Pedro Ismael da Silva

Molecular cloning, expression analysis and cellular localization of gomesin, an anti-microbial peptide from hemocytes of the spider Acanthoscurria gomesiana.

Gomesin is a cationic anti-microbial peptide of 18 amino acid residues isolated from the hemocytes of unchallenged tarantula spider Acanthoscurria gomesiana. This paper reports the first study of the processing and cellular location of an anti-microbial peptide (AMP) in spiders. Gomesin cDNA sequence analysis indicated that it is processed from a precursor containing a signal peptide (23 amino acid residues) and a negative C-terminal region (43 amino acid residues). The gomesin gene was constitutively transcribed in hemocytes and the gene product localized in hemocyte granules. The constitutive production of gomesin by a spider is discussed in the context of an ancient mechanism of AMP regulation and storage.

Specialization of the sting venom and skin mucus of Cathorops spixii reveals functional diversification of the toxins

Cathorops spixii is the most common venomous fish on the Brazilian coast. Apart from the involvement with defense against pathogens, the possible contribution of skin mucus components to the development of injuries caused by venomous fish species has not been investigated. Thus, the present study was conducted to gain a better understanding of the peptide and protein components of fish skin mucus and the sting venom from the catfish C. spixii. Our results show that sting venom and skin mucus have distinct constituents that distinguished them like structural proteins, chaperones, ion transport, carbohydrate metabolism, oxidoreductase, cell cycle and protein binding present in sting venom and like tropomyosin 3 isoform 2 and energy metabolim proteins in skin mucus. But in a group of common 13 proteins we identified and isolated a WAP65 protein. The peptide fractions caused more harmful effects, such as venular stasis, hemorrhage and changes in the arteriolar wall diameter, and the protein fractions produced a typical inflammatory process in post-capillary venules. And finally we showed for the first time the presence WAP65 in sting venom and skin mucus of C. spixii using LC/MS/MS and also we purified this protein in the sting venom. Wap65 shows inflammatory action, working at different doses inducing an increase in the number of leukocytes rolling and adhering to the endothelium.

Juruin: an antifungal peptide from the venom of the Amazonian Pink Toe spider, Avicularia juruensis, which contains the inhibitory cystine knot motif

The aim of this study was to screen the venom of the theraposid spider Avicularia juruensis for the identification of antimicrobial peptides (AMPs) which could be further used as prototypes for drug development. Eleven AMPs, named juruentoxins, with molecular weight ranging from 3.5 to 4.5 kDa, were identified by mass spectrometry after the soluble venom was separated by high performance liquid chromatography. Juruentoxins have a putative inhibitory cystine knot (ICK) motif, generally found in neurotoxins, which are also resistant to proteolysis. One juruentoxin that has 38 amino acid residues and three disulfide bonds were characterized, to which we proposed the name Juruin. Based on liquid growth inhibition assays, it has potent antifungal activity in the micromolar range. Importantly, Juruin lacks haemolytic activity on human erythrocytes at the antimicrobial concentrations. Based on the amino acid sequence, it is highly identical to the insecticidal peptides from the theraposid spiders Selenocosmia huwena, Chilobrachys jingzhao, and Haplopelma schmidti from China, indicating they belong to a group of conserved toxins which are likely to inhibit voltage-gated ion channels. Juruin is a cationic AMP, and Lys22 and Lys23 show maximum positive charge localization that might be important for receptor recognition. Although it shows marked sequence similarity to neurotoxic peptides, Juruin is a novel exciting molecule with potent antifungal activity, which could be used as a novel template for development of drugs against clinical resistant fungi strains.

Antimicrobial compounds from natural sources

Infectious diseases are one of the main causes of morbidity and mortality worldwide. Nowadays many infections are often caused by multi-resistant microorganisms resulting in difficult to treat diseases and, consequently, substantial increases in healthcare costs. The relative easy access to the antimicrobials and also the massive employment of these compounds for industrial purposes, including food production, have both strongly contributed to the progressive increase of resistant microorganisms. As a result, these multi-resistant microorganisms are reasserting themselves as worldwide threats. Research into natural products has demonstrated significant progress in the discovery of new compounds with antimicrobial activity. In fact, nature is a generous source of compounds with the potential to treat diseases, including infectious diseases. Among the known sources of natural compounds with valuable antimicrobial activity, we highlighted the medicinal plants and marine and terrestrial organisms, including fungi and bacteria. Nevertheless, there is still a vast fauna and flora that once systematically explored, could provide additional antimicrobial leads and new drugs. Thousands of natural products with the potential to act as antimicrobial compounds or as a structural lead compound still await further investigation. In this Research Topic Ebook, we present several scientific studies mainly focused on natural products with antimicrobial activity, which are the case of the natural antimicrobial peptides (AMPs) and host defense peptides (HDPs). This topic also includes recent studies on the roles of honey hydrogen peroxide in antimicrobial activity against resistant microbial strains, as well as the use of essential oils for food preservation. Such a wide and interesting topic also gave us an opportunity to include diverse sources, including plants, terrestrial and sea animals. Not to mention the interesting and unusual sources such as coal or lignite, which may provide future antimicrobial compounds candidates. The recent development of a patented process to GMP standards (PA107470/GB), rendering the obtainment of carbohydrate derived fulvic acid (CHD-FA), stimulated Sherry et al. (2012) to study and describe for the first time a highly effective novel antiseptic effect of fulvic acid with exquisite biofilm activity that acts by disrupting cell membranes. The antifungic peptide from Amazonian Pink Toe spider Juruin, described by Ayroza et al. (2012) is another outstanding example of the potential contribution of a systematic exploration of nature aiming to provide additional antimicrobial leads and drugs. In other words, nature is a generous source of compounds, with the potential to treat diseases, including infectious diseases. Studies exploiting the mechanism of action and the structure-activity aspects of these natural compounds may provide both additional antimicrobial leads and drugs, and also significant insight into potential possibilities to overcome the antimicrobial resistance.

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.

VdTX-1, a reversible nicotinic receptor antagonist isolated from venom of the spider Vitalius dubius (Theraphosidae).

Theraphosid spider venoms can block neurotransmission in vertebrate nerve-muscle preparations in vitro, but few of the components involved have been characterized. In this work, we describe the neuromuscular activity of venom from the Brazilian theraphosid Vitalius dubius and report the purification and pharmacological characterization of VdTX-1, a 728 Da toxin that blocks nicotinic receptors. Neuromuscular activity was assayed in chick biventer cervicis preparations and muscle responses to exogenous ACh and KCl were determined before and after incubation with venom or toxin. Changes in membrane resting potential were studied in mouse diaphragm muscle. The toxin was purified by a combination of filtration through Amicon® filters, cation exchange HPLC and RP-HPLC; toxin purity and mass were confirmed by mass spectrometry. Venom caused progressive neuromuscular blockade and muscle contracture; the blockade but not the contracture was reversible by washing. Venom attenuated contractures to exogenous ACh and KCl. Filtration yielded low (LM, <5 kDa) and high (HM, >5 kDa) fractions, with the latter reproducing the contracture seen in venom but with a slight and progressive twitch blockade. The LM fraction caused reversible blockade and attenuated contractures to ACh, but had no effect on contractures to KCl. VdTX-1 (728 Da) purified from the LM fraction was photosensitive and reduced the E(max) to ACh in biventer cervicis muscle without affecting the EC₅₀; VdTX-1 also abolished carbachol-induced depolarizations. V. dubius venom contains at least two components that affect vertebrate neurotransmission. One component, VdTX-1, blocks nicotinic receptors non-competitively to produce reversible blockade without muscle contracture.

The Musca domestica larval hexamerin is composed of multiple, similar polypeptides

The Musca domestica larval hexamerin (MdHex-L) is a hexameric glycoprotein with an apparent native molecular weight of 500 kDa. Seven different cDNAs that encode MdHex-L subunits were cloned and sequenced. Furthermore, amino acid sequences of isolated subunits were determined by the Edman degradation method and compared to the conceptual translation products derived from the cloned cDNAs. The obtained data indicate the existence of multiple forms of MdHex-L subunits and that these multiple forms may be grouped into three categories according to their percentages of nucleotide sequence identity.

The spider acylpolyamine Mygalin is a potent modulator of innate immune responses

Mygalin is an antibacterial molecule isolated from the hemocytes of the spider Acanthoscurria gomesiana. It was identified as bis-acylpolyamine spermidine. We evaluated the modulator effects of synthetic Mygalin in the innate immune response. We demonstrate that Mygalin induces IFN-γ synthesis by splenocytes increasing the nitrite secretion by splenocytes and macrophages. A specific inhibitor of iNOS abrogated Mygalin-induced nitrite production in macrophages independent of IFN-γ activation. In addition, Mygalin-activated macrophages produced TNF-α but not IL-1β, demonstrating that Mygalin does not act directly on the inflammasome. Furthermore, this compound did not affect spontaneous or Concanavalin A-induced proliferative responses by murine splenocytes and did not induce IL-5 or apoptosis of splenocytes or bone marrow-derived macrophages. These data provide evidence that Mygalin modulates the innate immune response by inducing IFN-γ and NO synthesis. The combined immune regulatory and antibacterial qualities of Mygalin should be explored as a strategy to enhance immune responses in infection.

Venom of the Phoneutria nigriventer spider alters the cell cycle, viability, and migration of cancer cells: BARRETO et al.

The mechanisms of cancer involve changes in multiple biological pathways. Multitarget molecules, which are components of animal venoms, are therefore a potential strategy for treating tumors. The objective of this study was to screen the effects of Phoneutria nigriventer spider venom (PnV) on tumor cell lines. Cultured human glioma (NG97), glioblastoma (U‐251) and cervix adenocarcinoma (HeLa) cells, and nontumor mouse fibroblasts (L929) were treated with low (14 µg/ml) and high (280 µg/ml) concentrations of PnV, and analyzed through assays for cell viability (thiazolyl blue tetrazolium blue), proliferation (carboxyfluorescein succinimidyl ester), death (annexin V/propidium iodide [Pi]), the cell cycle (Pi), and migration (wound healing and transwell assay). The venom decreased the viability of U‐251 cells, primarily by inducing cell death, and reduced the viability of NG97 cells, primarily by inhibiting the cell cycle. The migration of all the tumor cell lines was delayed when treated with venom. The venom significantly affected all the tumor cell lines studied, with no cytotoxic effect on normal cells (L929), although the nonglial tumor cell (HeLa) was less sensitive to PnV. The results of the current study suggest that PnV may be composed of peptides that are highly specific for the multiple targets involved in the hallmarks of cancer. Experiments are underway to identify these molecules.