Schistocins: Novel antimicrobial peptides encrypted in the Schistosoma mansoni Kunitz Inhibitor SmKI-1.

BACKGROUND: Here we describe a new class of cryptides (peptides encrypted within a larger protein) with antimicrobial properties, named schistocins, derived from SmKI-1, a key protein in Shistosoma mansoni survival. This is a multi-functional protein with biotechnological potential usage as a therapeutic molecule in inflammatory diseases and to control schistosomiasis. METHODS: We used our algorithm enCrypted, to perform an in silico proteolysis of SmKI-1 and a screening for potential antimicrobial activity. The selected peptides were chemically synthesized, tested in vitro and evaluated by both structural (CD, NMR) and biophysical (ITC) studies to access their structure-function relationship. RESULTS: EnCrypted was capable of predicting AMPs in SmKI-1. Our biophysical analyses described a membrane-induced conformational change from random coil-to-α-helix and a peptide-membrane equilibrium for all schistocins. Our structural data allowed us to suggest a well-known mode of peptide-membrane interaction in which electrostatic attraction between the cationic peptides and anionic membranes results in the bilayer disordering. Moreover, the NMR H/D exchange data with the higher entropic contribution observed for the peptide-membrane interaction showed that schistocins have different orientations upon the membrane. CONCLUSIONS: This work demonstrate the robustness for using the physicochemical features of predicted peptides in the identification of new bioactive cryptides. Besides, it demonstrates the relevance of combining these analyses with biophysical methods to understand the peptide-membrane affinity and improve further algorithms. GENERAL SIGNIFICANCE: Bioprospecting cryptides can be conducted through data mining of protein databases demonstrating the success of our strategy. The peptides-based agents derived from SmKI-1 might have high impact for system-biology and biotechnology.

Reverse transcriptase droplet digital PCR to identify the emerging vesicular virus Senecavirus A in biological samples.

Senecavirus A (SVA) belonging to the family Picornaviridae, genus Senecavirus was incidentally isolated in 2002 from the PER.C6 (transformed foetal retinoblast) cell line. However, currently, this virus is associated with vesicular disease in swine and it has been reported in countries such as the United States of America, Canada, China, Thailand and Colombia. In Brazil, the SVA was firstly reported in 2015 in outbreaks of vesicular disease in swine, clinically indistinguishable of Foot-and-mouth disease, a contagious viral disease that generates substantial economic losses. In the present work, it was standardized a diagnostic tool for SVA based on RNA reverse transcriptase droplet digital PCR (RT-ddPCR) using one-step and two-step approaches. Analytical sensitivity and specificity were done in parallel with real-time PCR, RT-qPCR (one-step and two-step) for comparison of sensitivity and specificity of both methods. In the standardization of RT-ddPCR, the double-quenched probe and the temperature gradient were crucial to reduce background and improve amplitude between positive and negative droplets. The limit of detection and analytical specificity of techniques of one-step techniques showed superior performance than two-step methods described here. Additionally, the results showed 94.2% concordance (p < 0.001) for RT-ddPCR and RT-qPCR using the one-step assay approach and biological samples from Brazilian outbreaks of Senecavirus A. However, ddRT-PCR had a better performance than RT-PCR when swine serum pools were tested. According to the results, the one-step RT-ddPCR and RT-qPCR is highlighted to be used as an auxiliary diagnostic tool for Senecavirus A and for viral RNA absolute quantification in biological samples (RT-ddPCR), being a useful tool for vesicular diseases control programs.

Morphological reassessment and molecular phylogenetic analyses of Amauroderma s.lat. raised new perspectives in the generic classification of the Ganodermataceae family.

Ganodermataceae is a remarkable group of polypore fungi, mainly characterized by particular double-walled basidiospores with a coloured endosporium ornamented with columns or crests, and a hyaline smooth exosporium. In order to establish an integrative morphological and molecular phylogenetic approach to clarify relationship of Neotropical Amauroderma s.lat. within the Ganodermataceae family, morphological analyses, including scanning electron microscopy, as well as a molecular phylogenetic approach based on one (ITS) and four loci (ITS-5.8S, LSU, TEF-1α and RPB1), were carried out. Ultrastructural analyses raised up a new character for Ganodermataceae systematics, i.e., the presence of perforation in the exosporium with holes that are connected with hollow columns of the endosporium. This character is considered as a synapomorphy in Foraminispora, a new genus proposed here to accommodate Porothelium rugosum (≡ Amauroderma sprucei). Furtadoa is proposed to accommodate species with monomitic context: F. biseptata, F. brasiliensis and F. corneri. Molecular phylogenetic analyses confirm that both genera grouped as strongly supported distinct lineages out of the Amauroderma s.str. clade.

Virtual screening reveals a viral-like polymerase inhibitor that complexes with the DNA polymerase of Moniliophthora perniciosa.

The filamentous fungus Moniliophthora perniciosa is a basidiomycota that causes the witches' broom disease in cocoa trees (Theobroma cacao L.). The mitochondrial DNA polymerase of M. perniciosa (MpmitDNApol) is classified within the B family of DNA polymerases, which can be found in viruses and cellular organelles. Using virtual screening processes, accessing KEGG, PubChem, and ZINC databases, we selected the 27 best putative nucleoside viral-like polymerase inhibitors to test against MpmitDNApol. We used Autodock Vina to perform docking simulations of the selected molecules and to return energy values in several ligand conformations. Then, we used Pymol v1.7.4.4 to check the stereochemistry of chiral carbons, hydrogen bonding receptors, absence or presence of hydrogen, sub and superstructure, numbers of rings, rotatable bonds, and donor groups. We selected the Entecavir Hydrate, a drug used to control hepatitis B; subsequently AMBER 14 was used to describe the behavior of polymerase-entecavir complex after setting up 3500 ps of simulation in water at a temperature of 300 K. From the simulation, a graph of Potential Energy was generated revealing that the ligand remains in the catalytic site after 3500 ps with a final energy of -612,587.4214 kcal/mol.

Phylogenetic analysis of DNA and RNA polymerases from a Moniliophthora perniciosa mitochondrial plasmid reveals probable lateral gene transfer.

The filamentous fungus Moniliophthora perniciosa is a hemibiotrophic basidiomycete that causes witches' broom disease of cacao (Theobroma cacao L.). Many fungal mitochondrial plasmids are DNA and RNA polymerase-encoding invertrons with terminal inverted repeats and 5'-linked proteins. The aim of this study was to carry out comparative and phylogenetic analyses of DNA and RNA polymerases for all known linear mitochondrial plasmids in fungi. We performed these analyses at both gene and protein levels and assessed differences between fungal and viral polymerases in order to test the lateral gene transfer (LGT) hypothesis. We analyzed all mitochondrial plasmids of the invertron type within the fungal clade, including five from Ascomycota, seven from Basidiomycota, and one from Chytridiomycota. All phylogenetic analyses generated similar tree topologies regardless of the methods and datasets used. It is likely that DNA and RNA polymerase genes were inserted into the mitochondrial genomes of the 13 fungal species examined in our study as a result of different LGT events. These findings are important for a better understanding of the evolutionary relationships between fungal mitochondrial plasmids.

DNA and RNA polymerase activity in a Moniliophthora perniciosa mitochondrial plasmid and self-defense against oxidative stress.

Moniliophthora perniciosa (Stahel) Aime and Phillips-Mora is a hemibiotrophic basidiomycete (Agaricales, Tricholomataceae) that causes witches' broom disease in cocoa (Theobroma cacao L.). This pathogen carries a stable integrated invertron-type linear plasmid in its mitochondrial genome that encodes viral-like DNA and RNA polymerases related to fungal senescence and longevity. After culturing the fungus and obtaining its various stages of development in triplicate, we carried out total RNA extraction and subsequent complementary DNA synthesis. To analyze DNA and RNA polymerase expression levels, we performed real-time reverse transcriptase polymerase chain reaction for various fungal phases of development. Our results showed that DNA and RNA polymerase gene expression in the primordium phase of M. perniciosa is related to a potential defense mechanism against T. cacao oxidative attack.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Multiclonal Leishmania braziliensis population structure and its clinical implication in a region of endemicity for American tegumentary leishmaniasis.

In Corte de Pedra (CP), northeastern Brazil, Leishmania braziliensis causes three distinct forms of American tegumentary leishmaniasis (ATL). To test the hypothesis that strain polymorphism may be involved in this disease spectrum and accurately characterize the parasite population structure in CP, we compared one L. major, two non-CP L. braziliensis, one CP L. amazonensis, and 45 CP L. braziliensis isolates, obtained over a 10-year period from localized cutaneous, mucosal, and disseminated leishmaniasis patients, with randomly amplified polymorphic DNA (RAPD). Electrophoretic profiles were mostly unique across species. All typing protocols revealed polymorphism among the 45 CP L. braziliensis isolates, which displayed eight different RAPD patterns and greater than 80% overall fingerprint identity, attesting to the adequacy of the tools to assess strain variability in CP's geographically limited population of parasites. The dendrogram based on the sum of RAPD profiles of each isolate unveiled nine discrete typing units clustered into five clades. Global positioning showed extensive overlap of these clades in CP, precluding geographic sequestration as the mechanism of the observed structuralization. Finally, all forms of ATL presented a statistically significant difference in their frequencies among the clades, suggesting that L. braziliensis genotypes may be accompanied by specific disease manifestation after infection.