The 43,000-Da glycoprotein (gp43) of Paracoccidioides brasiliensis is an immunodominant antigen for antibody-dependent and immune cellular responses in patients with paracoccidioidomycosis. In order to identify the peptide epitopes involved in the immunological reactivities of the gp43 and to obtain highly specific recombinant molecules for diagnosis of the infection, genomic and cDNA clones representing the entire coding region of the antigen were sequenced. The gp43 open reading frame was found in a 1,329-base pair fragment with 2 exons interrupted by an intron of 78 nucleotides. The gene is present in very few copies per genome, as indicated by Southern blotting and chromosomal megarestriction analysis. A single transcript of 1.5 kilobase pairs was verified in the yeast phase. The gene encodes a polypeptide of 416 amino acids (Mr 45,947) with a leader peptide of 35 residues; the mature protein has a single N-glycosylation site. The deduced amino acid sequence showed similarities of 56-58% with exo-1,3- beta-D-glucanases from Saccharomyces cerevisiae and Candida albicans. However, the gp43 is devoid of hydrolase activity and does not cross-react immunologically with the fungal glucanases. Internal and COOH-terminal gene fragments of the gp43 were expressed as recombinant fusion proteins, which reacted with antibodies elicited against the native antigen.

c Acetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD ؉ -dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation of Trypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion by T. cruzi at concentrations that did not affect host cell viability. In addition, in vivo infection was partially controlled upon administration of salermide. There are two sirtuins in T. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed in Escherichia coli with a 50% inhibitory concentration (IC 50 ) ؎ standard error of 1 ؎ 0.5 M. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

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We have identified telomerase activity in extracts of three evolutionarily diverse kinetoplastid species: Trypanosoma brucei, Leishmania major, and Leishmania tarentolae. Telomerase activity was initially detected in extracts from insect form cells of all three kinetoplastid species by using a modification of the one-tube telomere repeat amplification protocol [Kim, N., et al. The activity in T. brucei extracts was sufficiently robust to enable its detection in a direct assay of telomerase; enzyme processivity was found to be relatively low. The in vitro properties of telomerase suggest a possible templating domain sequence for the telomerase RNA of T. brucei. Telomerase activity is likely to contribute to telomere maintenance in these parasitic organisms and provides a new target for chemotherapeutic intervention.Chromosomal termini, or telomeres, in most eukaryotes consist of DNA-protein complexes that are essential for genomic integrity and cell viability. Part of the telomeric DNA that is lost during each round of cell replication is replaced primarily by the action of a ribonucleoprotein enzyme complex telomerase (1). Telomerase activity, first described in the ciliated protozoan Tetrahymena thermophila (1), is widely distributed among phylogenetically diverse eukaryotes including yeasts, amphibians, mammals, and plants, and was recently reported in the pathogenic malaria parasite Plasmodium falciparum (2-8). Telomerase adds dNTPs to the 3Ј end of the G-rich strand of chromosomal DNA by reverse transcription of a telomeric sequence template within the telomerase RNA subunit. Mutations in the template region of the telomerase RNA gene cause progressive shortening of the telomeres and cell senescence in T. thermophila (9). In mice, knock-out of telomerase RNA leads to increased telomere shortening, chromosomal aberrations, and sterility (10). The telomerase RNA subunit is generally difficult to identify because there is only limited sequence conservation at the level of secondary structure even between related species (ref. 11, and reviewed in ref. 12), necessitating more traditional biochemical approaches for its identification.The cloning and characterization of the protein catalytic subunit (telomerase reverse transcriptase, TERT) of Euplotes aediculatus and Saccharomyces cerevisiae telomerases by Lingner et al. (13) made possible the identification of telomerase reverse transcriptase homologues in Schizosaccharomyces pombe and humans (14) and the ciliates T. thermophila and Oxytricha trifallax (15,16). The amino acid sequences of these homologues indicate that the telomerase protein subunit is a specialized reverse transcriptase, evolutionarily most related to reverse transcriptases in the non-long terminal repeat retrotransposon family (ref.