On the Evolution of Hexose Transporters in Kinetoplastid Potozoans

Glucose, an almost universally used energy and carbon source, is processed through several well-known metabolic pathways, primarily glycolysis. Glucose uptake is considered to be the first step in glycolysis. In kinetoplastids, a protozoan group that includes relevant human pathogens, the importance of glucose uptake in different phases of the life cycles is well established, and hexose transporters have been proposed as targets for therapeutic drugs. However, little is known about the evolutionary history of these hexose transporters. Hexose transporters contain an intracellular N- and C- termini, and 12 transmembrane spans connected by alternate intracellular and extracellular loops. In the present work we tested the hypothesis that the evolutionary rate of the transmembrane span is different from that of the whole sequence and that it is possible to define evolutionary units inside the sequence. The phylogeny of whole molecules was compared to that of their transmembrane spans and the loops connecting the transmembrane spans. We show that the evolutionary units in these proteins primarily consist of clustered rather than individual transmembrane spans. These analyses demonstrate that there are evolutionary constraints on the organization of these proteins; more specifically, the order of the transmembrane spans along the protein is highly conserved. Finally, we defined a signature sequence for the identification of kinetoplastid hexose transporters

Actions of a Proline Analogue, L-Thiazolidine-4-Carboxylic Acid (T4C), on Trypanosoma cruzi

It is well established that L-proline has several roles in the biology of trypanosomatids. In Trypanosoma cruzi, the etiological agent of Chagas' disease, this amino acid is involved in energy metabolism, differentiation processes and resistance to osmotic stress. In this study, we analyzed the effects of interfering with L-proline metabolism on the viability and on other aspects of the T. cruzi life cycle using the proline analogue L- thiazolidine-4-carboxylic acid (T4C). The growth of epimastigotes was evaluated using different concentrations of T4C in standard culture conditions and at high temperature or acidic pH. We also evaluated possible interactions of this analogue with stress conditions such as those produced by nutrient starvation and oxidative stress. T4C showed a dose-response effect on epimastigote growth (IC50 = 0.89±0.02 mM at 28°C), and the inhibitory effect of this analogue was synergistic (p<0.05) with temperature (0.54±0.01 mM at 37°C). T4C significantly diminished parasite survival (p<0.05) in combination with nutrient starvation and oxidative stress conditions. Pre-incubation of the parasites with L-proline resulted in a protective effect against oxidative stress, but this was not seen in the presence of the drug. Finally, the trypomastigote bursting from infected mammalian cells was evaluated and found to be inhibited by up to 56% when cells were treated with non-toxic concentrations of T4C (between 1 and 10 mM). All these data together suggest that T4C could be an interesting therapeutic drug if combined with others that affect, for example, oxidative stress. The data also support the participation of proline metabolism in the resistance to oxidative stress

The C-terminal region of Trypanosoma cruzi MASPs is antigenic and secreted via exovesicles.

Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected and emerging tropical disease, endemic to South America and present in non-endemic regions due to human migration. The MASP multigene family is specific to T. cruzi, accounting for 6% of the parasite's genome and plays a key role in immune evasion. A common feature of MASPs is the presence of two conserved regions: an N-terminal region codifying for signal peptide and a C-terminal (C-term) region, which potentially acts as GPI-addition signal peptide. Our aim was the analysis of the presence of an immune response against the MASP C-term region. We found that this region is highly conserved, released via exovesicles (EVs) and has an associated immune response as revealed by epitope affinity mapping, IFA and inhibition of the complement lysis assays. We also demonstrate the presence of a fast IgM response in Balb/c mice infected with T. cruzi. Our results reveal the presence of non-canonical secreted peptides in EVs, which can subsequently be exposed to the immune system with a potential role in evading immune system targets in the parasite

Repertoire, Genealogy and Genomic Organization of Cruzipain and Homologous Genes in Trypanosoma cruzi, T. cruzi-Like and Other Trypanosome Species

Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches

MDL28170, a Calpain Inhibitor, Affects Trypanosoma cruzi Metacyclogenesis, Ultrastructure and Attachment to Rhodnius prolixus Midgut

BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas' disease. During the parasite life cycle, many molecules are involved in the differentiation process and infectivity. Peptidases are relevant for crucial steps of T. cruzi life cycle; as such, it is conceivable that they may participate in the metacyclogenesis and interaction with the invertebrate host. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we have investigated the effect of the calpain inhibitor MDL28170 on the attachment of T. cruzi epimastigotes to the luminal midgut surface of Rhodnius prolixus, as well as on the metacyclogenesis process and ultrastructure. MDL28170 treatment was capable of significantly reducing the number of bound epimastigotes to the luminal surface midgut of the insect. Once the cross-reactivity of the anti-Dm-calpain was assessed, it was possible to block calpain molecules by the antibody, leading to a significant reduction in the capacity of adhesion to the insect guts by T. cruzi. However, the antibodies were unable to interfere in metacyclogenesis, which was impaired by the calpain inhibitor presenting a significant reduction in the number of metacyclic trypomastigotes. The calpain inhibitor also promoted a direct effect against bloodstream trypomastigotes. Ultrastructural analysis of epimastigotes treated with the calpain inhibitor revealed disorganization in the reservosomes, Golgi and plasma membrane disruption. CONCLUSIONS/SIGNIFICANCE: The presence of calpain and calpain-like molecules in a wide range of organisms suggests that these proteins could be necessary for basic cellular functions. Herein, we demonstrated the effects of MDL28170 in crucial steps of the T. cruzi life cycle, such as attachment to the insect midgut and metacyclogenesis, as well as in parasite viability and morphology. Together with our previous findings, these results help to shed some light on the functions of T. cruzi calpains. Considering the potential roles of these molecules on the interaction with both invertebrate and vertebrate hosts, it is interesting to improve knowledge on these molecules in T. cruzi

Computational Identification of Uncharacterized Cruzain Binding Sites

Chagas disease, caused by the unicellular parasite Trypanosoma cruzi, claims 50,000 lives annually and is the leading cause of infectious myocarditis in the world. As current antichagastic therapies like nifurtimox and benznidazole are highly toxic, ineffective at parasite eradication, and subject to increasing resistance, novel therapeutics are urgently needed. Cruzain, the major cysteine protease of Trypanosoma cruzi, is one attractive drug target. In the current work, molecular dynamics simulations and a sequence alignment of a non-redundant, unbiased set of peptidase C1 family members are used to identify uncharacterized cruzain binding sites. The two sites identified may serve as targets for future pharmacological intervention

A Phosphoproteomic Approach towards the Understanding of the Role of TGF-β in Trypanosoma cruzi Biology

Transforming growth factor beta (TGF-β) plays a pivotal role in Chagas disease, not only in the development of chagasic cardiomyopathy, but also in many stages of the T. cruzi life cycle and survival in the host cell environment. The intracellular signaling pathways utilized by T. cruzi to regulate these mechanisms remain unknown. To identify parasite proteins involved in the TGF-β response, we utilized a combined approach of two-dimensional gel electrophoresis (2DE) analysis and mass spectrometry (MS) protein identification. Signaling via TGF-β is dependent on events of phosphorylation, which is one of the most relevant and ubiquitous post-translational modifications for the regulation of gene expression, and especially in trypanosomatids, since they lack several transcriptional control mechanisms. Here we show a kinetic view of T. cruzi epimastigotes (Y strain) incubated with TGF-β for 1, 5, 30 and 60 minutes, which promoted a remodeling of the parasite phosphorylation network and protein expression pattern. The altered molecules are involved in a variety of cellular processes, such as proteolysis, metabolism, heat shock response, cytoskeleton arrangement, oxidative stress regulation, translation and signal transduction. A total of 75 protein spots were up- or down-regulated more than twofold after TGF-β treatment, and from these, 42 were identified by mass spectrometry, including cruzipain–the major T. cruzi papain-like cysteine proteinase that plays an important role in invasion and participates in the escape mechanisms used by the parasite to evade the host immune system. In our study, we observed that TGF-β addition favored epimastigote proliferation, corroborating 2DE data in which proteins previously described to be involved in this process were positively stimulated by TGF-β