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Loxoscelism is the clinical condition triggered after the bite of spiders of the genus Loxosceles. The main species involved in accidents in South America are L. intermedia, L. laeta, and L. gaucho. The only specific treatment is the anti-Loxosceles serum produced with crude venoms. As phospholipases D (PLDs) trigger most of the effects observed in accidents, we developed and evaluated second-generation sera using mutated PLDs as antigens. Three isoforms of PLDs with site-directed mutations without biological activities were used for rabbit immunizations: D32A-E34A (L. gaucho), W230A (L. intermedia), and H12A-H47A (L. laeta). Sera were produced using crude venoms of three species of Loxosceles enriched with mutated recombinant PLDs (MIX) or using only mutated PLDs (REC). Immunizations stimulated the immune system from the second immunization with higher antibody production in the REC group. In vivo neutralization assays demonstrated that both sera reduced edema and dermonecrosis caused by Loxosceles intermedia crude venom. Follow-up of animals during the immunization protocols and in the neutralization assays demonstrated that the mutated proteins and the sera are safe. Results demonstrate the potential of using mutated recombinant PLDs in total or partial replacement of Loxosceles venoms in animal immunizations to produce anti-Loxosceles sera for treatments of Loxoscelism. [ABSTRACT FROM AUTHOR]

Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5-40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein, we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins. [ABSTRACT FROM AUTHOR]

Phospholipases-D (PLDs) found in Loxosceles spiders' venoms are responsible for the dermonecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents—L. gaucho and L. laeta—were recombinantly expressed and characterized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced. [ABSTRACT FROM AUTHOR]

Brown spider envenomation results in dermonecrosis with gravitational spreading characterized by a marked inflammatory reaction and with lower prevalence of systemic manifestations such as renal failure and hematological disturbances. Several toxins make up the venom of these species, and they are mainly peptides and proteins ranging from 5–40 kDa. The venoms have three major families of toxins: phospholipases-D, astacin-like metalloproteases, and the inhibitor cystine knot (ICK) peptides. Serine proteases, serpins, hyaluronidases, venom allergens, and a translationally controlled tumor protein (TCTP) are also present. Toxins hold essential biological properties that enable interactions with a range of distinct molecular targets. Therefore, the application of toxins as research tools and clinical products motivates repurposing their uses of interest. This review aims to discuss possibilities for brown spider venom toxins as putative models for designing molecules likely for therapeutics based on the status quo of brown spider venoms. Herein, we explore new possibilities for the venom components in the context of their biochemical and biological features, likewise their cellular targets, three-dimensional structures, and mechanisms of action. [ABSTRACT FROM AUTHOR]

Loxoscelism is the clinical condition triggered after the bite of spiders of the genus Loxosceles . The main species involved in accidents in South America are L. intermedia , L. laeta, and L. gaucho . The only specific treatment is the anti- Loxosceles serum produced with crude venoms. As phospholipases D (PLDs) trigger most of the effects observed in accidents, we developed and evaluated second-generation sera using mutated PLDs as antigens. Three isoforms of PLDs with site-directed mutations without biological activities were used for rabbit immunizations: D32A-E34A ( L. gaucho ), W230A ( L. intermedia ) , and H12A-H47A ( L. laeta ). Sera were produced using crude venoms of three species of Loxosceles enriched with mutated recombinant PLDs (MIX) or using only mutated PLDs (REC). Immunizations stimulated the immune system from the second immunization with higher antibody production in the REC group. In vivo neutralization assays demonstrated that both sera reduced edema and dermonecrosis caused by Loxosceles intermedia crude venom. Follow-up of animals during the immunization protocols and in the neutralization assays demonstrated that the mutated proteins and the sera are safe. Results demonstrate the potential of using mutated recombinant PLDs in total or partial replacement of Loxosceles venoms in animal immunizations to produce anti- Loxosceles sera for treatments of Loxoscelism.

Accidents caused by the bites of brown spiders ( Loxosceles ) generate a clinical condition that often includes a threatening necrotic skin lesion near the bite site along with a remarkable inflammatory response. Systemic disorders such as hemolysis, thrombocytopenia, and acute renal failure may occur, but are much less frequent than the local damage. It is already known that phospholipases D, highly expressed toxins in Loxosceles venom, can induce most of these injuries. However, this spider venom has a great range of toxins that probably act synergistically to enhance toxicity. The other protein classes remain poorly explored due to the difficulty in obtaining sufficient amounts of them for a thorough investigation. They include astacins (metalloproteases), serine proteases, knottins, translationally controlled tumor proteins (TCTP), hyaluronidases, allergens and serpins. It has already been shown that some of them, according to their characteristics, may participate to some extent in the development of loxoscelism. In addition, all of these toxins present potential application in several areas. The present review article summarizes information regarding some functional aspects of the protein classes listed above, discusses the directions that could be taken to materialize a comprehensive investigation on each of these toxins as well as highlights the importance of exploring the full venom repertoire., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Brown spider (genus Loxosceles ) venoms are mainly composed of protein toxins used for predation and defense. Bites of these spiders most commonly produce a local dermonecrotic lesion with gravitational spread, edema and hemorrhage, which together are defined as cutaneous loxoscelism. Systemic loxoscelism, such as hematological abnormalities and renal injury, are less frequent but more lethal. Some Loxosceles venom toxins have already been isolated and extensively studied, such as phospholipases D (PLDs), which have been recombinantly expressed and were proven to reproduce toxic activities associated to the whole venom. PLDs have a notable potential to be engineered and converted in non-toxic antigens to produce a new generation of antivenoms or vaccines. PLDs also can serve as tools to discover inhibitors to be used as therapeutic agents. Other Loxosceles toxins have been identified and functionally characterized, such as hyaluronidases, allergen factor, serpin, TCTP and knottins (ICK peptides). All these toxins were produced as recombinant molecules and are biologically active molecules that can be used as tools for the potential development of chemical candidates to tackle many medical and biological threats, acting, for instance, as antitumoral, insecticides, analgesic, antigens for allergy tests and biochemical reagents for cell studies. In addition, these recombinant toxins may be useful to develop a rational therapy for loxoscelism. This review summarizes the main candidates for the development of drugs and biotechnological inputs that have been described in Brown spider venoms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gremski, Matsubara, Polli, Antunes, Schluga, Justa, Minozzo, Wille, Senff-Ribeiro and Veiga.)