in cellular parasite content, as a 50 kDa reactive polypeptide, after probing with anti-cruzipain antibody. In addition, metallo-type peptidases belonging to the matrix metallopeptidase-9 (MMP-9) family were revealed, after Western blotting, as a 97 kDa protein band in cellular extract and an 85 kDa polypeptide in both cellular and secreted parasite extracts. The MMP-9-like activity present in cells and spent culture medium was immunoprecipitated by an anti-MMP-9 polyclonal antibody. The surface location of MMP-9-like proteins in T. cruzi was also evidenced by means of flow cytometry analysis. Furthermore, doxycycline that has direct MMP-9 inhibiting properties in vitro, inhibited MMP-9-like activities in gel zymography, immunoprecipitation and flow cytometry analyses. This is the first report of the presence of MMP-9-like molecules in T. cruzi. The presence of a matrix extracellular-degrading enzyme may play a role in the T. cruzi-host cell interaction, making this enzyme a potential target for future drug development against this pathogenic trypanosomatid.     

Leishmania infantum chagasi: A genome-based approach to identification of excreted/secreted proteins

The parasitic protozoan, Leishmania, survives in harsh environments within its mammalian and sand fly hosts. Secreted proteins likely play critical roles in the parasite’s interactions with its environment. As a preliminary identification of the spectrum of potential excreted/secreted (ES) proteins of Leishmania infantum chagasi (Lic), a causative agent of visceral leishmaniasis, we used standard algorithms to screen the annotated L. infantum genome for genes whose predicted protein products have an N-terminal signal peptide and lack transmembrane domains and membrane anchors. A suite of 181 candidate ES proteins were identified. These included several that were documented in the literature to be released by other Leishmania spp. Six candidate ES proteins were selected for further validation of their expression and release by different parasite stages. We found both amastigote-specific and promastigote-specific released proteins. The ES proteins of Lic are candidates for future studies of parasite virulence determinants and host protective immunity.     

Trypanosoma cruzi: multiple nucleoside diphosphate kinase isoforms in a single cell

Nucleoside diphosphate kinases (NDPKs) are multifunctional enzymes involved mainly in the conservation of nucleotides and deoxynucleotides at intracellular levels. Here we report the characterization of two NDPKs from the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease. TcNDPK1 and TcNDPK2 were biochemically characterized presenting different kinetic parameters and regulation mechanisms. NDPK activity was mainly detected in soluble fractions according to the digitonin extraction technique; however 20% of the activity remains insoluble at digitonin concentrations up to 5 mg ml⁻¹. TcNDPK1 is a short enzyme isoform, whereas TcNDPK2 is a long one containing a DM10 motif. In addition, two other putative NDPK genes (TcNPDK3 and TcNDPK4) were detected by data mining at the T. cruzi genome database. The large number and diversity of NDPK isoforms are in agreement with those previously observed for other T. cruzi phosphotransferases, such as adenylate kinases.     

Trypanosoma cruzi: in vitro activity of Epoxy-α-Lap, a derivative of α-lapachone, on trypomastigote and amastigote forms

Chagas disease is an endemic parasitic infection caused by Trypanosomacruzi that affects 18-20 million people in Central and South America. Recently we described the Epoxy-α-Lap, an oxyran derivative of α-lapachone, which presents a low toxicity profile and a high inhibitory activity against T.cruzi epimastigotes forms, the non-infective form of this parasite. In this work we described the trypanocidal effects of Epoxy-α-Lap on extracellular (trypomastigote) and intracellular (amastigote) infective forms of two T. cruzi strains (Y and Colombian) known by their different infective profile. Our results showed that Epoxy-α-Lap is lethal to trypomastigote Y and Colombian strains (97% and 84%, respectively). Interestingly, Epoxy-α-Lap also showed a trypanocidal effect in human macrophage infected with T. cruzi Y (85.6%) and Colombian (71.9%) strains amastigote forms. Similar effects were observed on T. cruzi amastigote infected Vero cells (96.4% and 95.0%, respectively). Our results pointed Epoxy-α-Lap as a potential candidate for Chagas disease chemotherapy since it presents trypanocidal activity on all T. cruzi forms with low) toxicity profile.     

Trypanosoma cruzi: Genetic diversity influences the profile of immunoglobulins during experimental infection

The clonal evolution model postulated for Trypanosoma cruzi predicts a correlation between the phylogenetic divergence of T. cruzi clonal genotypes and their biological properties. In the present study, the linkage between phylogenetic divergence of the parasite and IgG, IgG1, IgG2a and IgG2b response has been evaluated during the acute and chronic phases of the experimental infection. Eight laboratory-cloned stocks representative of this phylogenetic diversity and including the lineages T. cruzi I (genotypes 19 and 20), T. cruzi II (genotype 32) and T. cruzi (genotype 39) have been studied. The results showed that the pattern of humoral immune response was correlated with T. cruzi genotype, and that stocks included in genotype 20 were responsible for the high IgG response in the acute and chronic phases. Moreover, T. cruzi I lineage was more efficient in over-expressing all subclasses of specific anti-parasite IgG than either T. cruzi II or T. cruzi lineages. Curiously, the alteration in the pattern of antibodies induced by Benznidazole treatment was related to the phase of the infection but not to the genotype of the parasite. The data suggest that genotypes of T. cruzi are able to drive levels/subclasses of specific IgG, hence giving rise to further concerns about the sensitivity of serological assays in the diagnosis of human Chagas disease.