Proteomic and network analysis characterize stage-specific metabolism in Trypanosoma cruzi

<p>Abstract</p> <p>Background</p> <p><it>Trypanosoma cruzi </it>is a Kinetoplastid parasite of humans and is the cause of Chagas disease, a potentially lethal condition affecting the cardiovascular, gastrointestinal, and nervous systems of the human host. Constraint-based modeling has emerged in the last decade as a useful approach to integrating genomic and other high-throughput data sets with more traditional, experimental data acquired through decades of research and published in the literature.</p> <p>Results</p> <p>We present a validated, constraint-based model of the core metabolism of <it>Trypanosoma cruzi </it>strain CL Brener. The model includes four compartments (extracellular space, cytosol, mitochondrion, glycosome), 51 transport reactions, and 93 metabolic reactions covering carbohydrate, amino acid, and energy metabolism. In addition, we make use of several replicate high-throughput proteomic data sets to specifically examine metabolism of the morphological form of <it>T. cruzi </it>in the insect gut (epimastigote stage).</p> <p>Conclusion</p> <p>This work demonstrates the utility of constraint-based models for integrating various sources of data (e.g., genomics, primary biochemical literature, proteomics) to generate testable hypotheses. This model represents an approach for the systematic study of <it>T. cruzi </it>metabolism under a wide range of conditions and perturbations, and should eventually aid in the identification of urgently needed novel chemotherapeutic targets.</p

Systems analysis of host-parasite interactions.

Parasitic diseases caused by protozoan pathogens lead to hundreds of thousands of deaths per year in addition to substantial suffering and socioeconomic decline for millions of people worldwide. The lack of effective vaccines coupled with the widespread emergence of drug-resistant parasites necessitates that the research community take an active role in understanding host-parasite infection biology in order to develop improved therapeutics. Recent advances in next-generation sequencing and the rapid development of publicly accessible genomic databases for many human pathogens have facilitated the application of systems biology to the study of host-parasite interactions. Over the past decade, these technologies have led to the discovery of many important biological processes governing parasitic disease. The integration and interpretation of high-throughput -omic data will undoubtedly generate extraordinary insight into host-parasite interaction networks essential to navigate the intricacies of these complex systems. As systems analysis continues to build the foundation for our understanding of host-parasite biology, this will provide the framework necessary to drive drug discovery research forward and accelerate the development of new antiparasitic therapies

Biology of <em>Trypanosoma cruzi</em>

Trypanosoma cruzi, an important zoonotic protozoan that causes Chagas disease, affects at least 8 million people in Latin America. Chagas disease is an important life-long infection in humans that can be divided into distinct clinical stages: the acute phase, where patient symptoms can vary from asymptomatic to severe; the indeterminate form, which is usually asymptomatic; and the chronic phase, where cardiomyopathy and/or digestive megasyndromes appear. In addition to its medical importance, T. cruzi is an interesting biological model for studying processes such as: (1) cell differentiation, where a non-infective stage transforms into an infective one; (2) cell invasion, where the infective stages are able to penetrate into a mammalian host cell, where they multiply several times and thus amplify the infection; and (3) evasion from the immune system, using several mechanisms. This book, with 13 chapters, has been organized in four major sections: 1. "Basic Biology," 2. "Biochemistry and Molecular Biology," 3. "Parasite"Host Cell Interaction," and 4 "Chemotherapy." The chapters include basic biological information on the protozoan lifecycle, including new information on parasite genomics and proteomics. In addition, they analyze the interaction with host cells as well the immune response and evasion, ending with information on experimental chemotherapy against Chagas disease

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

Role of Proteomics in the Study of <em>Trypanosoma cruzi</em> Biology

Proteomics is the science that studies the proteome, which corresponds to the global expression of proteins at a given time under determined conditions. In the last 20 years, proteomics has emerged as a powerful tool that has allowed the study of proteins that are expressed in the cell under normal or altered conditions as well as post-translational modifications, such as phosphorylation, glycosidation, acetylation, and methylation, among others. In this chapter, we present the main contributions of proteomics to the knowledge of Trypanosoma cruzi biology. Proteomes of all T. cruzi life cycle stages, secretomes/exoproteomes, post-translational modifications such as phosphorylation or acetylation and immunomes, interactomes, and glycomes are described. The role of proteomics in the identification of new chemotherapeutic targets and potential vaccine candidates will also be discussed

Extracellular vesicles in parasitic diseases

Parasitic diseases affect billions of people and are considered a major public health issue. Close to 400 species are estimated to parasitize humans, of which around 90 are responsible for great clinical burden and mortality rates. Unfortunately, they are largely neglected as they are mainly endemic to poor regions. Of relevance to this review, there is accumulating evidence of the release of extracellular vesicles (EVs) in parasitic diseases, acting both in parasite–parasite inter-communication as well as in parasite–host interactions. EVs participate in the dissemination of the pathogen and play a role in the regulation of the host immune systems. Production of EVs from parasites or parasitized cells has been described for a number of parasitic infections. In this review, we provide the most relevant findings of the involvement of EVs in intercellular communication, modulation of immune responses, involvement in pathology, and their potential as new diagnostic tools and therapeutic agents in some of the major human parasitic pathogens

The ever unfolding story of cAMP signaling in trypanosomatids: vive la difference!

Kinetoplastids are unicellular, eukaryotic, flagellated protozoans containing the eponymous kinetoplast. Within this order, the family of trypanosomatids are responsible for some of the most serious human diseases, including Chagas disease (Trypanosoma cruzi), sleeping sickness (Trypanosoma brucei spp.), and leishmaniasis (Leishmania spp). Although cAMP is produced during the life cycle stages of these parasites, its signaling pathways are very different from those of mammals. The absence of G-protein-coupled receptors, the presence of structurally different adenylyl cyclases, the paucity of known cAMP effector proteins and the stringent need for regulation of cAMP in the small kinetoplastid cells all suggest a significantly different biochemical pathway and likely cell biology. However, each of the main kinetoplastid parasites express four class 1-type cyclic nucleotide-specific phosphodiesterases (PDEA-D), which have highly similar catalytic domains to that of human PDEs. To date, only TbrPDEB, expressed as two slightly different isoforms TbrPDEB1 and B2, has been found to be essential when ablated. Although the genomes contain reasonably well conserved genes for catalytic and regulatory domains of protein kinase A, these have been shown to have varied structural and functional roles in the different species. Recent discovery of a role of cAMP/AMP metabolism in a quorum-sensing signaling pathway in T. brucei, and the identification of downstream cAMP Response Proteins (CARPs) whose expression levels correlate with sensitivity to PDE inhibitors, suggests a complex signaling cascade. The interplay between the roles of these novel CARPs and the quorum-sensing signaling pathway on cell division and differentiation makes for intriguing cell biology and a new paradigm in cAMP signal transduction, as well as potential targets for trypanosomatid-specific cAMP pathway-based therapeutics

Kinetoplastid Genomics and Beyond

This book includes a collection of eight original research articles and three reviews covering a wide range of topics in the field of kinetoplastids. In addition, readers can find a compendium of molecular biology procedures and bioinformatics tools

Exocytosis and protein secretion in Trypanosoma

<p>Abstract</p> <p>Background</p> <p>Human African trypanosomiasis is a lethal disease caused by the extracellular parasite <it>Trypanosoma brucei</it>. The proteins secreted by <it>T. brucei </it>inhibit the maturation of dendritic cells and their ability to induce lymphocytic allogenic responses. To better understand the pathogenic process, we combined different approaches to characterize these secreted proteins.</p> <p>Results</p> <p>Overall, 444 proteins were identified using mass spectrometry, the largest parasite secretome described to date. Functional analysis of these proteins revealed a strong bias toward folding and degradation processes and to a lesser extent toward nucleotide metabolism. These features were shared by different strains of <it>T. brucei</it>, but distinguished the secretome from published <it>T. brucei </it>whole proteome or glycosome. In addition, several proteins had not been previously described in <it>Trypanosoma </it>and some constitute novel potential therapeutic targets or diagnostic markers. Interestingly, a high proportion of these secreted proteins are known to have alternative roles once secreted. Furthermore, bioinformatic analysis showed that a significant proportion of proteins in the secretome lack transit peptide and are probably not secreted through the classical sorting pathway. Membrane vesicles from secretion buffer and infested rat serum were purified on sucrose gradient and electron microscopy pictures have shown 50- to 100-nm vesicles budding from the coated plasma membrane. Mass spectrometry confirmed the presence of <it>Trypanosoma </it>proteins in these microvesicles, showing that an active exocytosis might occur beyond the flagellar pocket.</p> <p>Conclusions</p> <p>This study brings out several unexpected features of the secreted proteins and opens novel perspectives concerning the survival strategy of <it>Trypanosoma </it>as well as possible ways to control the disease. In addition, concordant lines of evidence support the original hypothesis of the involvement of microvesicle-like bodies in the survival strategy allowing <it>Trypanosoma </it>to exchange proteins at least between parasites and/or to manipulate the host immune system.</p