In this paper, comparative molecular studies between authentic Saccharomyces cerevisiae strains, related species, and the strain described as Saccharomyces boulardii were performed. The response of a S. boulardii strain and a S. cerevisiae strain (W303) to different stress conditions was also evaluated. The results obtained in this study show that S. boulardii is genetically very close or nearly identical to S. cerevisiae. Metabolically and physiologically, however, it shows a very different behavior, particularly in relation to growth yield and resistance to temperature and acidic stresses, which are important characteristics for a microorganism to be used as a probiotic.

BackgroundThe protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models.Methodology/Principal FindingsWe followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites.Conclusions/SignificanceOur results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector–human and vector–parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.

Leishmaniasis is a parasitic disease with a variety of clinical forms, which are related to the Leishmania species involved. In the murine model, Leishmania amazonensis causes chronic non-healing lesions in Leishmania braziliensis- or Leishmania major-resistant mouse strains. In this study, we investigated the involvement of the pathway of extracellular nucleotide hydrolysis, with special focus on the role of extracellular adenosine, in the establishment of Leishmania infection. Our results show that the more virulent parasite--L. amazonensis--hydrolyzes higher amounts of ATP, ADP and AMP than the two other species, probably due to the higher expression of membrane NTPDase. Corroborating the idea that increased production of adenosine is important to lesion development and establishment of tissue parasitism, we observed that increased 5'-nucleotidase activity in L. braziliensis or addition of adenosine at the moment of infection with this parasite resulted in an increase in lesion size and parasitism as well as a delay in lesion healing. Furthermore, inhibition of adenosine receptor A2B led to decreased lesion size and parasitism. Thus, our results suggest that the conversion of ATP, a molecule with pro-inflammatory activity, into adenosine, which possesses immunomodulatory properties, may contribute to the establishment of infection by Leishmania.

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Previous results from our laboratory and from the literature have implicated the expression of ecto-nucleotidases in the establishment of Leishmania infection. In the present study we evaluated the correlation between ecto-nucleotidasic activity and the infectivity of L. amazonensis promastigotes that were kept in culture for short or extended numbers of passages, a condition that is known to decrease parasite infectivity. We also analyzed the immune response associated with the infection by these parasites. As expected, we found that long-term cultured parasites induce the development of smaller lesions than the short-term cultured counterparts. Interestingly, long-term cultured parasites presented reduced ecto-nucleotidasic activity. In addition, cells recovered from animals infected with long-term cultured parasites produced higher amounts of IFN-gamma and have smaller parasite load, after 8weeks of infection. Furthermore, after 1week of infection, there is increased expression of the chemokine CCL2 mRNA in animals infected with short-term cultured parasites. Finally, infection of peritoneal macrophages by these parasites also shows marked differences. Thus, while short-term cultured parasites are able to infect a greater proportion of macrophages, cells infected by long-term cultured parasites express higher amounts of CXCL10 mRNA, which may activate these cells to kill the parasites. We suggest that the enzymes involved in metabolism of extracellular nucleotides may have an important role in infection by L. amazonensis, by acting directly in its adhesion to target cells and by modulating host cell chemokine production.

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.

In the present study, C57BL/6 mice were inoculated with metacyclic Leishmania amazonensis or L. braziliensis promastigotes. While these animals were capable of controlling the infection by L. braziliensis, they developed chronic lesions with elevated numbers of parasites when infected by L. amazonensis. The differences in parasite control were associated with a decreased production of IFN-gamma and TNF by lymph node cells from L. amazonensis-infected mice. Furthermore, these animals presented decreased spleen cell proliferation and activation of germinal centers. In addition, we compared the ability of these parasites to hydrolyze extracellular ATP and AMP. While the ATPase activity of both parasite species was similar, L. amazonensis promastigotes presented higher AMP hydrolytic activity. This increased activity may lead to an increased production of adenosine, which has been shown to present anti-inflammatory activity and may thus be involved in the establishment of the immunosuppression observed in mice infected by L. amazonensis.