Toxicological Characterization and Phospholipase D Activity of the Venom of the Spider Sicarius thomisoides

Envenomation by Loxosceles spiders (Sicariidae family) has been thoroughly documented. However, little is known about the potential toxicity of members from the Sicarius genus. Only the venom of the Brazilian Sicarius ornatus spider has been toxicologically characterized. In Chile, the Sicarius thomisoides species is widely distributed in desert and semidesert environments, and it is not considered a dangerous spider for humans. This study aimed to characterize the potential toxicity of the Chilean S. thomisoides spider. To do so, specimens of S. thomisoides were captured in the Atacama Desert, the venom was extracted, and the protein concentration was determined. Additionally, the venoms were analyzed by electrophoresis and Western blotting using anti-recombinant L. laeta PLD1 serum. Phospholipase D enzymatic activity was assessed, and the hemolytic and cytotoxic effects were evaluated and compared with those of the L. laeta venom. The S. thomisoides venom was able to hydrolyze sphingomyelin as well as induce complement-dependent hemolysis and the loss of viability of skin fibroblasts with a dermonecrotic effect of the venom in rabbits. The venom of S. thomisoides showed intraspecific variations, with a similar protein pattern as that of L. laeta venom at 32–35 kDa, recognized by serum anti-LlPLD1. In this context, we can conclude that the venom of Sicarius thomisoides is similar to Loxosceles laeta in many aspects, and the dermonecrotic toxin present in their venom could cause severe harm to humans; thus, precautions are necessary to avoid exposure to their bite.

Production of Highly Active Recombinant Dermonecrotic Toxin of Bordetella Pertussis

Pathogenic Bordetella bacteria release a neurotropic dermonecrotic toxin (DNT) that is endocytosed into animal cells and permanently activates the Rho family GTPases by polyamination or deamidation of the glutamine residues in their switch II regions (e.g., Gln63 of RhoA). DNT was found to enable high level colonization of the nasal cavity of pigs by B. bronchiseptica and the capacity of DNT to inhibit differentiation of nasal turbinate bone osteoblasts causes atrophic rhinitis in infected pigs. However, it remains unknown whether DNT plays any role also in virulence of the human pathogen B. pertussis and in pathogenesis of the whooping cough disease. We report a procedure for purification of large amounts of LPS-free recombinant DNT that exhibits a high biological activity on cells expressing the DNT receptors Cav3.1 and Cav3.2. Electron microscopy and single particle image analysis of negatively stained preparations revealed that the DNT molecule adopts a V-shaped structure with well-resolved protein domains. These results open the way to structure–function studies on DNT and its interactions with airway epithelial layers.

Bordetella Dermonecrotic Toxin Is a Neurotropic Virulence Factor That Uses CaV3.1 as the Cell Surface Receptor

ABSTRACT Dermonecrotic toxin (DNT) is one of the representative toxins produced by Bordetella pertussis, but its role in pertussis, B. pertussis infection, remains unknown. In this study, we identified the T-type voltage-gated Ca2+ channel CaV3.1 as the DNT receptor by CRISPR-Cas9-based genome-wide screening. As CaV3.1 is highly expressed in the nervous system, the neurotoxicity of DNT was examined. DNT affected cultured neural cells and caused flaccid paralysis in mice after intracerebral injection. No neurological symptoms were observed by intracerebral injection with the other major virulence factors of the organisms, pertussis toxin and adenylate cyclase toxin. These results indicate that DNT has aspects of the neurotropic virulence factor of B. pertussis. The possibility of the involvement of DNT in encephalopathy, which is a complication of pertussis, is also discussed. IMPORTANCE Bordetella pertussis, which causes pertussis, a contagious respiratory disease, produces three major protein toxins, pertussis toxin, adenylate cyclase toxin, and dermonecrotic toxin (DNT), for which molecular actions have been elucidated. The former two toxins are known to be involved in the emergence of some clinical symptoms and/or contribute to the establishment of bacterial infection. In contrast, the role of DNT in pertussis remains unclear. Our study shows that DNT affects neural cells through specific binding to the T-type voltage-gated Ca2+ channel that is highly expressed in the central nervous system and leads to neurological disorders in mice after intracerebral injection. These data raise the possibility of DNT as an etiological agent for pertussis encephalopathy, a severe complication of B. pertussis infection.

TCTP from Loxosceles Intermedia (Brown Spider) Venom Contributes to the Allergic and Inflammatory Response of Cutaneous Loxoscelism

LiTCTP is a toxin from the Translationally Controlled Tumor Protein (TCTP) family identified in Loxosceles brown spider venoms. These proteins are known as histamine-releasing factors (HRF). TCTPs participate in allergic and anaphylactic reactions, which suggest their potential role as therapeutic targets. The histaminergic effect of TCTP is related to its pro-inflammatory functions. An initial characterization of LiTCTP in animal models showed that this toxin can increase the microvascular permeability of skin vessels and induce paw edema in a dose-dependent manner. We evaluated the role of LiTCTP in vitro and in vivo in the inflammatory and allergic aspects that undergo the biological responses observed in Loxoscelism, the clinical condition after an accident with Loxosceles spiders. Our results showed LiTCTP recombinant toxin (LiRecTCTP) as an essential synergistic factor for the dermonecrotic toxin actions (LiRecDT1, known as the main toxin in the pathophysiology of Loxoscelism), revealing its contribution to the exacerbated inflammatory response clinically observed in envenomated patients.

CONSTRUCTION OF RECOMBINANT ATTENUATED BORDETELLA PERTUSSIS BACTERIA OF PTXP3 GENOTYPE

Aim. The construction of recombinant attenuated Bordetella pertussis bacteria of ptxP3 genotype and its genetic and biological stability characteristics. Materials and methods. During construction of recombinant attenuated bacteria of ptxP3 genotype virulent Bordetella pertussis bacteria of ptxP1 genotype were used as a recipient. PtxP1 genotype bacteria are used for whole cell pertussis (wP) vaccine production in Russia. Mutant bacteria B. pertussis 475 were received by crossing-over between chromosome comprising native copy of target sequence and its mutant copy in recombinant suicide plasmid transferred in recipient bacteria by conjugation. Genetically engineered construction of recombinant plasmids was conducted. The structure of modified chromosome locus of attenuated bacteria was determined by PCR and amplification fragments sequence. The stability of structure and characteristics of attenuated bacteria was defined after 15 passages of strains on culture medium and 5 passages in mice. Results. Isogenic attenuated ptxP1 B. pertussis 4M and ptxP3 B. pertussis 4MKS were constructed. These bacteria produce non-toxic pertussis toxin (PT) and do not produce dermonecrotic toxin (DNT). The promoter region of ptx operon contains mutation, typical for «new» genotype of circulating virulent bacteria and increasing PT production. The structure of modified DNA fragments and characteristics of attenuated bacteria did not change while storing and after passages on culture medium and in mice. Conclusion. Recombinant attenuated bacteria B. pertussis 4MKS of «new» ptxP3 genotype are constructed. Application perspectiveness of genetically engineered modification of isogenic B.pertussis bacteria for pertussis vaccines development is shown.

Health advantages of transition to batch management system in farrow-to-finish pig herds

Sow batch management systems have become more popular due to advantages in labour planning, piglet batch sizes, all-in all-out practices and health management. The present study investigated the potential health advantages of 10 selected farrow-to-finish pig herds before and after transition from a one week batch management system to a four or five week batch management system. Five different animal categories (gilts, sows, piglets, growers and finishers) were sampled at three time points (T0, T1 and T2) before and after transition to a four or five week batch management system. Different matrices of the animals were collected: blood, nasal swabs and faeces. Several economically important diseases were monitored through serology: Lawsonia intracellularis, Porcine Reproductive and Respiratory Syndrome virus (PRRSv), Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae; and PCR-testing: Pasteurella multocida dermonecrotic toxin (DNT) and Brachyspira species, especially the major pathogenic Brachyspira hyodysenteriae. Following serological analysis, the percentage of positive animals per category and sampling occasion were calculated. Health improvement based on serology was defined as the reduction in the percentage of positive animals for a specific disease in a specified animal category. All samples were negative for P. multocida DNT and B. hyodysenteriae. Little to no improvement could be observed for PRRSv. For L. intracellularis an improvement could be observed in piglets (71%) and growers (56%; P < 0.05). For both of the respiratory pathogens, M. hyopneumoniae and A. pleuropneumoniae, significant improvement was observed in finishers (34 and 24%, respectively). In growers, only M. hyopneumoniae showed a significant improvement (34%). In conclusion, the transition from a one week batch management system to a four or five week batch management system in the present herds resulted in a reduction of the percentage of seropositive animals for three of the monitored economically important diseases: L. intracellularis, M. hyopneumoniae and A. pleuropneumoniae.