RPA-1 from Leishmania amazonensis (LaRPA-1) structurally differs from other eukaryote RPA-1 and interacts with telomeric DNA via its N-terminal OB-fold domain.

Replication protein A-1 (RPA-1) is a single-stranded DNA-binding protein involved in DNA metabolism. We previously demonstrated the interaction between LaRPA-1 and telomeric DNA. Here, we expressed and purified truncated mutants of LaRPA-1 and used circular dichroism measurements and molecular dynamics simulations to demonstrate that the tertiary structure of LaRPA-1 differs from human and yeast RPA-1. LaRPA-1 interacts with telomeric ssDNA via its N-terminal OB-fold domain, whereas RPA from higher eukaryotes show different binding modes to ssDNA. Our results show that LaRPA-1 is evolutionary distinct from other RPA-1 proteins and can potentially be used for targeting trypanosomatid telomeres.

Sir2-Related Protein 1 from Leishmania amazonensis is a glycosylated NAD+-dependent deacetylase.

Sirtuin proteins form a family of NAD+-dependent protein deacetylases that are considered potential drug targets against parasites. Here, we present the first characterization of a sirtuin orthologue from Leishmania amazonensis, an aetiological agent of American tegumentary leishmaniasis that has been the subject of many studies focused in the development of therapeutic approaches. The protein has high sequence identity with other Kinetoplastid Silent information regulator 2 Related Protein 1 (Sir2RP1) and was named LaSir2RP1. The gene exists as a single copy, encoding a monomeric protein (LaSir2RP1) of approximately 41 kDa that has NAD+-dependent deacetylase activity. LaSir2RP1 was immunodetected in total protein extracts, in cytoplasmic granules, and in the secreted material of both promastigotes and lesion-derived amastigotes. Analysis of both lectin­affinity purified promastigote and amastigote extracts revealed the presence of a major enriched protein of approximately 66 kDa that was recognized by an anti-LaSir2RP1 serum, suggesting that a parasite sirtuin could be glycosylated in vivo.

Leishmania amazonensis: partial purification and study of the biochemical properties of the telomerase reverse transcriptase activity from promastigote-stage.

Telomeres are protein-DNA complexes that protect chromosome ends from degradation and fusion. In Leishmania spp., telomeric DNA comprises a conserved TTAGGG repeat and is maintained by telomerase. Telomerase is a multisubunit enzymatic complex that ensures the complete DNA replication by adding new telomeric repeats to the G-rich strand. In this report we aimed to purify and study the biochemical properties of Leishmania amazonensis telomerase. In a first trial we used affinity chromatography with antisense 2'-O-methyl oligonucleotide without success since the Leishmania telomerase, similarly to Trypanosoma cruzi enzyme, was not eluted by competition, but instead, it remained bound to the column. Partially purified L. amazonensis telomerase activity was achieved by fractionation of extracts on complementary ion exchange and Heparin columns. Further purification of these fractions on a G-rich telomeric DNA affinity chromatography enriched for telomerase activity. The knowledge of telomerase characteristics in Leishmania could help to develop new strategies to overcome leishmaniasis.

DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis.

Replication protein A (RPA) is a single-stranded DNA-binding protein that has been implicated in DNA metabolism and telomere maintenance. Subunit 1 of RPA from Leishmania amazonensis (LaRPA-1) has previously been affinity-purified on a column containing a G-rich telomeric DNA. LaRPA-1 binds and co-localizes with parasite telomeres in vivo. Here we describe the purification and characterization of native recombinant LaRPA-1 (rLaRPA-1). The protein was initially re-solubilized from inclusion bodies by using urea. After dialysis, rLaRPA-1 was soluble but contaminated with DNA, which was removed by an anion-exchange chromatography of the protein solubilized in urea. However, rLaRPA-1 precipitated after dialysis to remove urea. To investigate whether the contaminating DNA was involved in chaperoning the refolding of rLaRPA-1, salmon sperm DNA or heparin was added to the solution before dialysis. The addition of either of these substances prevented the precipitation of rLaRPA-1. The resulting rLaRPA-1 was soluble, correctly folded, and able to bind telomeric DNA. This is the first report showing the characterization of rLaRPA1 and of the importance of additives in chaperoning the refolding of this protein. The availability of rLaRPA-1 should be helpful in assessing the importance of this protein as a potential drug target.

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA.

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres.

LaRbp38: a Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs.

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.

Genomic organization of telomeric and subtelomeric sequences of Leishmania (Leishmania) amazonensis.

Telomeres are DNA-protein complexes that protect linear chromosomes from degradation and fusions. Telomeric DNA is repetitive and G-rich, and protrudes towards the end of the chromosomes as 3'G-overhangs. In Leishmania spp., sequences adjacent to telomeres comprise the Leishmania conserved telomere associated sequences (LCTAS) that are around 100 bp long and contain two conserved sequence elements (CSB1 and CSB2), in addition to non-conserved sequences. The aim of this work was to study the genomic organization of Leishmania (Leishmania) amazonensis telomeric/subtelomeric sequences. Leishmania amazonensis chromosomes were separated in a single Pulsed Field Gel Electrophoresis (PFGE) gel as 25 ethidium bromide-stained bands. All of the bands hybridized with the telomeric probe (5'-TTAGGG-3')3 and with probes generated from the conserved subtelomeric elements (CSB1, CSB2). Terminal restriction fragments (TRF) of L. amazonensis chromosomes were analyzed by hybridizing restriction digested genomic DNA and chromosomal DNA separated in 2D-PFGE with the telomeric probe. The L. amazonensis TRF was estimated to be approximately 3.3 kb long and the telomeres were polymorphic and ranged in size from 0.2 to 1.0 kb. Afa I restriction sites within the conserved CSB1 elements released the telomeres from the rest of the chromosome. Bal 31-sensitive analysis confirmed the presence of terminal Afa I restriction sites and served to differentiate telomeric fragments from interstitial internal sequences. The size of the L. amazonensis 3' G-overhang was estimated by non-denaturing Southern blotting to be approximately 12 nt long. Using similar approaches, the subtelomeric domains CSB1 and CSB2 were found to be present in a low copy number compared to telomeres and were organized in blocks of 0.3-1.5 kb flanked by Hinf I and Hae III restriction sites. A model for the organization of L. amazonensis chromosomal ends is provided.

The sensitivity, specificity and efficiency values of some serological tests used in the diagnosis of paracoccidioidomycosis

O presente trabalho avalia a sensibilidade, especificidade e eficiencia da imunodifusao dupla (ID), contraimunoeletroforese (CIE), reacao de fixacao de complemento (FC) e imunofluorescencia indireta (IFI) no diagnostico da paracoccidioidomicose. Os pacientes portadores da micose, virgens de tratamento, tiveram o diagnostico confirmado por exame micologico e/ou histopatologico. Utilizou-se como antigenos o filtrado de cultura da fase leveduriforme do Paracoccidioides brasiliensis para os testes de ID, CIE, FC e suspensao de celulas leveduriformes de "pool" de cepas do mesmo fungo para o teste de IFI. O estudo foi realizado em 4 grupos de individuos: 46 com paracoccidioidomicose ativa (sem tratamento), 22 com outras micoses profundas, 30 com outras doencas infecciosas (tuberculose e leishmaniose tegumentar) e 47 controles normais. Os valores de sensibilidade, especificidade e eficiencia foram obtidos de acordo com a metodologia utilizada por GALEN & GAMBINO (1975). Os resultados revelaram que os testes de precipitacao em gel de agar e agarose, representados pela ID e CIE foram os melhores, apresentando maior sensibilidade (91,3 por cento e 95,6 por cento, respectivamente), maxima especificidade (100 por cento) e os maiores valores de eficiencia quando comparados a FC e IFI...

Utilização de aminoácidos no estudo do crescimento do Paracoccidioides brasiliensis: influencia sobre o dimorfismo / Utilization of aminoacids in the study of the growth of Paracoccidioides brasiliensis. Influence on dimorphism

Utilizamos 15 amostras de Pacoccidioides brasiliensis nas formas miceliana (M) e leveduriforme (L), cultivadas em meio minimo (MM) e adaptadas ao mesmo meio suplementado com a solucao de aminoacidos (MMS). Para a realizacao do estudo auxologico das amostras, foram preparadas solucoes complementares das quais foram retirados um aminoacido de cada vez. Nove amostras foram prototroficas nas formas M e/ou L e as demais auxotroficas para os diferentes aminoacidos e bases nitrogenadas. A heterogeneidade dos resultados apresentados nao permitiu a caracterizacao auxologica das 15 amostras de P. brasiliensis estudadas. Nenhum dos compostos nitrogenados demonstrou ser essencial para o crescimento ou para a manuntencao da morfogenese do fungo. Alteracoes morfologicas (macro e microscopicas) tambem foram observadas, mas somente entre as amostras prototroficas, sugerindo a ativacao de um mecanismo de adaptacao desenvolvido pelo fungo mediante a ausencia de substratos nitrogenados no meio de cultura (MM).