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 approximately 850 mucin-encoding genes comprise approximately 1% of the parasite genome and approximately 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 enzyme Activation Induced Deaminase (AID) triggers antibody diversification in B-cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively diffuse into the nucleus, despite its small size, its nuclear entry requiring active import mediated by a conformational nuclear localization sequence (NLS). We also identify a determinant for AID cytoplasmic retention in its Cterminus, which hampers diffusion to the nucleus, competes with nuclear import and is critical for maintaining the predominantly cytoplasmic localization of AID in steady-state conditions. Blocking nuclear import alters the balance between these processes in favor of cytoplasmic retention, resulting in reduced isotype class switching.3

A thick coat of mucin-like glycoproteins covers the surface of Trypanosoma cruzi and plays a crucial role in parasite protection and infectivity and host immunomodulation. The appealing candidate genes coding for the mucins of the mammal-dwelling stages define a heterogeneous family termed TcMUC, which comprises up to 700 members, thus precluding a genetic approach to address the protein core identity. Here, we demonstrate by multiple approaches that the TcMUC II genes code for the majority of trypomastigote mucins. These molecules display a variable, non-repetitive, highly O-glycosylated central domain, followed by a short conserved C terminus and a glycosylphosphatidylinositol anchor. A simultaneous expression of multiple TcMUC II gene products was observed. Moreover, the C terminus of TcMUC II mucins, but not their central domain, elicited strong antibody responses in patients with Chagas' disease and T. crusi infected animals. This highly diverse coat of mucins may represent a refined parasite strategy to elude the mammalian host immune system.Trypanosoma cruzi is the etiologic agent of Chagas' disease, which is of major medical and economical significance in Latin America (1). The T. cruzi life cycle involves distinct stages in both the mammalian host and the hematophagous insect vector (2). Within the insect, two major developmental forms can be observed: replicative epimastigotes and metacyclic trypomastigotes. The latter form brings the infection into humans when released on the skin or mucosa after the insect blood meal. Following cell invasion, metacyclic trypomastigotes differentiate into amastigotes, which, after several divisions, transform into cell-derived trypomastigotes, which are then released into the bloodstream. This stage is able to invade a wide variety of nucleated cells, thus propagating the infection.A thick coat of glycoproteins covers the surface of all these developmental stages (3-11). The major protein components of this coat have been identified as glycosylphosphatidylinositol (GPI) 1 -anchored molecules enriched in Thr, Ser, and Pro residues that serve as a scaffold for the extensive addition of O-glycans (5, 8 -10, 12). This particular feature enables their classification as mucin-like proteins by analogy to mammalian mucins (13). Mucins play a key role in parasite protection and infectivity and modulation of the host immune response throughout the T. cruzi life cycle (14 -16). The mucin coat of the cell-derived trypomastigotes (tGPI-mucins) is composed of an undefined mixture of molecules ranging from 60 to 220 kDa (6, 7, 9) and sharing the stage-specific, sialic acid-containing epitope Ssp-3, critical for mammalian cell attachment/invasion (17, 18). tGPI-mucins or their GPI moieties are potent inducers of nitric oxide and pro-inflammatory cytokines by macrophages (15,19). Major protective lytic antibodies directed against ␣-galactosyl epitopes present in tGPI-mucins have been described in sera from chronic Chagas' disease patients (6, 7, 9, 11).Recently, substantial informatio...

Trypanosomatids are protozoan micro-organisms that cause serious health problems in humans and domestic animals. In addition to their medical relevance, these pathogens have novel biological structures and processes. From nuclear DNA transcription to mRNA translation, trypanosomes use unusual mechanisms to control gene expression. For example, transcription by RNAPII (RNA polymerase II) is polycistronic, and only a few transcription initiation sites have been identified so far. The sequences present in the polycistronic units code for proteins having unrelated functions, that is, not involved in a similar metabolic pathway. Owing to these biological constraints, these micro-organisms regulate gene expression mostly by post-transcriptional events. Consequently, the function of proteins that recognize RNA elements preferentially at the 3' UTR (untranslated region) of transcripts is central. It was recently shown that mRNP (messenger ribonucleoprotein) complexes are organized within post-transcriptional operons to co-ordinately regulate gene expression of functionally linked transcripts. In the present chapter we will focus on particular characteristics of gene expression in the so-called TriTryp parasites: Trypanosoma cruzi, Trypanosoma brucei and Leishmania major.

Histone post-translational modification, mediated by histone acetyltransferases and deacetylases, is one of the most studied factors affecting gene expression. Recent data showing differential histone acetylation states during the Trypanosoma cruzi cell cycle suggest a role for epigenetics in the control of this process. As a starting point to study the role of histone deacetylases in the control of gene expression and the consequences of their inhibition and activation in the biology of T. cruzi, two inhibitors for different histone deacetylases: trichostatin A for class I/II and sirtinol for class III and the activator resveratrol for class III, were tested on proliferative and infective forms of this parasite. The two inhibitors tested caused histone hyperacetylation whereas resveratrol showed the opposite effect on both parasite forms, indicating that a biologically active in vivo level of these compounds was achieved. Histone deacetylase inhibitors caused life stage-specific effects, increasing trypomastigotes infectivity and blocking metacyclogenesis. Moreover, these inhibitors affected specific transcript levels, with sirtinol causing the most pronounced change. On the other hand, resveratrol showed strong anti-parasitic effects. This compound diminished epimastigotes growth, promoted metacyclogenesis, reduced in vitro infection and blocked differentiation and/or replication of intracellular amastigotes. In conclusion, the data presented here supports the notion that these compounds can modulate T. cruzi gene expression, differentiation, infection and histones deacetylase activity. Furthermore, among the compounds tested in this study, the results point to Resveratrol as promising trypanocidal drug candidate.

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A total of 1,921 expressed sequence tags (ESTs) were obtained from bloodstream trypomastigotes of Trypanosoma carassii, a parasite of economic importance due to its high prevalence in fish farms. Analysis of the data set allowed us to identify a trans-sialidase (TS)-like gene and three ESTs coding for putative mucin-like genes. TS activity was detected in cell extracts of bloodstream trypomastigotes. We have also used the sequence information obtained to identify genes that have not been previously described in trypanosomatids. (Additional information on these ESTs can be found at http://genoma.unsam.edu.ar/projects/tca.)Trypanosoma carassii infects a variety of cyprinid fish, such as carp, goldfish, crucian carp, and tench, as well as some members of noncyprinid families. The prevalence in nature is very high and may approach 100% in densely populated fish cultures.Unlike the stercorarian trypanosome Trypanosoma cruzi, T. carassii does not appear to have intracellular stages within the infected host (16). However, T. carassii trypomastigotes show a carbohydrate-rich coat of glycosyl-phosphatidylinositol (GPI)-anchored mucin-like proteins, which are biochemically similar to the mucin coat present in T. cruzi (13,17). The carbohydrate moiety of T. carassii mucins contains sialic acid, a monosaccharide that in other trypanosomes is transferred from host glycoconjugates to parasite surface molecules by trans-sialidase (TS) (9). TS is a modified sialidase, unique to a few trypanosomatids, that instead of hydrolyzing sialic acid is highly efficient in transferring the monosaccharide to an acceptor molecule. In T. cruzi, the main acceptors are mucins. Sialylated mucins are considered to be essential for the survival of the parasite in the mammalian host (20).A small-scale expressed sequence tag (EST) sequencing project for the blood stage of T. carassii was undertaken. Among others, genes encoding proteins homologous to trypanosome TS and putative mucin core proteins were identified. We also detected, for the first time, TS activity in T. carassii. The sequence information obtained was used to search for genes that have not been described previously in other trypanosomatids.Starting with poly(A) ϩ RNA obtained from blood trypomastigotes, we constructed an oriented cDNA library in the pSport1 vector (Amersham Pharmacia Biotech). Randomly selected clones were sequenced from the 5Ј end to generate 1,921 5Ј ESTs with Ͼ100 bp of good-quality, nonvector sequence. The average length for the whole data set was 390 bp.