Kinetoplastid parasites are responsible for serious diseases in humans and livestock such as Chagas disease and sleeping sickness (caused by Trypanosoma cruzi and Trypanosoma brucei, respectively), and the different forms of cutaneous, mucocutaneous and visceral leishmaniasis (produced by Leishmania spp). The limited number of antiparasitic drugs available together with the emergence of resistance underscores the need for new therapeutic agents with novel mechanisms of action. The use of agents binding to surface glycans has been recently suggested as a new approach to antitrypanosomal design and a series of peptidic and non-peptidic carbohydrate-binding agents have been identified as antiparasitics showing efficacy in animal models of sleeping sickness. Here we provide an overview of the nature of surface glycans in three kinetoplastid parasites, T. cruzi, T. brucei and Leishmania. Their role in virulence and host cell invasion is highlighted with the aim of identifying specific glycan–lectin interactions and carbohydrate functions that may be the target of novel carbohydrate-binding agents with therapeutic applications.

Uridylate residues (Us) are inserted and deleted at precise positions in mitochondrial transcripts of Trypanosoma brucei. These sequence changes are determined by interactions with small guide RNAs (gRNAs) that are complementary to edited sequence. Adenylate (A) and guanylate (G) residues in gRNAs across from editing sites pair with inserted Us. We evaluate whether sequence bias exists in the bases surrounding insertion sites. Upon analyzing all reported insertion sites in T. brucei, we find that the predicted base pairs flanking insertion sites show a strong bias. Specifically, guiding As and Gs tend to be flanked by cytosine residues and Us. This bias is expected if precise base-pair interactions at the editing site determine the number of inserted Us.

In trypanosomatids, the majority of the guide (g) RNAs that provide the information for U-insertion/deletion RNA editing are encoded by minicircles that are catenated into large networks. In contrast, in the distantly related cryptobiid Trypanoplasma borreli, gRNA genes appear to reside in large 180-kb noncatenated DNA circles. To shed light on the evolutionary history and function of the minicircle network, we have analyzed minicircle organization in the free-living bodonid Bodo saltans, which is more closely related to trypanosomatids than T. borreli. We identified 1.4-kb circular DNAs as the B. saltans equivalent of minicircles via sequence analysis of 4 complete minicircles, 14 minicircle fragments, and 14 gRNAs. We show that each minicircle harbors two gRNA gene cassettes of opposite polarity residing in variable regions of about 200 nt in otherwise highly conserved molecules. In the conserved region, B. saltans minicircles contain a putative bent helix sequence and a degenerate dodecamer motif (CSB-3). Electron microscopy, sedimentation, and gel electrophoresis analyses showed no evidence for the existence of large minicircle networks in B. saltans, the large majority of the minicircles being present as circular and linear monomers (85–90%) with small amounts of catenated dimers and trimers. Our results provide the first example of a kinetoplastid species with noncatenated, gRNA gene-containing minicircles, which implies that the creation of minicircles and minicircle networks are separate evolutionary events.

The gp63 gene encoding the major surface antigen of Leishmania infantum has been cloned and sequenced. In spite of the overall sequence homology with the gp63 genes from other Leishmania species, particularly with the constitutively expressed Leishmania chagasi Gp63 gene, the carboxy-terminal ends of these genes are clearly divergent (62% homology). To study the prevalence of anti-gp63 antibodies in the sera from dogs with visceral leishmaniasis, a recombinant L. infantum gp63 protein was expressed in Escherichia coli. It was found that 100% of the sera from these dogs recognized the recombinant gp63 protein, suggesting that it must function as a potent B cell immunogen during natural canine visceral leishmaniasis. However, heterogeneity in the level of response was observed. Fine mapping of the antigenic determinants was performed by means of 6 overlapping subfragments of the gp63 protein and by the use of a library of synthetic peptides. The data showed that there is some degree of immunological restriction in the recognition of the protein since reactivity was observed preferentially against the most divergent region. The epitope mapping of this region showed 2 immunodominant peptides the response to which seems to be preferentially of the IgG2 type.