Leishmania infection in bats from a non-endemic region of Leishmaniasis in Brazil.

Leishmaniasis is a complex of zoonotic diseases caused by parasites of the genus Leishmania, which can develop in domestic as well as wild animals and humans throughout the world. Currently, this disease is spreading in rural and urban areas of non-endemic regions in Brazil. Recently, bats have gained epidemiological significance in leishmaniasis due to its close relationship with human settlements. In this study, we investigated the presence of Leishmania spp. DNA in blood samples from 448 bats belonging to four families representing 20 species that were captured in the Triangulo Mineiro and Alto Paranaiba areas of Minas Gerais State (non-endemic areas for leishmaniasis), Brazil. Leishmania spp. DNA was detected in 8·0% of the blood samples, 41·6% of which were Leishmania infantum, 38·9% Leishmania amazonensis and 19·4% Leishmania braziliensis. No positive correlation was found between Leishmania spp. and bat food source. The species with more infection rates were the insectivorous bats Eumops perotis; 22·2% (4/18) of which tested positive for Leishmania DNA. The presence of Leishmania in the bat blood samples, as observed in this study, represents epidemiological importance due to the absence of Leishmaniasis cases in the region.

Leishmania major phosphoglycerate kinase transcript and protein stability contributes to differences in isoform expression levels.

Leishmania contains two phosphoglycerate kinase (PGK) genes, PGKB and PGKC, which code for the cytosolic and glycosomal isoforms of the enzyme, respectively. Although differences in PGKB and PGKC transcript and protein levels and isoform activities have been well documented, the mechanisms of control of both transcript and protein abundance have not been described to date. To better understand the regulation of Leishmania PGK expression, we investigated the stabilities of both PGK transcripts using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) in combination with transcription and trans-splicing inhibitors. Cells were treated with sinefungin and actinomycin D, and RNA decay kinetics were assessed. In addition, immunoblotting and protein synthesis inhibition by cycloheximide were employed to evaluate protein steady states and degradation. We observed increased stabilities of both PGKB mRNA and protein compared with the glycosomal isoform (PGKC). Our results indicate that both post-transcriptional and post-translational events contribute to the distinct expression levels of the PGKB and PGKC isoforms in Leishmania major.

Gene identification and comparative molecular modeling of a Trypanosoma rangeli major surface protease.

Trypanosoma rangeli is a hemoflagellate parasite which is able to infect humans. Distinct from Trypanosoma cruzi, the causative agent of Chagas disease, T. rangeli is non-pathogenic to the vertebrate host. The manner by which the T. rangeli interacts with the host is still unknown, but it certainly depends on the surface molecules. Major surface proteins (MSP) are GPI-anchored, zinc-dependent metalloproteases present in the surface of all trypanosomatids studied so far, which are implicated as virulence factors in pathogenic trypanosomatids, such as Leishmania spp and T. cruzi. The aims of this work were to generate the complete sequence of a T. rangeli MSP (TrMSP) gene and to determine the 3D-structure of the predicted protein by homology modeling. The plasmid bearing a complete copy of a TrMSP gene was completely sequenced and the predicted protein was modeled using Modeller software. Results indicate that TrMSP open reading frame (ORF) codes for a predicted 588 amino acid protein and shows all elements required for its posttranslational processing. Multiple sequence alignment of TrMSP with other trypanosomatids' MSPs showed an extensive conservation of the N-terminal and central regions and a more divergent C-terminal region. Leishmania major MSP (LmMSP), which had its crystal structure previously determined, has an overall 35% identity with TrMSP. This identity allowed the comparative molecular modeling of TrMSP, which demonstrated a high degree of structural conservation between MSPs from other trypanosomatids (TrypMSPs). All modeled MSPs have a conserved folding pattern, apart from structural divergences in the C-domain and discrete differences of charge and topology in the catalytic cleft, and present the same geometry of the canonical HEXXH zinc-binding motif. The determination of surface charges of the molecules revealed that TrMSP is a predominantly positive protein, whereas LmMSP and Trypanosoma cruzi MSP (TcMSP) are negative proteins, suggesting that substrates recognized by TcMSP and LmMSP could not interact with TrMSP. Moreover, the comparison between TrMSP and TcMSP protein sequences has revealed 45 non-neutral amino acid substitutions, which can be further assessed through protein engineering. The characteristics of TrMSP could explain, at least in part, the lack of pathogenicity of T. rangeli to humans and point to the necessity of identifying the biological targets of this enzyme.

Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains.

BACKGROUND: Corynebacterium pseudotuberculosis, a gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. METHODOLOGY AND FINDINGS: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. CONCLUSIONS: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829.

Polymorphisms in the 18S rDNA gene of Cystoisospora belli and clinical features of cystoisosporosis in HIV-infected patients.

Intraspecific variability among Cystoisospora belli isolates and its clinical implications in human cystoisosporosis have not been established. In this study, the restriction fragment length polymorphisms in a 1.8-kb amplicon of the small subunit ribosomal DNA (SSU rDNA) of the parasite was investigated in 20 C. belli-positive stool samples obtained from 15 HIV-infected patients. Diarrheic syndrome was observed in all patients with cystoisosporosis and the number of diarrheic episodes per patient during hospitalization ranged from 1 to 26 (mean of 9.64 ± 9.30), with a mean duration of 2 to 12 days (mean of 5.90 ± 3 days). Three restriction profiles (RF) were generated with MboII digestion, which were named RFI, RFII, and RFIII. Two isolates obtained from a patient with extraintestinal cystoisosporosis showed distinct restriction profiles with MboII. This study demonstrates that patients can be infected with different C. belli genotypes, and this information may be useful for identifying new C. belli genotypes infecting humans.

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.

Human urine stimulates in vitro growth of Trypanosoma cruzi and Trypanosoma rangeli.

Previous studies conducted in Leishmania led us to test the hypothesis that addition of human urine (HU) to the Liver Infusion Tryptose (LIT) medium would stimulate the in vitro growth of Trypanosoma cruzi and Trypanosoma rangeli strains. Herein, we show that the addition of 3% HU to LIT medium (LIT-HU3) significantly stimulated the growth of all the T. rangeli strains studied when compared with the parasite growth in conventional LIT medium (p<0.05), and it was equivalent to the growth observed in LIT supplemented with fetal calf serum (FCS) in two parasite strains. Four out of the six T. cruzi strains analyzed showed a significant increase in parasite multiplication in LIT-HU3 (p<0.05). However, two parasite strains presented good growth in both LIT and LIT-HU, suggesting differences in the parasite's ability to grow in vitro. Furthermore, we have not observed differences in T. cruzi growth in LIT-HU3 and LIT supplemented with heat-denatured HU and in the metacyclogenesis of parasite strains cultured in LIT-HU3. These results allow concluding that the addition of HU to LIT medium stimulates the in vitro growth of T. rangeli and T. cruzi and can replace FCS as a supplement in culture medium.

Characterization of LST-R533: uncovering a novel repetitive element in Leishmania.

We have previously isolated and sequenced a novel repetitive element, now named LST-R533, which is present in four different regions of one extremity of Leishmania major chromosome 20. The repeats are polymorphic in size, ranging from 367 to 533 bp and contain an internal 81 bp sequence with highly conserved segments (14-81 bp long) dispersed throughout the parasite's genome. These sequences were not found in coding regions of any predicted gene in L. major Friedlin genome, but are part of untranslated regions of some Leishmania transcripts. Analysis of the 81 bp sequence revealed significant degrees of identity with retrotransposons described in several other organisms. The presence of the sequence in other species from genus Leishmania was determined by Southern hybridisation and DNA sequencing. This analysis indicated the conservation of the 81-nucleotide element in all the Leishmania species evaluated. No sequences corresponding to LST-R533 or the 81 bp element were found on either Trypanosoma brucei or Trypanosoma cruzi databanks.

The effect of location and direction of an episomal gene on the restoration of a phenotype by functional complementation in Leishmania.

The feasibility of genetic manipulation in trypanosomatids has allowed the introduction of molecular approaches for the investigation of gene function. The development of cosmids that carry approximately 40 kb of insert and can be easily introduced in trypanosomatids greatly increased the possibility of gene rescue by functional complementation in these parasites. Although functional complementation is widely used, some of its aspects, such as differential levels of expression along the insert clone, are not clear. We have used the DHFRTS gene as a tool to better understand the mechanisms of transcription of genes present in episomes and the results obtained via functional complementation in Leishmania. This gene was chosen not only because its inactivation in the parasite generates an easily recoverable phenotype, auxotrophy for thymidine (TdR), but also because null mutants are already available. The null mutant available contained two resistance markers (neomycin phosphotransferase-NEO and hygromycin phosphotransferase-HYG), and the loss of heterozygosity (LOH) was induced to recover clones sensitive to hygromycin B, which were necessary for the rescue of transfectants. Analyses of the Leishmania clones confirmed the loss of the HYG gene associated with unanticipated genomic rearrangements. A LOH clone was transfected with cosmids containing the DHFRTS gene in several distinct contexts in order to evaluate the levels of expression of the complementing gene. Results presented here show that the lost phenotype is rescued, irrespective to the DHFRTS location or direction of transcription indicating that functional complementation can be achieved without concern for the position of the complementing gene in a cosmid.