Analysis of NGS data from Peruvian Loxosceles laeta spider venom gland reveals toxin diversity.

Accidents involving spiders from the genus Loxosceles cause medical emergencies in several countries of South America. The species Loxosceles laeta is ubiquitously present in Peru and is responsible for severe accidents in this country. To further characterize L. laeta venom components and to unveil possible variations in the Peruvian population, we provide an overview of the toxins-related transcripts present in the venom gland of Peruvian L. laeta. A dataset from a cDNA library previously sequenced by MiSeq sequencer (Illumina) was re-analyzed and the obtained data was compared with available sequences from Loxosceles toxins. Phospholipase-D represent the majority (69,28 %) of the transcripts related to venom toxins, followed by metalloproteases (20,72 %), sicaritoxins (6,03 %), serine-proteases (2,28 %), hyaluronidases (1,80 %) and Translationally Controlled Tumor Protein (TCTP) (0,56 %). New sequences of phospholipases D,sicaritoxins, hyaluronidase, TCTP and serine proteinases were described. Differences between the here-described toxin sequences and others, previously identified in venom glands from other spiders, were visualized upon sequence alignments. In addition, an in vitro hyaluronidase activity assay was also performed to complement comparisons between Peruvian and Brazilian L. laeta venom enzymatic activities, revealing a superior activity in the venom from Brazilian specimens. These new data provide a molecular basis that can help to explain the difference in toxicity among L. laeta venoms from different countries in South America.

An Integrative View of the Phyllosphere Mycobiome of Native Rubber Trees in the Brazilian Amazon.

The rubber tree, Hevea brasiliensis, is a neotropical Amazonian species. Despite its high economic value and fungi associated with native individuals, in its original area in Brazil, it has been scarcely investigated and only using culture-dependent methods. Herein, we integrated in silico approaches with novel field/experimental approaches and a case study of shotgun metagenomics and small RNA metatranscriptomics of an adult individual. Scientific literature, host fungus, and DNA databases are biased to fungal taxa, and are mainly related to rubber tree diseases and in non-native ecosystems. Metabarcoding retrieved specific phyllospheric core fungal communities of all individuals, adults, plantlets, and leaves of the same plant, unravelling hierarchical structured core mycobiomes. Basidiomycotan yeast-like fungi that display the potential to produce antifungal compounds and a complex of non-invasive ectophytic parasites (Sooty Blotch and Flyspeck fungi) co-occurred in all samples, encompassing the strictest core mycobiome. The case study of the same adult tree (previously studied using culture-dependent approach) analyzed by amplicon, shotgun metagenomics, and small RNA transcriptomics revealed a high relative abundance of insect parasite-pathogens, anaerobic fungi and a high expression of Trichoderma (a fungal genus long reported as dominant in healthy wild rubber trees), respectively. Altogether, our study unravels new and intriguing information/hypotheses of the foliar mycobiome of native H. brasiliensis, which may also occur in other native Amazonian trees.

Exploring antibiotic resistance in environmental integron-cassettes through intI-attC amplicons deep sequencing.

INTRODUCTION: Freshwater ecosystems provide propitious conditions for the acquisition and spread of antibiotic resistance genes (ARGs), and integrons play an important role in this process. MATERIAL AND METHODS: In the present study, the diversity of putative environmental integron-cassettes, as well as their potential bacterial hosts in the Velhas River (Brazil), was explored through intI-attC and 16S rRNA amplicons deep sequencing. RESULTS AND DISCUSSION: ORFs related to different biological processes were observed, from DNA integration to oxidation-reduction. ARGs-cassettes were mainly associated with class 1 mobile integrons carried by pathogenic Gammaproteobacteria, and possibly sedentary chromosomal integrons hosted by Proteobacteria and Actinobacteria. Two putative novel ARG-cassettes homologs to fosB3 and novA were detected. Regarding 16SrRNA gene analysis, taxonomic and functional profiles unveiled Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria as dominant phyla. Betaproteobacteria, Alphaproteobacteria, and Actinobacteria classes were the main contributors for KEGG orthologs associated with resistance. CONCLUSIONS: Overall, these results provide new information about environmental integrons as a source of resistance determinants outside clinical settings and the bacterial community in the Velhas River.

Complete mitochondrial genome sequence of the high altitude Brazilian treefrog Pithecopus megacephalus (Anura, Phyllomedusidae).

The first complete mitochondrial genome (mtDNA) for the family Phyllomedusidae (genus Pithecopus) is presented. It is a circular molecule with 17713 pb including 13 protein coding genes, 22 tRNA genes, two rRNA genes, and a control region (D-loop). Pithecopus megacephalus was close to the only other phyllomedusid whose complete mtDNA sequence is available, but had the cytb gene 147 pb smaller.

Mapping Alterations Induced by Long-Term Axenic Cultivation of Leishmania amazonensis Promastigotes With a Multiplatform Metabolomic Fingerprint Approach.

Leishmaniases are widespread neglected diseases with an incidence of 1.6 million new cases and 40 thousand deaths per year. Leishmania parasites may show distinct, species-specific patterns of virulence that lead to different clinical manifestations. It is well known that successive in vitro passages (SIVP) lead to the attenuation of virulence, but neither the metabolism nor the pathways involved in these processes are well understood. Herein, promastigotes of a virulent L. amazonensis strain recently isolated from mice was compared to SIVP derived and attenuated promastigotes, submitted to 10, 40, and 60 axenic passages and named R10, R40, and R60, respectively. In vitro assays and in vivo tests were performed to characterize and confirmed the attenuation profiles. A metabolomic fingerprint comparison of R0, R10, and R60 was performed by means of capillary electrophoresis, liquid and gas chromatography coupled to mass spectrometry. To validate the metabolomic data, qPCR for selected loci, flow cytometry to measure aPS exposure, sensitivity to antimony tartrate and ROS production assays were conducted. The 65 identified metabolites were clustered in biochemical categories and mapped in eight metabolic pathways: ABC transporters; fatty acid biosynthesis; glycine, serine and threonine metabolism; ß-alanine metabolism; glutathione metabolism; oxidative phosphorylation; glycerophospholipid metabolism and lysine degradation. The obtained metabolomic data correlated with previous proteomic findings of the SVIP parasites and the gene expression of 13 selected targets. Late SIVP cultures were more sensitive to SbIII produced more ROS and exposed less phosphatidylserine in their surface. The correspondent pathways were connected to build a biochemical map of the most significant alterations involved with the process of attenuation of L. amazonensis. Overall, the reported data pointed out to a very dynamic and continuous metabolic reprogramming process, accompanied by changes in energetic, lipid and redox metabolisms, membrane remodeling and reshaping of parasite-host cells interactions, causing impacts in chemotaxis, host inflammatory responses and infectivity at the early stages of infection.

Phenotypic characteristics and transcriptome profile of Cryptococcus gattii biofilm.

In this study, we characterized Cryptococcus gattii biofilm formation in vitro. There was an increase in the density of metabolically active sessile cells up to 72 h of biofilm formation on polystyrene and glass surfaces. Scanning electron microscopy and confocal laser scanning microscopy analysis revealed that in the early stage of biofilm formation, yeast cells adhered to the abiotic surface as a monolayer. After 12 h, extracellular fibrils were observed projecting from C. gattii cells, connecting the yeast cells to each other and to the abiotic surface; mature biofilm consisted of a dense network of cells deeply encased in an extracellular polymeric matrix. These features were also observed in biofilms formed on polyvinyl chloride and silicone catheter surfaces. We used RNA-Seq-based transcriptome analysis to identify changes in gene expression associated with C. gattii biofilm at 48 h compared to the free-floating planktonic cells. Differential expression analysis showed that 97 and 224 transcripts were up-regulated and down-regulated in biofilm, respectively. Among the biological processes, the highest enriched term showed that the transcripts were associated with cellular metabolic processes, macromolecule biosynthetic processes and translation.

CPP-Ts: a new intracellular calcium channel modulator and a promising tool for drug delivery in cancer cells.

Scorpion sting envenoming impacts millions of people worldwide, with cardiac effects being one of the main causes of death on victims. Here we describe the first Ca2+ channel toxin present in Tityus serrulatus (Ts) venom, a cell penetrating peptide (CPP) named CPP-Ts. We show that CPP-Ts increases intracellular Ca2+ release through the activation of nuclear InsP3R of cardiomyocytes, thereby causing an increase in the contraction frequency of these cells. Besides proposing a novel subfamily of Ca2+ active toxins, we investigated its potential use as a drug delivery system targeting cancer cell nucleus using CPP-Ts's nuclear-targeting property. To this end, we prepared a synthetic CPP-Ts sub peptide14-39 lacking pharmacological activity which was directed to the nucleus of specific cancer cell lines. This research identifies a novel subfamily of Ca2+ active toxins and provides new insights into biotechnological applications of animal venoms.

Insights into the Genome Sequence of Chromobacterium amazonense Isolated from a Tropical Freshwater Lake.

Members of the genus Chromobacterium have been isolated from geographically diverse ecosystems and exhibit considerable metabolic flexibility, as well as biotechnological and pathogenic properties in some species. This study reports the draft assembly and detailed sequence analysis of Chromobacterium amazonense strain 56AF. The de novo-assembled genome is 4,556,707 bp in size and contains 4294 protein-coding and 95 RNA genes, including 88 tRNA, six rRNA, and one tmRNA operon. A repertoire of genes implicated in virulence, for example, hemolysin, hemolytic enterotoxins, colicin V, lytic proteins, and Nudix hydrolases, is present. The genome also contains a collection of genes of biotechnological interest, including esterases, lipase, auxins, chitinases, phytoene synthase and phytoene desaturase, polyhydroxyalkanoates, violacein, plastocyanin/azurin, and detoxifying compounds. Importantly, unlike other Chromobacterium species, the 56AF genome contains genes for pore-forming toxin alpha-hemolysin, a type IV secretion system, among others. The analysis of the C. amazonense strain 56AF genome reveals the versatility, adaptability, and biotechnological potential of this bacterium. This study provides molecular information that may pave the way for further comparative genomics and functional studies involving Chromobacterium-related isolates and improves our understanding of the global genomic diversity of Chromobacterium species.

Immunization with a nontoxic naturally occurring Clostridium perfringens alpha toxin induces neutralizing antibodies in rabbits.

Clostridium perfringens alpha toxin, encoded by plc gene, has been implicated in gas gangrene, a life threatening infection. Vaccination is considered one of the best solutions against Clostridium infections. Although studies have identified many low quality clostridial vaccines, the use of recombinant proteins has been considered a promising alternative. Previously, a naturally occurring alpha toxin isoform (αAV1b) was identified with a mutation at residue 11 (His/Tyr), which can affect its enzymatic activity. The aim of the present study was to evaluate whether the mutation in the αAV1b isoform could result in an inactive toxin and was able to induce protection against the native alpha toxin. We used recombinant protein techniques to determine whether this mutation in αAV1b could result in an inactive toxin compared to the active isoform, αZ23. Rabbits were immunized with the recombinant toxins (αAV1b and αZ23) and with native alpha toxin. αAV1b showed no enzymatic and hemolytic activities. ELISA titration assays showed a high titer of both anti-recombinant toxin (anti-rec-αAV1b and anti-rec-αZ23) antibodies against the native alpha toxin. The alpha antitoxin titer detected in the rabbits' serum pool was 24.0 IU/mL for both recombinant toxins. These results demonstrate that the inactive naturally mutated αAV1b is able to induce an immune response, and suggest it can be considered as a target for the development of a commercial vaccine against C. perfringens alpha toxin.

Leporinus elongatus (Characiformes, Anostomidae): complete mtDNA sequence of an economically important fish from the Paraná and La Plata river basins.

Leporinus elongatus is an important commercial fish found in the La Plata and Paraná River basins. Next-generation sequencing was used to sequence the mitochondrial DNA (mtDNA) of L. elongatus. The mtDNA was assembled using the CLC Workbench software v. 9.0 and subsequently aligned to other 10 complete fish mitochondrial sequences to enable phylogenetic analysis using MEGA 7.0. The complete mtDNA molecule had 16,784 bp and its GC content was 43%. The mtDNA structure was similar to that of other vertebrates: two ribosomal RNA, 22 transfer RNA, 13 protein-coding genes, and a D-loop region containing 1115 bp. Phylogenetic analysis yielded a tree with high bootstrap value that was coherent with the current phylogeny proposed for Characiformes.

Complete mitochondrial genome sequence of the high-altitude Brazilian tree frog Bokermannohyla alvarengai (Anura, Hylidae).

The first complete mitochondrial genome (mtDNA) for the genus Bokermannohyla (Hylidae) is presented. The mtDNA contains 17,325 bp and is similar in size, gene content, and gene location to other hylid mitochondrial genomes described, with 2 rRNA, 22 tRNA, and 13 PCGs. The control region (D-loop) is shorter than in mtDNAs of hylids from Asia. A phylogenetic tree based on homologous genes did not corroborate the monophyly of Hylidae neither the recently proposed monophyly of Hyla and Dryophytes.

Complete mitochondrial genome of Sporophila maximiliani (Ave, Passeriformes).

Sporophila maximiliani, commonly known as Great-billed Seed-Finch or 'bicudo', is a trafficked bird in Brazil due to the species' beauty and singing, which is appreciated by breeders and collectors. Generally, the Environmental Military Police and IBAMA maintain enforcement actions, rescue work, and seizure of illegally traded of 'bicudo' specimens. The genomic DNA of one specimen was sequenced on MiSeq (Illumina) sequencer. The reads obtained were analyzed, trimmed, and de novo assembled using CLC Workbench® v9.0 (CLC Bio-Qiagen). The mitochondrial genome of S. maximiliani consisted of 16,765 base pairs, 2 ribosomal RNA, 22 transporter RNA, 13 protein-coding genes, and 1 control region. The molecular phylogeny demonstrated that the mitochondrial genome of S. maximiliani diverged from others related Passeriformes.

A MLST Clade 2 Clostridium difficile strain with a variant TcdB induces severe inflammatory and oxidative response associated with mucosal disruption.

The epidemiology of Clostridium difficile infections is highly dynamic as new strains continue to emerge worldwide. Here we present a detailed analysis of a new C. difficile strain (ICC-45) recovered from a cancer patient in Brazil that died from severe diarrhea. A polyphasic approach assigned a new PCR-ribotype and PFGE macrorestriction pattern to strain ICC-45, which is toxigenic (tcdA(+), tcdB(+) and ctdB(+)) and classified as ST41 from MLST Clade 2 and toxinotype IXb. Strain ICC-45 encodes for a variant TcdB that induces a distinct CPE in agreement with its toxinotype. Unlike epidemic NAP1/027 strains, which are also classified to MLST Clade 2, strain ICC-45 is susceptible to fluoroquinolones and does not overproduce toxins TcdA and TcdB. However, supernatants from strain ICC-45 and a NAP1/027 strain produced similar expression of pro-inflammatory cytokines, epithelial damage, and oxidative stress response in the mouse ileal loop model. These results highlight inflammation and oxidative stress as common features in the pathogenesis of C. difficile Clade 2 strains. Finally, this work contributes to the description of differences in virulence among various C. difficile strains.

Cardiovascular-Active Venom Toxins: An Overview.

Animal venoms are a mixture of bioactive compounds produced as weapons and used primarily to immobilize and kill preys. As a result of the high potency and specificity for various physiological targets, many toxins from animal venoms have emerged as possible drugs for the medication of diverse disorders, including cardiovascular diseases. Captopril, which inhibits the angiotensin-converting enzyme (ACE), was the first successful venom-based drug and a notable example of rational drug design. Since captopril was developed, many studies have discovered novel bradykinin-potentiating peptides (BPPs) with actions on the cardiovascular system. Natriuretic peptides (NPs) have also been found in animal venoms and used as template to design new drugs with applications in cardiovascular diseases. Among the anti-arrhythmic peptides, GsMTx-4 was discovered to be a toxin that selectively inhibits the stretch-activated cation channels (SACs), which are involved in atrial fibrillation. The present review describes the main components isolated from animal venoms that act on the cardiovascular system and presents a brief summary of venomous animals and their venom apparatuses.

Complete mitochondrial genome sequence of Prochilodus lineatus (Characiformes, Prochilodontidae).

Prochilodus lineatus (curimba) is an important Brazilian freshwater migratory fish with substantial economic importance in fishing. The complete mitochondrial genome of P. lineatus is 16,699 bp and contains 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and one control region (D-loop). The mtDNA of P. lineatus is the first mitogenome of the Prochilodontidae family to be fully characterized. All of the PCGs in the mtDNA use the standard ATG start codon, with the exception of Cox1 that utilizes the GTG start codon. Six of the 13 PCGs contain TAA stop codons, two contain the incomplete stop codon TA- (Atp6 and Nd6), and five contain the incomplete stop codon T- - (Nd2, Cox2, Nd3, Nd4 and Cytb).

Complete mitochondrial DNA of Hoplias intermedius (Günther, 1864) (Ostariophysi: Characiformes: Erythrinidae).

Mitochondrial complete genome (mtDNA) of Hoplias intermedius, commonly known as "traírão" is presented. DNA from a muscle tissue sample was sequenced by next-generation sequencing. To corroborate the results, phylogenetic analysis was performed with MEGA6 to compare the complete mitogenome of H. intermedius with genomes of other Characiformes species. The mtDNA molecule was circular and 16,629 bp in length. Gene content included 22 tRNA genes, 2 rRNA genes, 13 protein-coding genes and a D-loop noncoding region. All genes had ATG as the start codon. Six genes carried complete stop codons of AGG (COI), TGA (ND6) and TAA (ND1, ATPase 8, ND4L and ND5), while seven genes had incomplete stop codons. Phylogenetic relationships with other Ostariophysi species placed H. intermedius together with H. malabaricus as a monophyletic group belonging to the order Characiformes.

Pimelodus maculatus (Siluriformes, Pimelodidae): complete mtDNA sequence of an economically important fish from the São Francisco river basin.

Pimelodus maculatus is an important commercial fish found in the São Francisco and Paraná river basins. NGS was used to sequence the mtDNA of P. maculatus. The mtDNA was annotated and aligned with that of 25 other fish species to enable phylogenetic analysis. The complete mtDNA molecule had 16,561 bp and its GC content was 43.7%; the structure was similar to that of other vertebrates: 2 rRNA, 22 tRNA, 13 protein-coding genes, and a D-loop region containing 914 bp. Phylogenetic analysis yielded a tree with a high bootstrap coefficient that was coherent with the actual phylogeny of the species.

Whole venom of Loxosceles similis activates caspases-3, -6, -7, and -9 in human primary skin fibroblasts.

Spiders of the Loxosceles genus represent a risk to human health due to the systemic and necrotic effects of their bites. The main symptoms of these bites vary from dermonecrosis, observed in the majority of cases, to occasional systemic hemolysis and coagulopathy. Although the systemic effects are well characterized, the mechanisms of cell death triggered by the venom of these spiders are poorly characterized. In this study, we investigated the cell death mechanisms induced by the whole venom of the spider Loxosceles similis in human skin fibroblasts. Our results show that the venom initiates an apoptotic process and a caspase cascade involving the initiator caspase-9 and the effector caspases-3, -6, and -7.

Molecular, immunological, and biological characterization of Tityus serrulatus venom hyaluronidase: new insights into its role in envenomation.

BACKGROUND: Scorpionism is a public health problem in Brazil, and Tityus serrulatus (Ts) is primarily responsible for severe accidents. The main toxic components of Ts venom are low-molecular-weight neurotoxins; however, the venom also contains poorly characterized high-molecular-weight enzymes. Hyaluronidase is one such enzyme that has been poorly characterized. METHODS AND PRINCIPAL FINDINGS: We examined clones from a cDNA library of the Ts venom gland and described two novel isoforms of hyaluronidase, TsHyal-1 and TsHyal-2. The isoforms are 83% identical, and alignment of their predicted amino acid sequences with other hyaluronidases showed conserved residues between evolutionarily distant organisms. We performed gel filtration followed by reversed-phase chromatography to purify native hyaluronidase from Ts venom. Purified native Ts hyaluronidase was used to produce anti-hyaluronidase serum in rabbits. As little as 0.94 µl of anti-hyaluronidase serum neutralized 1 LD50 (13.2 µg) of Ts venom hyaluronidase activity in vitro. In vivo neutralization assays showed that 121.6 µl of anti-hyaluronidase serum inhibited mouse death 100%, whereas 60.8 µl and 15.2 µl of serum delayed mouse death. Inhibition of death was also achieved by using the hyaluronidase pharmacological inhibitor aristolochic acid. Addition of native Ts hyaluronidase (0.418 µg) to pre-neutralized Ts venom (13.2 µg venom+0.94 µl anti-hyaluronidase serum) reversed mouse survival. We used the SPOT method to map TsHyal-1 and TsHyal-2 epitopes. More peptides were recognized by anti-hyaluronidase serum in TsHyal-1 than in TsHyal-2. Epitopes common to both isoforms included active site residues. CONCLUSIONS: Hyaluronidase inhibition and immunoneutralization reduced the toxic effects of Ts venom. Our results have implications in scorpionism therapy and challenge the notion that only neurotoxins are important to the envenoming process.