Computational analysis of the interaction between transcription factors and the predicted secreted proteome of the yeast Kluyveromyces lactis

<p>Abstract</p> <p>Background</p> <p>Protein secretion is a cell translocation process of major biological and technological significance. The secretion and downstream processing of proteins by recombinant cells is of great commercial interest. The yeast <it>Kluyveromyces lactis </it>is considered a promising host for heterologous protein production. Because yeasts naturally do not secrete as many proteins as filamentous fungi, they can produce secreted recombinant proteins with few contaminants in the medium. An ideal system to address the secretion of a desired protein could be exploited among the native proteins in certain physiological conditions. By applying algorithms to the completed <it>K. lactis </it>genome sequence, such a system could be selected. To this end, we predicted protein subcellular locations and correlated the resulting extracellular secretome with the transcription factors that modulate the cellular response to a particular environmental stimulus.</p> <p>Results</p> <p>To explore the potential <it>Kluyveromyces lactis </it>extracellular secretome, four computational prediction algorithms were applied to 5076 predicted <it>K. lactis </it>proteins from the genome database. SignalP v3 identified 418 proteins with N-terminal signal peptides. From these 418 proteins, the Phobius algorithm predicted that 176 proteins have no transmembrane domains, and the big-PI Predictor identified 150 proteins as having no glycosylphosphatidylinositol (GPI) modification sites. WoLF PSORT predicted that the <it>K. lactis </it>secretome consists of 109 putative proteins, excluding subcellular targeting. The transcription regulators of the putative extracellular proteins were investigated by searching for DNA binding sites in their putative promoters. The conditions to favor expression were obtained by searching Gene Ontology terms and using graph theory.</p> <p>Conclusion</p> <p>A public database of <it>K. lactis </it>secreted proteins and their transcription factors are presented. It consists of 109 ORFs and 23 transcription factors. A graph created from this database shows 134 nodes and 884 edges, suggesting a vast number of relationships to be validated experimentally. Most of the transcription factors are related to responses to stress such as drug, acid and heat resistance, as well as nitrogen limitation, and may be useful for inducing maximal expression of potential extracellular proteins.</p

Tripping over emerging pathogens around the world: A phylogeographical approach for determining the epidemiology of Porcine circovirus-2 (PCV-2), considering global trading

AbstractPorcine circovirus-2 (PCV-2) is an emerging virus associated with a number of different syndromes in pigs known as Porcine Circovirus Associated Diseases (PCVAD). Since its identification and characterization in the early 1990s, PCV-2 has achieved a worldwide distribution, becoming endemic in most pig-producing countries, and is currently considered as the main cause of losses on pig farms. In this study, we analyzed the main routes of the spread of PCV-2 between pig-producing countries using phylogenetic and phylogeographical approaches. A search for PCV-2 genome sequences in GenBank was performed, and the 420 PCV-2 sequences obtained were grouped into haplotypes (group of sequences that showed 100% identity), based on the infinite sites model of genome evolution. A phylogenetic hypothesis was inferred by Bayesian Inference for the classification of viral strains and a haplotype network was constructed by Median Joining to predict the geographical distribution of and genealogical relationships between haplotypes. In order to establish an epidemiological and economic context in these analyses, we considered all information about PCV-2 sequences available in GenBank, including papers published on viral isolation, and live pig trading statistics available on the UN Comtrade database (http://comtrade.un.org/). In these analyses, we identified a strong correlation between the means of PCV-2 dispersal predicted by the haplotype network and the statistics on the international trading of live pigs. This correlation provides a new perspective on the epidemiology of PCV-2, highlighting the importance of the movement of animals around the world in the emergence of new pathogens, and showing the need for effective sanitary barriers when trading live animals

Protective effect of ions against cell death induced by acid stress in Saccharomyces

Saccharomyces boulardii is a probiotic used to prevent or treat antibiotic-induced gastrointestinal disorders and acute enteritis. For probiotics to be effective they must first be able to survive the harsh gastrointestinal environment. In this work, we show that S. boulardii displayed the greatest tolerance to simulated gastric environments compared with several Saccharomyces cerevisiae strains tested. Under these conditions, a pH 2.0 was the main factor responsible for decreased cell viability. Importantly, the addition of low concentrations of sodium chloride (NaCl) protected cells in acidic conditions more effectively than other salts. In the absence of S. boulardii mutants, the protective effects of Na 1 in yeast viability in acidic conditions was tested using S. cerevisiae Na 1 -ATPases (ena1-4), Na 1 /H 1 antiporter (nha1D) and Na 1 /H 1 antiporter prevacuolar (nhx1D) null mutants, respectively. Moreover, we provide evidence suggesting that this protection is determined by the plasma membrane potential, once altered by low pH and low NaCl concentrations. Additionally, the absence or low expression/activity of Ena proteins seems to be closely related to the basal membrane potential of the cells

Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, an endemic zoonosis present in some countries of South and Central Americas. The World Health Organization estimates that 100 million people are at risk of acquiring this disease. The infection affects mainly muscle tissues in the heart and digestive tract. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed, which makes a strong case for the development of new drugs to treat the disease. In this work we evaluate a family of proteins called Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) as new chemotherapy target to block T. cruzi infection in mammalian cells and in mice. We have used inhibitors and antibodies against this protein and demonstrated that T. cruzi Ecto-NTPDases act as facilitators of infection in mammalian cells and virulence factors in mice model. Two of the drugs used in this study (Suramin and Gadolinium) are currently used for other diseases in humans, supporting the possibility of their use in the treatment of Chagas disease

The Genome of Anopheles darlingi, the main neotropical Malaria vector

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 a