Leishmania infantum ecto-nucleoside triphosphate diphosphohydrolase-2 is an apyrase involved in macrophage infection and expressed in infected dogs.

BACKGROUND: Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum) is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa) have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection. METHODOLOGY/PRINCIPAL FINDINGS: We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP  =  UDP> ADP> UTP  =  ATP) in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis. CONCLUSIONS/SIGNIFICANCE: In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in naturally infected dogs. Additionally, the blockade of NTPDases led to lower levels of in vitro adhesion and infection, suggesting that these proteins are possible targets for rational drug design.

Predicting the proteins of Angomonas deanei, Strigomonas culicis and their respective endosymbionts reveals new aspects of the trypanosomatidae family.

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.

Molecular characterization of beta-tubulin from Phakopsora pachyrhizi, the causal agent of Asian soybean rust.

ß-tubulins are structural components of microtubules and the targets of benzimidazole fungicides used to control many diseases of agricultural importance. Intron polymorphisms in the intron-rich genes of these proteins have been used in phylogeographic investigations of phytopathogenic fungi. In this work, we sequenced 2764 nucleotides of the ß-tubulin gene (Pp tubB) in samples of Phakopsora pachyrhizi collected from seven soybean fields in Brazil. Pp tubB contained an open reading frame of 1341 nucleotides, including nine exons and eight introns. Exon length varied from 14 to 880 nucleotides, whereas intron length varied from 76 to 102 nucleotides. The presence of only four polymorphic sites limited the usefulness of Pp tubB for phylogeographic studies in P. pachyrhizi. The gene structures of Pp tubB and orthologous ß-tubulin genes of Melampsora lini and Uromyces viciae-fabae were highly conserved. The amino acid substitutions in ß-tubulin proteins associated with the onset of benzimidazole resistance in model organisms, especially at His (6) , Glu (198) and Phe (200) , were absent from the predicted sequence of the P. pachyrhizi ß-tubulin protein.

Molecular characterization of beta-tubulin from Phakopsora pachyrhizi, the causal agent of Asian soybean rust

β-tubulins are structural components of microtubules and the targets of benzimidazole fungicides used to control many diseases of agricultural importance. Intron polymorphisms in the intron-rich genes of these proteins have been used in phylogeographic investigations of phytopathogenic fungi. In this work, we sequenced 2764 nucleotides of the β-tubulin gene (Pp tubB) in samples of Phakopsora pachyrhizi collected from seven soybean fields in Brazil. Pp tubB contained an open reading frame of 1341 nucleotides, including nine exons and eight introns. Exon length varied from 14 to 880 nucleotides, whereas intron length varied from 76 to 102 nucleotides. The presence of only four polymorphic sites limited the usefulness of Pp tubB for phylogeographic studies in P. pachyrhizi. The gene structures of Pp tubB and orthologous β-tubulin genes of Melampsora lini and Uromyces viciae-fabae were highly conserved. The amino acid substitutions in β-tubulin proteins associated with the onset of benzimidazole resistance in model organisms, especially at His6, Glu198 and Phe200, were absent from the predicted sequence of the P. pachyrhizi β-tubulin protein.