Multiomics Profiling of Toxins in the Venom of the Amazonian Spider Acanthoscurria juruenicola.

Acanthoscurria juruenicola is an Amazonian spider described for the first time almost a century ago. However, little is known about their venom composition. Here, we present a multiomics characterization of A. juruenicola venom by a combination of transcriptomics, proteomics, and peptidomics approaches. Transcriptomics of female venom glands resulted in 93,979 unique assembled mRNA transcript encoding proteins. A total of 92 proteins were identified in the venom by mass spectrometry, including 14 mature cysteine-rich peptides (CRPs). Quantitative analysis showed that CRPs, cysteine-rich secretory proteins, metalloproteases, carbonic anhydrases, and hyaluronidase comprise >90% of the venom proteome. Relative quantification of venom toxins was performed by DIA and DDA, revealing converging profiles of female and male specimens by both methods. Biochemical assays confirmed the presence of active hyaluronidases, phospholipases, and proteases in the venom. Moreover, the venom promoted in vivo paralytic activities in crickets, consistent with the high concentration of CRPs. Overall, we report a comprehensive analysis of the arsenal of toxins of A. juruenicola and highlight their potential biotechnological and pharmacological applications. Mass spectrometry data were deposited to the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD013149 and via the MassIVE repository with the dataset identifier MSV000087777.

An integrated analysis of mRNA and sRNA transcriptional profiles in Coffea arabica L. roots: insights on nitrogen starvation responses.

Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.

Systemic toxicity of snake venom metalloproteinases: Multi-omics analyses of kidney and blood plasma disturbances in a mouse model.

Snakebite envenomation is classified as a Neglected Tropical Disease. Bothrops jararaca venom induces kidney injury and coagulopathy. HF3, a hemorrhagic metalloproteinase of B. jararaca venom, participates in the envenomation pathogenesis. We evaluated the effects of HF3 in mouse kidney and blood plasma after injection in the thigh muscle, mimicking a snakebite. Transcriptomic analysis showed differential expression of 31 and 137 genes related to kidney pathology after 2 h and 6 h, respectively. However, only subtle changes were observed in kidney proteome, with differential abundance of 15 proteins after 6 h, including kidney injury markers. N-terminomic analysis of kidney proteins showed 420 proteinase-generated peptides compatible with meprin specificity, indicating activation of host proteinases. Plasma analysis revealed differential abundance of 90 and 219 proteins, respectively, after 2 h and 6 h, including coagulation-cascade and complement-system components, and creatine-kinase, whereas a semi-specific search of N-terminal peptides indicated activation of endogenous proteinases. HF3 promoted host reactions, altering the gene expression and the proteolytic profile of kidney tissue, and inducing plasma proteome imbalance driven by changes in abundance and proteolysis. The overall response of the mouse underscores the systemic action of a hemorrhagic toxin that transcends local tissue damage and is related to known venom-induced systemic effects.

Rondonin: antimicrobial properties and mechanism of action.

Infectious diseases are among the major causes of death in the human population. A wide variety of organisms produce antimicrobial peptides (AMPs) as part of their first line of defense. A peptide from Acanthoscurria rondoniae plasma, rondonin-with antifungal activity, a molecular mass of 1236 Da and primary sequence IIIQYEGHKH-was previously studied (UniProt accession number B3EWP8). It showed identity with the C terminus of subunit 'D' of the hemocyanin of the Aphonopelma hentzi spider. This result led us to propose a new pathway of the immune system of arachnids that suggests a new function to hemocyanin: production of antimicrobial peptides. Rondonin does not interact with model membranes and was able to bind to yeast nucleic acids but not bacteria. It was not cytotoxic against mammalian cells. The antifungal activity of rondonin is pH-dependent and peaks at pH ˜ 4-5. The peptide presents synergism with gomesin (spider hemocyte antimicrobial peptide-UniProtKB-P82358) against human yeast pathogens, suggesting a new potential alternative treatment option. Antiviral activity was detected against RNA viruses, measles, H1N1, and encephalomyocarditis. This is the first report of an arthropod hemocyanin fragment with activity against human viruses. Currently, it is vital to invest in the search for natural and synthetic antimicrobial compounds that, above all, present alternative mechanisms of action to first-choice antimicrobials.

A Multiomics Approach Unravels New Toxins With Possible In Silico Antimicrobial, Antiviral, and Antitumoral Activities in the Venom of Acanthoscurria rondoniae.

The Araneae order is considered one of the most successful groups among venomous animals in the world. An important factor for this success is the production of venoms, a refined biological fluid rich in proteins, short peptides and cysteine-rich peptides (CRPs). These toxins may present pharmacologically relevant biological actions, as antimicrobial, antiviral and anticancer activities, for instance. Therefore, there is an increasing interest in the exploration of venom toxins for therapeutic reasons, such as drug development. However, the process of peptide sequencing and mainly the evaluation of potential biological activities of these peptides are laborious, considering the low yield of venom extraction and the high variability of toxins present in spider venoms. Here we show a robust methodology for identification, sequencing, and initial screening of potential bioactive peptides found in the venom of Acanthoscurria rondoniae. This methodology consists in a multiomics approach involving proteomics, peptidomics and transcriptomics analyses allied to in silico predictions of antibacterial, antifungal, antiviral, and anticancer activities. Through the application of this strategy, a total of 92,889 venom gland transcripts were assembled and 84 novel toxins were identified at the protein level, including seven short peptides and 10 fully sequenced CRPs (belonging to seven toxin families). In silico analysis suggests that seven CRPs families may have potential antimicrobial or antiviral activities, while two CRPs and four short peptides are potentially anticancer. Taken together, our results demonstrate an effective multiomics strategy for the discovery of new toxins and in silico screening of potential bioactivities. This strategy may be useful in toxin discovery, as well as in the screening of possible activities for the vast diversity of molecules produced by venomous animals.

Peptidomics of Acanthoscurria gomesiana spider venom reveals new toxins with potential antimicrobial activity.

Acanthoscurria gomesiana is a Brazilian spider from the Theraphosidae family inhabiting regions of Southeastern Brazil. Potent antimicrobial peptides as gomesin and acanthoscurrin have been discovered from the spider hemolymph in previous works. Spider venoms are also recognized as sources of biologically active peptides, however the venom peptidome of A. gomesiana remained unexplored to date. In this work, a MS-based workflow was applied to the investigation of the spider venom peptidome. Data-independent and data-dependent LC-MS/MS acquisitions of intact peptides and of peptides submitted to multiple enzyme digestions, followed by automated chromatographic alignment, de novo analysis, database and homology searches with manual validations showed that the venom is composed by <165 features, with masses ranging from 0.4-15.8kDa. From digestions, 135 peptides were identified from 17 proteins, including three new mature peptides: U1-TRTX-Agm1a, U1-TRTX-Agm2a and U1-TRTX-Agm3a, containing 3, 4 and 3 disulfide bonds, respectively. The toxins U1-TRTX-Agm1a differed by only one amino acid from U1-TRTX-Ap1a from A. paulensis and U1-TRTX-Agm2a was derived from the genicutoxin-D1 precursor from A. geniculata. These toxins have potential applications as antimicrobial agents, as the peptide fraction of A. gomesiana showed activity against Escherichia coli, Enterobacter cloacae and Candida albicans strains. MS data are available via ProteomeXchange Consortium with identifier PXD003884. BIOLOGICAL SIGNIFICANCE: Biological fluids of the Acanthoscurria gomesiana spider are sources of active molecules, as is the case of antimicrobial peptides and acylpolyamines found in the hemolymphs. The venom is also a potential source of toxins with pharmacological and biotechnological applications. However, to our knowledge no A. gomesiana venom toxin structure has been determined to date. Using a combination of high resolution mass spectrometry, transcriptomics and bioinformatics, we employed a workflow to fully sequence, determine the number of disulfide bonds of mature peptides and we found new potential antimicrobial peptides. This workflow is suitable for complete peptide toxin sequencing when handling limited amount of venom samples and can accelerate the discovery of peptides with potential biotechnological applications.

Development of molecular assays for the identification of the 11 Eimeria species of the domestic rabbit (Oryctolagus cuniculus).

Coccidiosis are the major parasitic diseases in poultry and other domestic animals including the domestic rabbit (Oryctolagus cuniculus). Eleven distinct Eimeria species have been identified in this host, but no PCR-based method has been developed so far for unequivocal species differentiation. In this work, we describe the development of molecular diagnostic assays that allow for the detection and discrimination of the 11 Eimeria species that infect rabbits. We determined the nucleotide sequences of the ITS1 ribosomal DNAs and designed species-specific primers for each species. We performed specificity tests of the assays using heterologous sets of primers and DNA samples, and no cross-specific bands were observed. We obtained a detection limit varying from 500fg to 1pg, which corresponds approximately to 0.8-1.7 sporulated oocysts, respectively. The test reported here showed good reproducibility and presented a consistent sensitivity with three different brands of amplification enzymes. These novel diagnostic assays will permit population surveys to be performed with high sensitivity and specificity, thus contributing to a better understanding of the epidemiology of this important group of coccidian parasites.