Alberto C.C. Frasch

Trypanosoma cruzi surface mucins: host-dependent coat diversity

The surface of the protozoan parasite Trypanosoma cruzi is covered in mucins, which contribute to parasite protection and to the establishment of a persistent infection. Their importance is highlighted by the fact that the ∼850 mucin-encoding genes comprise ∼1% of the parasite genome and ∼6% of all predicted T. cruzi genes. The coordinate expression of a large repertoire of mucins containing variable regions in the mammal-dwelling stages of the T. cruzi life cycle suggests a possible strategy to thwart the host immune response. Here, we discuss the expression profiling of T. cruzi mucins, the mechanisms leading to the acquisition of mucin diversity and the possible consequences of a mosaic surface coat in the interplay between parasite and host.

The crystal structure and mode of action of trans-sialidase, a key enzyme in Trypanosoma cruzi pathogenesis

Trans-sialidases (TS) are GPI-anchored surface enzymes expressed in specific developmental stages of trypanosome parasites like Trypanosoma cruzi, the etiologic agent of Chagas disease, and T. brucei, the causative agent of sleeping sickness. TS catalyzes the transfer of sialic acid residues from host to parasite glycoconjugates through a transglycosidase reaction that appears to be critical for T. cruzi survival and cell invasion capability. We report here the structure of the T. cruzi trans-sialidase, alone and in complex with sugar ligands. Sialic acid binding is shown to trigger a conformational switch that modulates the affinity for the acceptor substrate and concomitantly creates the conditions for efficient transglycosylation. The structure provides a framework for the structure-based design of novel inhibitors with potential therapeutic applications.

Regulation of Hippocampal Gene Expression Is Conserved in Two Species Subjected to Different Stressors and Antidepressant Treatments

BACKGROUND Chronic stress has significant effects on hippocampal structure and function. We have previously identified nerve growth factor (NGF), membrane glycoprotein 6a (M6a), the guanine nucleotide binding protein (G protein) alpha q polypeptide (GNAQ), and CDC-like kinase 1 (CLK-1) as genes regulated by psychosocial stress and clomipramine treatment in the hippocampus of tree shrews. These genes encode proteins involved in neurite outgrowth. METHODS To analyze whether regulation of the above-mentioned genes is conserved between different species, stressors, and antidepressant drugs, we subjected mice to repeated restraint stress and tianeptine treatment and measured hippocampal messenger RNA (mRNA) levels by real time reverse transcription polymerase chain reaction (RT-PCR). RESULTS Chronically stressed mice displayed a reduction in transcript levels for NGF, M6a, GNAQ, and CLK-1. In addition, other genes implicated in neuronal plasticity, such as brain-derived neurotrophic factor (BDNF), cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), protein kinase C (PKC), neural cell adhesion molecule (NCAM), and synapsin I were downregulated in stressed mice. Tianeptine treatment reversed the stress effects for the genes analyzed. Alterations in gene expression were dependent on the duration of the stress treatment and, in some cases, were only observed in male mice. CONCLUSIONS These results suggest that genes involved in neurite remodeling are one of the main targets for regulation by chronic stress. The finding that this regulation is conserved in different stress models and antidepressant treatments highlights the biological relevance of the genes analyzed and suggests that they might be involved in stress-related disorders.

The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is encoded by multiple polymorphic tandemly organized genes located on different chromosomes

We demonstrate that cruzipain, the major cysteine proteinase of Trypanosoma cruzi epimastigotes, is encoded by a large number of tandemly arranged genes. Restriction enzyme analysis of 20 clones containing complete repeat units of the gene, as well as sequencing of 2 of these clones, and comparison with previously published partial sequences, indicated that the sequence is conserved among the repeat units, although polymorphisms clearly exist. The repeat units contain an intergenic region of 528 bp and coding regions for pre- and pro-enzyme, a central domain and a C-terminal extension. The predicted amino acid sequences of these regions indicated a sequence identity of 30, 60, 70 and 36%, respectively, when the T. cruzi sequence was compared with the sequence of a similar cysteine proteinase from Trypanosoma brucei. Studies by pulsed field gel electrophoresis, complemented with restriction analysis, indicated that the clusters are located on 2-4 different chromosomes in several parasite isolates.

Structural basis of sialyltransferase activity in trypanosomal sialidases

The intracellular parasite Trypanosoma cruzi, the etiological agent of Chagas disease, sheds a developmentally regulated surface trans‐sialidase, which is involved in key aspects of parasite–host cell interactions. Although it shares a common active site architecture with bacterial neuraminidases, the T.cruzi enzyme behaves as a highly efficient sialyltransferase. Here we report the crystal structure of the closely related Trypanosoma rangeli sialidase and its complex with inhibitor. The enzyme folds into two distinct domains: a catalytic β‐propeller fold tightly associated with a lectin‐like domain. Comparison with the modeled structure of T.cruzi trans‐sialidase and mutagenesis experiments allowed the identification of amino acid substitutions within the active site cleft that modulate sialyltransferase activity and suggest the presence of a distinct binding site for the acceptor carbohydrate. The structures of the Trypanosoma enzymes illustrate how a glycosidase scaffold can achieve efficient glycosyltransferase activity and provide a framework for structure‐based drug design.

Identification of the gene(s) coding for the trans-sialidase of Trypanosoma cruzi.

The gene(s) encoding the Trypanosoma cruzi shed‐acute‐phase‐antigen (SAPA) has a 5′ end encoding a region containing two totally and two partially conserved Ser‐X‐Asp‐X‐Gly‐X‐Thr‐Trp motifs which are present in bacterial neuraminidases, and a 3′ end encoding tandemly repeated units of 12 amino acids. It is now reported that 54–87% of the total neuraminidase activity present in the parasite could be immunoprecipitated with polyclonal or monoclonal antibodies against the repeated amino acid units of SAPA. These immunoprecipitates also had greater than 80% of the trans‐sialidase activity of the parasite. SAPA used sialyllactose, fetuin and 4‐methylumbelliferyl‐sialic acid as substrate donors. In the presence of a suitable acceptor molecule (lactose) the sialic acid residues were transferred to the disaccharide, whereas in the absence of acceptors the residues were transferred to water. If relatively inefficient acceptors (maltose or cellobiose) were added to the incubation mixtures, the sialic acid units were transferred both to the disaccharides and to water. It is concluded that a major T. cruzi antigen has both the trans‐sialidase and the neuraminidase activities of the parasite. Both activities are probably located on the N‐terminus of SAPA since antibodies directed against the C‐terminus, which contains the repeated amino acid units, do not affect the enzymatic activities.

Identification of genes regulated by chronic psychosocial stress and antidepressant treatment in the hippocampus

Analysis of differentially expressed genes in the brain is a promising tool for elucidating pathological mechanisms that lead to central nervous disorders. Stress is known to be involved in the development of psychopathologies such as depression. In the present study, we searched for differentially expressed genes in the hippocampal formation after chronic psychosocial stress and after treatment with the antidepressant clomipramine. Experiments were conducted in male tree shrews, a valid psychosocial stress model in which antidepressant drugs prevent diverse effects of stress. Because many effects of stress have been attributed to the stress‐induced elevation in glucocorticoids, we screened two subtractive hippocampal cDNA libraries generated from RNA of chronic cortisol‐treated animals. Using real‐time PCR to measure mRNA amounts, we identified five sequences whose expression levels differed between stressed animals and controls. Transcript levels of four of them, nerve growth factor (NGF), membrane glycoprotein 6a (M6a), CDC‐like kinase 1 (CLK‐1) and G‐protein alpha q (GNAQ) were reduced by chronic psychosocial stress. Reduced amounts of these genes, which are all related to processes of cell differentiation, is in agreement with previous findings showing a retraction of dendrites and an impairment of neurogenesis in the hippocampal formation after chronic stress. An additional expressed sequence that was also regulated by stress could not be assigned to any known gene. Treatment with the antidepressant clomipramine prevented stress effects on expression of M6a, CLK‐1, GNAQ and the novel sequence, but showed no effect on NGF stress‐induced down‐regulation. These findings support the concept that depressive disorders are accompanied by processes of neuronal dedifferentiation, at least in the hippocampal formation, and that antidepressants prevent these processes.

RNA-Binding Domain Proteins in Kinetoplastids: a Comparative Analysis

ABSTRACT RNA-binding proteins are important in many aspects of RNA processing, function, and destruction. One class of such proteins contains the RNA recognition motif (RRM), which consists of about 90 amino acid residues, including the canonical RNP1 octapeptide: (K/R)G(F/Y)(G/A)FVX(F/Y). We used a variety of homology searches to classify all of the RRM proteins of the three kinetoplastids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. All three organisms have similar sets of RRM-containing protein orthologues, suggesting common posttranscriptional processing and regulatory pathways. Of the 75 RRM proteins identified in T. brucei, only 13 had clear homologues in other eukaryotes, although 8 more could be given putative functional assignments. A comparison with the 18 RRM proteins of the obligate intracellular parasite Encephalitozoon cuniculi revealed just 3 RRM proteins which appear to be conserved at the primary sequence level throughout eukaryotic evolution: poly(A) binding protein, the rRNA-processing protein MRD1, and the nuclear cap binding protein.

Recruitment of mRNAs to cytoplasmic ribonucleoprotein granules in trypanosomes.

Trypanosomes are outstanding examples of the importance of mRNA metabolism in the regulation of gene expression, as these unicellular eukaryotes mostly control protein synthesis by post‐transcriptional mechanisms. Here, we show that mRNA metabolism in these organisms involves recruitment of mRNAs and proteins to microscopically visible ribonucleoprotein granules in the cytoplasm. These structures engage transcripts that are being translated and protect mRNAs from degradation. Analysis of the protein composition of trypanosomal mRNA granules indicated that they contain orthologous proteins to those present in P bodies and stress granules from metazoan organisms. Formation of mRNA granules was observed after carbon‐source deprivation of parasites in axenic culture. More important, mRNA granules are formed naturally in trypanosomes present in the intestinal tract of the insect vector. We suggest that trypanosomes make use of mRNA granules for transient transcript protection as a strategy to cope with periods of starvation that they have to face during their complex life cycles.

A single tyrosine differentiates active and inactive Trypanosoma cruzi trans-sialidases

Several genes encode members of the Trypanosoma cruzi (Tc) trans-sialidase (TS) family. These proteins contain an enzymatic domain on the N terminus, the only one required for TS activity, and an antigenic domain (SAPA (shed acute phase antigen) amino acid (aa) repeats) on the C terminus. Only some members of this glycoprotein family are enzymatically active. The complete sequence of two clones encoding the enzymatic domain of active and inactive protein from each of two Tc strains has now been obtained. Comparison of these sequences showed a limited divergence among them: 20 out of the 642 deduced aa in the enzymatic domain were found to differ. From these 20 aa, only one was found to be essential for enzymatic activity. A Tyr342 residue is deduced in both active proteins while a His342 is present in both inactive ones. This naturally occurring Tyr342-->His substitution completely abolished the TS activity. In addition to Tyr342, a second deduced aa, Pro231, was found to be necessary for full enzymatic TS activity; a Pro231-->Ala change rendered the TS protein partially active. Fourteen aa residues, including Tyr342, out of the 16 aa in the active site of a sialidase from Salmonella typhimurium are present at the same or very similar positions in the Tc TS.