Christian Macagnan Probst

Profiling the Trypanosoma cruzi Phosphoproteome

Protein phosphorylation is a reversible post-translational modification essential for the regulation of several signal transduction pathways and biological processes in the living cell. Therefore, the identification of protein phosphorylation sites is crucial to understand cell signaling control at the molecular level. Based on mass spectrometry, recent studies have reported the large-scale mapping of phosphorylation sites in various eukaryotes and prokaryotes. However, little is known about the impact of phosphorylation in protozoan parasites. To in depth characterize the phosphoproteome of Trypanosoma cruzi, a parasite of the Kinetoplastida class, protein samples from cells at different phases of the metacyclogenesis – differentiation process of the parasites from non-infective epimastigotes to infective metacyclic trypomastigotes - were enriched for phosphopeptides using TiO2 chromatography and analyzed on an LTQ-Orbitrap mass spectrometer. In total, 1,671 proteins were identified, including 753 phosphoproteins, containing a total of 2,572 phosphorylation sites. The distribution of phosphorylated residues was 2,162 (84.1%) on serine, 384 (14.9%) on threonine and 26 (1.0%) on tyrosine. Here, we also report several consensus phosphorylation sequence motifs and as some of these conserved groups have enriched biological functions, we can infer the regulation by protein kinases of this functions. To our knowledge, our phosphoproteome is the most comprehensive dataset identified until now for Kinetoplastida species. Here we also were able to extract biological information and infer groups of sites phosphorylated by the same protein kinase. To make our data accessible to the scientific community, we uploaded our study to the data repositories PHOSIDA, Proteome Commons and TriTrypDB enabling researchers to access information about the phosphorylation sites identified here.

Association between Vitamin D Receptor Gene Polymorphisms and Susceptibility to Chronic Kidney Disease and Periodontitis

Background/Aims: Chronic kidney disease (CKD) and periodontitis (PD) are serious public-health concerns. Vitamin D is a fat-soluble steroid hormone that interacts with its nuclear receptor (VDR) to regulate a variety of biological processes, such as bone metabolism, immune response modulation and transcription of several genes involved in CKD and PD disease mechanisms. The aim of this work was to investigate the association between polymorphisms in the VDR gene and end-stage renal disease (ESRD) and PD. Methods: 222 subjects with and without ESRD (in hemodialysis) were divided into groups with and without PD. Polymorphisms TaqI and BsmI in the VDR gene were analyzed by PCR restriction fragment length polymorphism. The significance of differences in allele, genotype and haplotype frequencies between groups was assessed by the χ2 test (p value <0.05) and odds ratio (OR). Results: Allele G was associated with protection against ESRD: groups without versus with ESRD (GG) × (GA+AA): OR = 2.5, 95% CI = 1.4–4.6, p = 0.00; (G × A): OR = 1.5, 95% CI = 1.0–2.3, p = 0.02; (TG + CG) × (TA + CA): OR = 1.5, 95% CI = 1.0–2.3, p = 0.02. No association was observed between the study polymorphisms and susceptibility to or protection against PD. Conclusion: Allele G of the VDR BsmI polymorphism was associated with protection against ESRD.

Trypanosoma cruzi Response to Sterol Biosynthesis Inhibitors: Morphophysiological Alterations Leading to Cell Death

The protozoan parasite Trypanosoma cruzi displays similarities to fungi in terms of its sterol lipid biosynthesis, as ergosterol and other 24-alkylated sterols are its principal endogenous sterols. The sterol pathway is thus a potential drug target for the treatment of Chagas disease. We describe here a comparative study of the growth inhibition, ultrastructural and physiological changes leading to the death of T. cruzi cells following treatment with the sterol biosynthesis inhibitors (SBIs) ketoconazole and lovastatin. We first calculated the drug concentration inhibiting epimastigote growth by 50% (EC50/72 h) or killing all cells within 24 hours (EC100/24 h). Incubation with inhibitors at the EC50/72 h resulted in interesting morphological changes: intense proliferation of the inner mitochondrial membrane, which was corroborated by flow cytometry and confocal microscopy of the parasites stained with rhodamine 123, and strong swelling of the reservosomes, which was confirmed by acridine orange staining. These changes to the mitochondria and reservosomes may reflect the involvement of these organelles in ergosterol biosynthesis or the progressive autophagic process culminating in cell lysis after 6 to 7 days of treatment with SBIs at the EC50/72 h. By contrast, treatment with SBIs at the EC100/24 h resulted in rapid cell death with a necrotic phenotype: time-dependent cytosolic calcium overload, mitochondrial depolarization and reservosome membrane permeabilization (RMP), culminating in cell lysis after a few hours of drug exposure. We provide the first demonstration that RMP constitutes the “point of no return” in the cell death cascade, and propose a model for the necrotic cell death of T. cruzi. Thus, SBIs trigger cell death by different mechanisms, depending on the dose used, in T. cruzi. These findings shed new light on ergosterol biosynthesis and the mechanisms of programmed cell death in this ancient protozoan parasite.

Quantitative proteomics of Trypanosoma cruzi during metacyclogenesis

Trypanosoma cruzi is the etiologic agent of Chagas disease, which is estimated to affect over eight million people around the world. Trypanosoma cruzi has a complex life cycle, involving insect and mammalian hosts and four distinct developmental stages: epimastigotes, metacyclic trypomastigotes, amastigotes, and bloodstream trypomastigotes. Metacyclogenesis is the process by which T. cruzi epimastigotes differentiate into metacyclic trypomastigotes and acquire infectivity, and involves differential gene expression associated with acquisition of virulence. In T. cruzi, gene expression regulation is achieved mainly posttranscriptionally. Therefore, proteomics‐based approaches are extremely useful for gaining a better understanding of the changes that occur in the stage‐regulated gene expression program of the parasite at the molecular level. Here, we performed an in‐depth quantitative MS‐based proteomic study of T. cruzi metacyclogenesis and quantified almost 3000 proteins expressed during the process of differentiation. To the best of our knowledge, this work is the most comprehensive quantitative proteomics study of different cell populations of T. cruzi available so far. We identified relevant proteins and pathways involved in the parasites differentiation and infectivity acquisition, opening new perspectives for further studies that could, ultimately, lead to the identification of new targets for chemotherapy.

Association of IL1 gene polymorphisms with chronic periodontitis in Brazilians

UNLABELLED Chronic periodontal disease (PD) is an infectious immune-inflammatory illness. Polymorphisms in IL1 genes play a role in inflammatory diseases through the modulation of cytokine levels. OBJECTIVE this study aimed to investigate the association between polymorphisms in the IL1 gene cluster and chronic periodontitis in a Brazilian population. DESIGN a sample of 113 subjects over 25 years (mean age 41.2) were grouped into: 44 healthy individuals, 31 subjects with moderate and 38 with severe periodontitis. DNA was obtained through a mouthwash and oral mucosa scraping. PCR-RFLP was used to identify the following polymorphisms: IL1A C-889T (rs1800587), IL1B C-511T (rs16944), IL1B C+3954T (rs11436340), IL1RN intron 2 (rs2234663). Differences in the allele/genotype/haplotype frequencies were assessed by Chi-square test (p<0.05). The risk associated with alleles, genotypes and haplotypes was calculated as odds ratio (OR) with 95% confidence intervals (CI). RESULTS neither IL1A (C-889T) nor IL1B (C+3954T) polymorphisms was associated with chronic PD. Allele T for IL1B (C-511T) only associated with PD in the group of blacks and mulattos. Moreover, genotype 2/2 for IL1RN (intron 2) was associated with severe PD. CONCLUSIONS genotype 2/2 of IL1RN for the whole Brazilian population and allele T of IL1B (C-511T) in a subgroup of Afro-Americans and mulattos were suggested as putative risk indicators for chronic periodontitis.

Differential gene expression in Trypanosoma cruzi populations susceptible and resistant to benznidazole

Differential gene expression in three pairs of Trypanosoma cruzi populations or clones susceptible or resistant to benznidazole (BZ) was investigated by differential display (DD) and representation of differential expression (RDE). GenBank searches of 14 genes selected by DD showed that four sequences corresponded to different hypothetical proteins and the others were very similar to T. cruzi genes encoding mucin (TcMUC), dihydrolipoamide dehydrogenase (TcLipDH), the hexose transporter (TcHT), or a ribosomal protein. Sequence analysis was performed on 34 clones obtained by RDE; approximately half of these clones encoded 14 different hypothetical proteins and the other half encoded proteins involved with stress response, antioxidant defence, metabolism, transporter proteins, surface proteins, ribosomal proteins and others. The mRNA levels of eight T. cruzi genes obtained by RDE and DD were analysed by northern blotting to confirm the differential expression of these sequences. For six of the eight genes, TcLipDH, TcHT, TcFeSOD-A (iron superoxide dismutase-A), TcHSP70, TcHSP100 (heat shock protein) and Tc52 (thiol-transferase), mRNA levels in the drug-resistant T. cruzi population were at least twice those in the susceptible population. Further analysis of TcHSP70 showed that although the levels of TcHSP70 mRNA were four-fold higher in T. cruzi BZ-resistant population, no corresponding increase was observed in the levels of TcHSP70 protein expression. The results suggest that TcHSP70 is not directly associated with the T. cruzi drug resistance phenotype.

Predicting the proteins of angomonas deanei, strigomonas culicis and their respective endosymbionts reveals new aspects of the trypanosomatidae family

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.

Protein and mRNA content of TcDHH1‐containing mRNPs in Trypanosoma cruzi

In trypanosomatids, the regulation of gene expression occurs mainly at the post‐transcriptional level. Previous studies have revealed nontranslated mRNA in the Trypanosoma cruzi cytoplasm. Previously, we have identified and cloned the TcDHH1 protein, a DEAD box RNA helicase. It has been reported that Dhh1 is involved in multiple RNA‐related processes in various eukaryotes. It has also been reported to accumulate in stress granules and processing bodies of yeast, animal cells, Trypanosoma brucei and T. cruzi. TcDHH1 is localized to discrete cytoplasmic foci that vary depending on the life cycle status and nutritional conditions. To study the composition of mRNPs containing TcDHH1, we carried out immunoprecipitation assays with anti‐TcDHH1 using epimastigote lysates. The protein content of mRNPs was determined by MS and pre‐immune serum was used as control. We also carried out a ribonomic approach to identify the mRNAs present within the TcDHH1 immunoprecipitated complexes. For this purpose, competitive microarray hybridizations were performed against negative controls, the nonprecipitated fraction. Our results showed that mRNAs associated with TcDHH1 in the epimastigote stage are those mainly expressed in the other forms of the T. cruzi life cycle. These data suggest that mRNPs containing TcDHH1 are involved in mRNA metabolism, regulating the expression of at least epimastigote‐specific genes.

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 1185bp long and encodes a 44.3kDa 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.

A high-throughput cloning system for reverse genetics in Trypanosoma cruzi

BackgroundThe three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.ResultsWe constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (Tc Rab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed.ConclusionsWe constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.