Jeronimo C. Ruiz

Comparative genomic analysis of three Leishmania species that cause diverse human disease.

Leishmania parasites cause a broad spectrum of clinical disease. Here we report the sequencing of the genomes of two species of Leishmania: Leishmania infantum and Leishmania braziliensis. The comparison of these sequences with the published genome of Leishmania major reveals marked conservation of synteny and identifies only ∼200 genes with a differential distribution between the three species. L. braziliensis, contrary to Leishmania species examined so far, possesses components of a putative RNA-mediated interference pathway, telomere-associated transposable elements and spliced leader–associated SLACS retrotransposons. We show that pseudogene formation and gene loss are the principal forces shaping the different genomes. Genes that are differentially distributed between the species encode proteins implicated in host-pathogen interactions and parasite survival in the macrophage.

The Leishmania genome project: new insights into gene organization and function

Abstract. The sequencing of Leishmania major Friedlin chromosome 1 (Chr1), Chr3, and Chr4 has been completed, and several other chromosomes are well underway. The complete genome sequence should be available by 2003. Over 1,000 full-length new genes have been identified, with the majority (~75%) having unknown function. Many of these may be Leishmania (or kinetoplastid) specific. Most interestingly, the genes are organized into large (>100–500xa0kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a divergent manner, i.e., the mRNAs for the two sets of genes are both transcribed towards the telomeres. Nuclear run-on analysis suggests that transcription is initiated in both directions within the divergent region. Chr3 and Chr4 contain two convergent clusters, with a single divergent gene at one telomere of Chr3. Sequence analysis of several genes from the LD1 region of Chr35 indicates a high degree of sequence conservation between L. major and L. donovani/L. infantum within protein-coding open reading frames (ORFs), with a lower degree of conservation within the non-coding regions. Immunization of mice with recombinant antigen from two of these genes, BT1 (formerly ORFG) and ORFF, results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.

Subproteomic analysis of soluble proteins of the microsomal fraction from two Leishmania species

Parasites of the genus Leishmania are the causative agents of a range of clinical manifestations collectively known as Leishmaniasis, a disease that affects 12 million people worldwide. With the aim of identifying potential secreted protein targets for further characterization, we have applied two-dimensional gel electrophoresis and mass spectrometry methods to study the soluble protein content of the microsomal fraction from two Leishmania species, Leishmania L. major and L. L. amazonensis. MALDI-TOF peptide mass fingerprint analysis of 33 protein spots from L. L. amazonensis and 41 protein spots from L. L. major identified 14 proteins from each sample could be unambiguously assigned. These proteins include the nucleotide diphosphate kinase (NDKb), a calpain-like protease, a tryparedoxin peroxidase (TXNPx) and a small GTP-binding Rab1-protein, all of which have a potential functional involvement with secretion pathways and/or environmental responses of the parasite. These results complement ongoing genomic studies in Leishmania, and are relevant to further understanding of host/parasite interactions.

Organization of H locus conserved repeats in Leishmania (Viannia) braziliensis correlates with lack of gene amplification and drug resistance

Resistance to antimonials is a major problem when treating visceral leishmaniasis in India and has already been described for New World parasites. Clinical response to meglumine antimoniate in patients infected with parasites of the Viannia sub-genus can be widely variable, suggesting the presence of mechanisms of drug resistance. In this work, we have compared L. major and L. braziliensis mutants selected in different drugs. The cross-resistance profiles of some cell lines resembled those of mutants bearing H locus amplicons. However, amplified episomal molecules were exclusively detected in L. major mutants. The analysis of the L. braziliensis H region revealed a strong conservation of gene synteny. The typical intergenic repeats that are believed to mediate the amplification of the H locus in species of the Leishmania sub-genus are partially conserved in the Viannia species. The conservation of these non-coding elements in equivalent positions in both species is indicative of their relevance within this locus. The absence of amplicons in L. braziliensis suggests that this species may not favour extra-chromosomal gene amplification as a source of phenotypic heterogeneity and fitness maintenance in changing environments.

The Comparative Genomics and Phylogenomics of Leishmania amazonensis Parasite

Leishmaniasis is an infectious disease caused by Leishmania species. Leishmania amazonensis is a New World Leishmania species belonging to the Mexicana complex, which is able to cause all types of leishmaniasis infections. The L. amazonensis reference strain MHOM/BR/1973/M2269 was sequenced identifying 8,802 codifying sequences (CDS), most of them of hypothetical function. Comparative analysis using six Leishmania species showed a core set of 7,016 orthologs. L. amazonensis and Leishmania mexicana share the largest number of distinct orthologs, while Leishmania braziliensis presented the largest number of inparalogs. Additionally, phylogenomic analysis confirmed the taxonomic position for L. amazonensis within the “Mexicana complex”, reinforcing understanding of the split of New and Old World Leishmania. Potential non-homologous isofunctional enzymes (NISE) were identified between L. amazonensis and Homo sapiens that could provide new drug targets for development.

New Trypanosoma cruzi Repeated Element That Shows Site Specificity for Insertion

ABSTRACT A new family of site-specific repeated elements identified in Trypanosoma cruzi, which we named TcTREZO, is described here. TcTREZO appears to be a composite repeated element, since three subregions may be defined within it on the basis of sequence similarities with other T. cruzi sequences. Analysis of the distribution of TcTREZO in the genome clearly indicates that it displays site specificity for insertion. Most TcTREZO elements are flanked by conserved sequences. There is a highly conserved 68-bp sequence at the 5′ end of the element and a sequence domain of ∼500 bp without a well-defined borderline at the 3′ end. Northern blot hybridization and reverse transcriptase PCR analyses showed that TcTREZO transcripts are expressed as oligo(A)-terminated transcripts whose length corresponds to the unit size of the element (1.6 kb). Transcripts of ∼0.2 kb derived from a small part of TcTREZO are also detected in steady-state RNA. TcTREZO transcripts are unspliced and not translated. The copy number of TcTREZO sequences was estimated to be ∼173 copies per haploid genome. TcTREZO appears to have been assembled by insertions of sequences into a progenitor element. Once associated with each other, these subunits were amplified as a new transposable element. TcTREZO shows site specificity for insertion, suggesting that a sequence-specific endonuclease could be responsible for its insertion at a unique site.

Characterisation of three chromosomal ends of Leishmania major reveals transcriptional activity across arrays of reiterated and unique sequences

The 36 chromosomes of the parasite Leishmania major range in size from 200 kb to approximately 2.5 Mb and variation between homologues seems to be restricted to the telomeric and subtelomeric regions. We have isolated three cosmids carrying the telomere hexameric repeat and assigned them to the extreme location of chromosomes 3, 7 and 20. When considering the distribution of repetitive sequences, Southern analysis of the three chromosomal ends indicated the existence of at least two classes of chromosomal extremities: one of them is composed almost exclusively of unique sequences and the other is characterised by patches of both reiterated and unique sequences. We devised a transfection-based strategy that allowed the determination of a map of transcripts in each of the regions examined. Sequencing of the chromosome 20 cosmid revealed the existence of a novel class of reiterated sequence, LST-R378, and 10 ORFs drawing a map of putative genes compatible with the map of transcripts.

Genome Survey Sequence Analysis and Identification of Homologs of Major Surface Protease (gp63) Genes in Trypanosoma rangeli

In this study, 222 genome survey sequences were generated for Trypanosoma rangeli strain P07 isolated from an opossum (Didelphis albiventris) in Minas Gerais State, Brazil. T. rangeli sequences were compared by BLASTX (Basic Local Alignment Search Tool X) analysis with the assembled contigs of Leishmania braziliensis, Leishmania infantum, Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Results revealed that 82% (182/222) of the sequences were associated with predicted proteins described, whereas 18% (40/222) of the sequences did not show significant identity with sequences deposited in databases, suggesting that they may represent T. rangeli-specific sequences. Among the 182 predicted sequences, 179 (80.6%) had the highest similarity with T. cruzi, 2 (0.9%) with T. brucei, and 1 (0.5%) with L. braziliensis. Computer analysis permitted the identification of members of various gene families described for trypanosomatids in the genome of T. rangeli, such as trans-sialidases, mucin-associated surface proteins, and major surface proteases (MSP or gp63). This is the first report identifying sequences of the MSP family in T. rangeli. Multiple sequence alignments showed that the predicted MSP of T. rangeli presented the typical characteristics of metalloproteases, such as the presence of the HEXXH motif, which corresponds to a region previously associated with the catalytic site of the enzyme, and various cysteine and proline residues, which are conserved among MSPs of different trypanosomatid species. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of MSP transcripts in epimastigote forms of T. rangeli.

In silico identification of GPI-anchored proteins in Paracoccidioides

Glycosylphosphatidylinositol-anchored proteins (GPI-proteins) are widely found in eukaryotic organisms. In fungi, GPI-proteins are thought to be involved in diverse cellular mechanisms such as cell wall biosynthesis and cell wall remodeling, adhesion, antigenicity, and virulence. The conserved structural domains of GPI-protein allow the utilization of in silico prediction approach to identify this class of proteins using a genome-wide analysis. We used different previously characterized algorithms to search for genes that encode predicted GPI-proteins in the genome of P. brasiliensis and P. lutzii, thermal dimorphic fungi that causes paracoccidioidomycosis (PCM). By using these methods, 98 GPI-proteins were found in P. brasiliensis with orthologs in P. lutzii. A series of 28 GPI-proteins were classified in functional categories (such as glycoside hydrolases, chitin-processing proteins, and proteins involved in the biogenesis of the cell wall). Furthermore, 70 GPI-proteins exhibited homology with hypothetical conserved proteins of unknown function. These data will be an important resource for the future analysis of GPI-proteins in Paracoccidioides spp.