Signaling and host cell invasion by Trypanosoma cruzi

Signal transduction events triggered in mammalian host cells by the obligate intracellular parasite Trypanosoma cruzi are required for invasion. Infective T. cruzi trypomastigotes elicit Ca2+ signaling in mammalian host cells and activate transforming growth factor-beta receptor signaling pathways. The elevation of Ca2+ in T. cruzi, induced by host-cell contact, is also required for invasion, extending the concept of host-pathogen 'cross-talk' to invasive protozoan pathogens.     

Invasion of MDCK epithelial cells with altered expression of Rho GTPases by Trypanosoma cruzi amastigotes and metacyclic trypomastigotes of strains from the two major phylogenetic lineages

In order to invade mammalian cells, Trypanosoma cruzi infective forms cause distinct rearrangements of membrane and host cell cytoskeletal components. Rho GTPases have been shown to regulate three separate signal transduction pathways, linking plasma membrane receptors to the assembly of distinct actin filament structures. Here, we examined the role of Rho GTPases on the interaction between different T. cruzi infective forms of strains from the two major phylogenetic lineages with nonpolarized MDCK cells transfected with different Rho GTPase constructs. We compared the infectivity of amastigotes isolated from infected cells (intracellular amastigotes) with forms generated from the axenic differentiation of trypomastigotes (extracellular amastigotes), and also with metacyclic trypomastigotes. No detectable effect of GTPase expression was observed on metacyclic trypomastigote invasion and parasites of Y and CL (T. cruzi II) strains invaded to similar degrees all MDCK transfectants, and were more infective than either G or Tulahuen (T. cruzi I) strains. Intracellular amastigotes were complement sensitive and showed very low infectivity towards the different transfectants regardless of the parasite strain. Complement-resistant T. cruzi I extracellular amastigotes, especially of the G strain, were more infective than T. cruzi II parasites, particularly for the Rac1V12 constitutively active GTPase transfectant. The fact that in Rac1N17 dominant-negative cells, the invasion of G strain extracellular amastigotes was specifically inhibited suggested an important role for Rac1 in this process.     

Characterization of transsulfuration and cysteine biosynthetic pathways in the protozoan hemoflagellate, Trypanosoma cruzi. Isolation and molecular characterization of cystathionine beta-synthase and serine acetyltransferase from Trypanosoma

Sulfur-containing amino acids play an important role in a variety of cellular functions such as protein synthesis, methylation, and polyamine and glutathione synthesis. We cloned and characterized cDNA encoding cystathionine beta-synthase (CBS), which is a key enzyme of transsulfuration pathway, from a hemoflagellate protozoan parasite Trypanosoma cruzi. T. cruzi CBS, unlike mammalian CBS, lacks the regulatory carboxyl terminus, does not contain heme, and is not activated by S-adenosylmethionine. T. cruzi CBS mRNA is expressed as at least six independent isotypes with sequence microheterogeneity from tandemly linked multicopy genes. The enzyme forms a homotetramer and, in addition to CBS activity, the enzyme has serine sulfhydrylase and cysteine synthase (CS) activities in vitro. Expression of the T. cruzi CBS in Saccharomyces cerevisiae and Escherichia coli demonstrates that the CBS and CS activities are functional in vivo. Enzymatic studies on T. cruzi extracts indicate that there is an additional CS enzyme and stage-specific control of CBS and CS expression. We also cloned and characterized cDNA encoding serine acetyltransferase (SAT), a key enzyme in the sulfate assimilatory cysteine biosynthetic pathway. Dissimilar to bacterial and plant SAT, a recombinant T. cruzi SAT showed allosteric inhibition by l-cysteine, l-cystine, and, to a lesser extent, glutathione. Together, these studies demonstrate the T. cruzi is a unique protist in possessing both transsulfuration and sulfur assimilatory pathways.     

Molecular cloning and expression of the catalytic subunit of protein kinase A from Trypanosoma cruzi

The activation of protein kinase A (cyclic adenosine monophosphate-dependent protein kinase) by cyclic adenosine monophosphate is believed to play an important role in regulating the growth and differentiation of Trypanosoma cruzi. A PCR using degenerate oligonucleotide primers against conserved motifs in the VIb and VIII subdomains of the ACG family of serine/threonine protein kinases was utilised to amplify regions corresponding to the parasite homologue of the protein kinase A catalytic subunit. This putative protein kinase A fragment was used to isolate the entire gene from T. cruzi genomic libraries. The deduced 329 amino acid sequence of this gene contained all of the signature motifs of known protein kinase A catalytic subunit proteins. The recombinant protein expressed in Escherichia coli was shown to phosphorylate Kemptide, a synthetic peptide substrate of protein kinase A, in a protein kinase inhibitor (PKI)-inhibitory manner. Immunoprecipitation with polyclonal antisera raised against recombinant protein of this gene was able to pull-down PKI-inhibitory phosphotransferase activity from epimastigote lysates. Immunoblot and Northern blot analyses, in combination with enzyme activity assays, revealed that this gene was a stage-regulated enzyme in T. cruzi with higher levels and activity being present in epimastigotes compared with amastigotes or trypomastigotes. Overall these studies indicate that the cloned gene encodes an authentic protein kinase A catalytic subunit from T. cruzi and are the first demonstration of PKI-inhibitory phosphotransferase activity in an expressed protozoan protein kinase A catalytic subunit.     

Macrophage signaling by glycosylphosphatidylinositol-anchored mucin-like glycoproteins derived from Trypanosoma cruzi trypomastigotes

Activation of cells from the innate immune system has an important role in host resistance to early infection with the intracellular protozoan parasite, Trypanosoma cruzi. Here we review the studies that have identified and structurally characterized the glycosylphosphatidylinositol (GPI) anchors, as parasite molecules responsible for the activation of cells from the macrophage lineage. We also cover the studies that have identified the receptor, signaling pathways as well as the array of genes expressed in macrophages that are activated by these glycoconjugates. We discuss the possible implications of such response on the host resistance to T. cruzi infection and the pathogenesis of Chagas disease.     

In silico reconstruction of the amino acid metabolic pathways of Trypanosoma cruzi

Trypanosoma cruzi is the epidemiological agent of Chagas' disease, affecting most of Central and South America, constituting a significant health and socio-economic problem. The parasite has a metabolism largely based on the consumption of amino acids, which participate in a diversity of metabolic pathways, leading to many crucial compounds for the survival of this parasite. Study of its enzymes has the potential to disclose new therapeutic targets and foster the development of new drugs. In this study, we employed computational approaches to reconstruct in silico the amino acid metabolic pathways of T. cruzi, aiming to link genomic information with functional information. For that, protein sequences from 570 EC classes belonging to 25 different pathways in general amino acid metabolism were downloaded from KEGG. A subset of 471 EC classes had at least one sequence deposited. Clustering of the proteins belonging to each EC class was performed using a similarity-based approach implemented in the tool AnEnPi. Reconstruction of the metabolic pathways comprising the amino acid metabolism of T. cruzi was performed by analyzing the output of BLASTP, using as query the dataset of predicted proteins of T. cruzi against all sequences of each individual cluster. This approach allowed us to identify 764 T. cruzi proteins probably involved in the metabolism of amino acids as well as the identification of several putative cases of analogy. Furthermore, we were able to identify several enzymatic activities of T. cruzi that were not previously included in KEGG.     

Determination and characterization of new benzimidazoles with activity against Trypanosoma cruzi by UV spectroscopy and HPLC

This work presents the development of analytical methodologies by UV spectrophotometry and HPLC to characterize five nitroarylbenzimidazole derivatives with activity against Trypanosoma cruzi: NB, BNB, PNB, PMNB and PCNB. Both methodologies exhibit adequate repeatabilities and reproducibilities (CV<2%) and recoveries higher than 98%. The ionization constants (pK(a)), lipophilicity (log P) and effective permeability (Pe) are reported. The five compounds present an inhibitory effect on the T. cruzi growth (epimastigotes) at 1-100 microM concentration range in an order rank of PMNB>PCNB>PNB>BNB>NB. Additionally, cyclic voltammetric data reveal that the nitroarylbenzimidazole derivatives might sustain their effects on growth and oxygen uptake on T. cruzi epimastigotes.     

Inositolphosphoceramide metabolism in Trypanosoma cruzi as compared with other trypanosomatids

Chagas disease is caused by Trypanosoma cruzi and is endemic to North, Central and South American countries. Current therapy against this disease is only partially effective and produces adverse side effects. Studies on the metabolic pathways of T. cruzi, in particular those with no equivalent in mammalian cells, might identify targets for the development of new drugs. Ceramide is metabolized to inositolphosphoceramide (IPC) in T. cruzi and other kinetoplastid protists whereas in mammals it is mainly incorporated into sphingomyelin. In T. cruzi, in contrast to Trypanosoma brucei and Leishmania spp., IPC functions as lipid anchor constituent of glycoproteins and free glycosylinositolphospholipids (GIPLs). Inhibition of IPC and GIPLs biosynthesis impairs differentiation of trypomastigotes into the intracellular amastigote forms. The gene encoding IPC synthase in T. cruzi has been identified and the enzyme has been expressed in a cell-free system. The enzyme involved in IPC degradation and the remodelases responsible for the incorporation of ceramide into free GIPLs or into the glycosylphosphatidylinositols anchoring glycoproteins, and in fatty acid modifications of these molecules of T. cruzi have been understudied. Inositolphosphoceramide metabolism and remodeling could be exploited as targets for Chagas disease chemotherapy.     

Study of Trypanosoma cruzi epimastigote cell death by NMR-visible mobile lipid analysis

Cell death mechanisms in Trypanosoma cruzi have not been disclosed in detail though different conventional techniques have been used in the classification of parasite-cell death type. Nuclear magnetic resonance (NMR) has successfully been used as a tool to evaluate the onset of apoptosis in a number of higher eukaryote-cell models analysing the ratio of CH(2)/CH(3) integration from the visible mobile lipids (VML). Surprisingly, this versatile non-invasive spectroscopy technique has never been employed with this purpose in T. cruzi. In the present study it is shown that under different parasite death-conditions the ratio CH(2)/CH(3) varied drastically. Thus, T. cruzi epimastigotes in apoptotic conditions increase significantly this ratio while in necrotic as well as in autophagic situations the parasites maintain the VML, CH(2)/CH(3) ratio, in normal values. Additionally, other VML markers commonly used in these studies, such as the change in the region of methyl-choline moiety, -N(+)(CH(3))(3), exhibited different particular patterns according to the type of cell death. Our results suggest that the (1)H NMR-VML technique is an adequate tool to discriminate different T. cruzi death pathways.     

Cell signalling and Trypanosoma cruzi invasion

Mammalian cell invasion by the protozoan pathogen Trypanosoma cruzi is critical to its survival in the host. To promote its entry into a wide variety of non-professional phagocytic cells, infective trypomastigotes exploit an arsenal of heterogenous surface glycoproteins, secreted proteases and signalling agonists to actively manipulate multiple host cell signalling pathways. Signals initiated in the parasite upon contact with mammalian cells also function as critical regulators of the invasion process. Whereas the full spectrum of cellular responses modulated by T. cruzi is not yet known, mounting evidence suggests that these pathways impinge on a number of cellular processes, in particular the ubiquitous wound-repair mechanism exploited for lysosome-mediated parasite entry. Furthermore, differential engagement of host cell signalling pathways in a cell type-specific manner and modulation of host cell gene expression by T. cruzi are becoming recognized as essential determinants of infectivity and intracellular survival by this pathogen.     

Sialyl-Tn antigen expression and O-linked GalNAc-Thr synthesis by Trypanosoma cruzi

Most Trypanosoma cruzi O-glycans are linked to Thr/Ser residues via N-acetylglucosamine. We report that the mucin-type carcinoma-associated sialyl-Tn antigen (NeuAc-GalNAc-O-Ser/Thr) is expressed by T. cruzi. A specific MAb allowed us to localize the antigen on the surface of epimastigotes and to identify reactive components in parasite lysates (32, 60, and 94kDa). In addition, ppGalNAc-T activity was characterized in epimastigotes, and direct evidence was obtained for the in vitro incorporation of GalNAc to a synthetic peptide derived from a T. cruzi mucin. These results add an as yet unknown complexity to the pathways of O-glycan biosynthesis in this protozoan parasite.     

Defining the role of a FYVE domain in the localization and activity of a cAMP phosphodiesterase implicated in osmoregulation in Trypanosoma cruzi

Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Trypanosoma cruzi, the causative agent of Chagas disease, encodes four different PDE families. One of these PDEs, T. cruzi PDE C2 (TcrPDEC2) has been characterized as a FYVE domain containing protein. Here, we report a novel role for TcrPDEC2 in osmoregulation in T. cruzi and reveal the relevance of its FYVE domain. Our data show that treatment of epimastigotes with TcrPDEC2 inhibitors improves their regulatory volume decrease, whereas cells overexpressing this enzyme are unaffected by the same inhibitors. Consistent with these results, TcrPDEC2 localizes to the contractile vacuole complex, showing strong labelling in the region corresponding to the spongiome. Furthermore, transgenic parasites overexpressing a truncated version of TcrPDEC2 without the FYVE domain show a failure in its targeting to the contractile vacuole complex and a marked decrease in PDE activity, supporting the importance of this domain to the localization and activity of TcrPDEC2. Taking together, the results here presented are consistent with the importance of the cyclic AMP signalling pathway in regulatory volume decrease and implicate TcrPDEC2 as a specifically localized PDE involved in osmoregulation in T. cruzi.     

Cyclic AMP signaling in trypanosomatids

Curative interference with signal transduction pathways is a spectacularly successful concept in many domains of modern pharmacology; indeed, the 'wonder drug' Viagra is but a humble inhibitor of a cyclic GMP (cGMP)-specific phosphodiesterase and, thus, interferes with cGMP-signaling in a strategic organ. In fact, about half of the 100 most successful drugs currently on the market act through modulating cellular signal transduction. Despite these encouraging findings, signal transduction pathways as potential drug targets in trypanosomatids have remained largely unexplored. However, what little is known indicates that adenylyl cyclases of trypanosomatids, and probably other enzymes of the cyclic nucleotide signaling pathways, are significantly different from their mammalian counterparts. Here, Christina Naula and Thomas Seebeck summarize what is known about cAMP signal transduction in trypanosomatids.     

Mechanisms of Trypanosoma cruzi persistence in Chagas disease

Trypanosoma cruzi infection leads to development of chronic Chagas disease. In this article, we provide an update on the current knowledge of the mechanisms employed by the parasite to gain entry into the host cells and establish persistent infection despite activation of a potent immune response by the host. Recent studies point to a number of T. cruzi molecules that interact with host cell receptors to promote parasite invasion of the diverse host cells. T. cruzi expresses an antioxidant system and thromboxane A(2) to evade phagosomal oxidative assault and suppress the host's ability to clear parasites. Additional studies suggest that besides cardiac and smooth muscle cells that are the major target of T. cruzi infection, adipocytes and adipose tissue serve as reservoirs from where T. cruzi can recrudesce and cause disease decades later. Further, T. cruzi employs at least four strategies to maintain a symbiotic-like relationship with the host, and ensure consistent supply of nutrients for its own survival and long-term persistence. Ongoing and future research will continue to help refining the models of T. cruzi invasion and persistence in diverse tissues and organs in the host.     

Cyclic AMP and adenylate cyclase activators stimulate Trypanosoma cruzi differentiation

A chemically defined in vitro differentiating condition was used to study the potential role of cyclic AMP (cAMP) and adenylate cyclase activators on the transformation of Trypanosoma cruzi epimastigotes to the infective metacyclic trypomastigotes (metacyclogenesis). It was observed that both addition of cAMP analogs or adenylate cyclase activators to the differentiating medium stimulated the transformation of epimastigotes to metacyclic trypomastigotes. These results were further corroborated by showing that inhibitors of cAMP phosphodiesterase were stimulatory while activators of this enzyme inhibited the metacyclogenesis process. On the other hand, inhibitors of calmodulin inhibited the transformation of epimastigotes to metacyclic trypomastigotes, suggesting that T. cruzi adenylate cyclase might be activated by calmodulin. In addition, the results strongly suggest that guanine nucleotide binding proteins are involved in T. cruzi adenylate cyclase activation. This system may be useful for studying cell differentiation mechanisms in eukaryotes.     

The Trypanosoma cruzi protease cruzain mediates immune evasion

Trypanosoma cruzi is the causative agent of Chagas' disease. Novel chemotherapy with the drug K11777 targets the major cysteine protease cruzain and disrupts amastigote intracellular development. Nevertheless, the biological role of the protease in infection and pathogenesis remains unclear as cruzain gene knockout failed due to genetic redundancy. A role for the T. cruzi cysteine protease cruzain in immune evasion was elucidated in a comparative study of parental wild type- and cruzain-deficient parasites. Wild type T. cruzi did not activate host macrophages during early infection (<60 min) and no increase in ～P iκB was detected. The signaling factor NF-κB P65 colocalized with cruzain on the cell surface of intracellular wild type parasites, and was proteolytically cleaved. No significant IL-12 expression occurred in macrophages infected with wild type T. cruzi and treated with LPS and BFA, confirming impairment of macrophage activation pathways. In contrast, cruzain-deficient parasites induced macrophage activation, detectable iκB phosphorylation, and nuclear NF-κB P65 localization. These parasites were unable to develop intracellularly and survive within macrophages. IL 12 expression levels in macrophages infected with cruzain-deficient T. cruzi were comparable to LPS activated controls. Thus cruzain hinders macrophage activation during the early (<60 min) stages of infection, by interruption of the NF-κB P65 mediated signaling pathway. These early events allow T. cruzi survival and replication, and may lead to the spread of infection in acute Chagas' disease.     

Animals as reservoir hosts of human trypanosomes

Present knowledge on reservoir hosts of Trypanosoma rhodesiense, T. gambiense and T. brucei in Africa and T. cruzi and T. rangeli in America and experimental transmission studies of T. cruzi in mammalian hosts and in lizards is discussed. The difficulty in differentiating the African species of human trypanosomes, which appear not to be host specific, is a major obstacle to epizootiological studies.