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.

New Sulphated Flavonoids from Wissadula periplocifolia (L.) C. Presl (Malvaceae).

Wissadula periplocifolia (L.) C. Presl (Malvaceae) is commonly used in Brazil to treat bee stings and as an antiseptic. The antioxidant properties of its extracts have been previously demonstrated, thus justifying a phytochemical investigation for its bioactive phenolic constituents. This has yielded five new sulphated flavonoids: 8-O-sulphate isoscutellarein (yannin) (1a); 4'-O-methyl-7-O-sulphate isoscutellarein (beltraonin) (1b); 7-O-sulphate acacetin (wissadulin) (2a); 4'-O-methyl-8-O-sulphate isoscutellarein (caicoine) (2b) and 3'-O-methyl-8-O-sulphate hypolaetin (pedroin) (3b) along with the known flavonoids 7,4'-di-O-methyl-8-O-sulphate isoscutellarein (4), acacetin, apigenin, isoscutellarein, 4'-O-methyl isoscutellarein, 7,4'-di-O-methylisoscutellarein, astragalin and tiliroside. The compounds were isolated by column chromatography and identified by NMR (¹H, (13)C, HMQC, HMBC and COSY) and LC-HRMS. A cell based assay was carried out to evaluate the preliminary cytotoxic properties of the flavonoids against UVW glioma and PC-3M prostate cancer cells as well as non-tumour cell lines. The obtained results showed that acacetin, tiliroside, a mixture of acacetin + apigenin and the sulphated flavonoids 2a + 2b exhibited inhibitory activity against at least one of the cell lines tested. Among the tested flavonoids acacetin and tiliroside showed lower IC50 values, presenting promising antitumor effects.

Molecular cloning and functional characterization of recombinant Loxtox from Loxosceles similis venom.

Loxoscelism is a recognized public health problem in Brazil, but the venom from Loxosceles similis, which is widespread in Brazil due to its adaptability to the urban environment, remains poorly characterized. Loxtox is a family of phospholipase D enzymes (PLDs), which are the major components of Loxosceles venom and are responsible for the clinical effects of loxoscelism. Loxtox toxins correspond to 15% of L. similis venom gland transcripts, but the Loxtox family of L. similis has yet to be fully described. In this study, we cloned and functionally characterized recLoxtox s1A and recLoxtox s11A. These recombinant toxins exhibited different in vitro activities depending on pH, and recLoxtox s1A had more intense effects on rabbit skin than did recLoxtox s11A in vivo. Both recombinant toxins were used in immunization protocols, and mapping of their epitopes revealed different immunological reactions for the produced immune serums. Additionally, polyclonal antibodies raised against recLoxtox s1A had greater capacity to significantly reduce the in vitro and in vivo effects of L. similis venom. In summary, we obtained and characterized two novel Loxtox isoforms from L. similis venom, which may be valuable biotechnological and immunological tools against loxoscelism.

From taxonomy to molecular characterization of brown spider venom: An overview focused on Loxosceles similis.

Loxosceles spp. (Araneae, Sicariidae), known as brown spiders, are distributed in temperate and tropical regions worldwide. Accidents caused by these spiders are known as loxoscelism and constitute a public health problem, especially in Brazil. The present review describes the taxonomy, distribution, and ecological profile of brown spiders, as well as the molecular and biochemical aspects of Loxosceles venom. Additionally, it presents an overview on L. similis, a species found in the Southeastern region of Brazil. In this region, the number of Loxosceles accidents has been increasing in the past few years, thus calling attention to its raising importance as a medically relevant spider species in Brazil.

Inhibition of Tityus serrulatus venom hyaluronidase affects venom biodistribution.

BACKGROUND: The hyaluronidase enzyme is generally known as a spreading factor in animal venoms. Although its activity has been demonstrated in several organisms, a deeper knowledge about hyaluronidase and the venom spreading process from the bite/sting site until its elimination from the victim's body is still in need. Herein, we further pursued the goal of demonstrating the effects of inhibition of T. serrulatus venom (TsV) hyaluronidase on venom biodistribution. METHODS AND PRINCIPAL FINDINGS: We used technetium-99m radiolabeled Tityus serrulatus venom (99mTc-TsV) to evaluate the venom distribution kinetics in mice. To understand the hyaluronidase's role in the venom's biodistribution, 99mTc-TsV was immunoneutralized with specific anti-T.serrulatus hyaluronidase serum. Venom biodistribution was monitored by scintigraphic images of treated animals and by measuring radioactivity levels in tissues as heart, liver, lungs, spleen, thyroid, and kidneys. In general, results revealed that hyaluronidase inhibition delays venom components distribution, when compared to the non-neutralized 99mTc-TsV control group. Scintigraphic images showed that the majority of the immunoneutralized venom is retained at the injection site, whereas non-treated venom is quickly biodistributed throughout the animal's body. At the first 30 min, concentration peaks are observed in the heart, liver, lungs, spleen, and thyroid, which gradually decreases over time. On the other hand, immunoneutralized 99mTc-TsV takes 240 min to reach high concentrations in the organs. A higher concentration of immunoneutralized 99mTc-TsV was observed in the kidneys in comparison with the non-treated venom. Further, in situ neutralization of 99mTc-TsV by anti-T.serrulatus hyaluronidase serum at zero, ten, and 30 min post venom injection showed that late inhibition of hyaluronidase can still affect venom biodistribution. In this assay, immunoneutralized 99mTc-TsV was accumulated in the bloodstream until 120 or 240 min after TsV injection, depending on anti-hyaluronidase administration time. Altogether, our data show that immunoneutralization of hyaluronidase prevents venom spreading from the injection site. CONCLUSIONS: By comparing TsV biodistribution in the absence or presence of anti-hyaluronidase serum, the results obtained in the present work show that hyaluronidase has a key role not only in the venom spreading from the inoculation point to the bloodstream, but also in venom biodistribution from the bloodstream to target organs. Our findings demonstrate that hyaluronidase is indeed an important spreading factor of TsV and its inhibition can be used as a novel first-aid strategy in envenoming.

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.

Description of Loxtox protein family and identification of a new group of Phospholipases D from Loxosceles similis venom gland.

Envenoming resulting from Loxosceles spider bites (loxoscelism) is a recognized public health problem in Brazil. However, the pathophysiology of loxoscelism caused by L. similis bites, which is widespread in Brazil, remains poorly understood. In the present work, the RNA sequencing (RNA-Seq - Next Generation sequencing - NGS) of the L. similis venom gland was performed to identify and analyze the sequences of the key component phospholipase D. The sequences were aligned based on their classical domains, and a phylogenetic tree was constructed. In the bioinformatics analysis, 23 complete sequences of phospholipase D proteins were found and classified as Loxtox proteins, as they contained the characteristic domains of phospholipase D: the active site, the Mg(2+)-binding domain, and the catalytic loop. Three phospholipase D sequences with non-canonical domains were also found in this work. They were analyzed separately and named PLDs from L. similis (PLD-Ls). This study is the first to characterize phospholipase D sequences from Loxosceles spiders by RNA-Seq. These results contribute new knowledge about the composition of L. similis venom, revealing novel tools that could be used for pharmacological, immunological, and biotechnological applications.

Survey of microbial enzymes in soil, water, and plant microenvironments.

Detection of microbial enzymes in natural environments is important to understand biochemical activities and to verify the biotechnological potential of the microorganisms. In the present report, 346 isolates from soil, water, and plants were screened for enzyme production (caseinase, gelatinase, amylase, carboxymethyl cellulase, and esterase). Our results showed that 89.6% of isolates produced at least one tested enzyme. A predominance of amylase in soil samples, carboxymethyl cellulase in plants, as well as esterase and gelatinase in water was observed. Interesting enzymatic profiles were found in some microenvironments, suggesting specificity of available nutrients and/or natural selection. This study revealed the potential of microorganisms present in water, soil, and plant to produce important enzymes for biotechnological exploration. A predominance of certain enzymes was found, depending on the type of environmental sample. The distribution of microbial enzymes in soil, water and plants has been little exploited in previous reports.

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.