Loxosceles spider envenomation results in dermonecrosis, principally due to phospholipases D (PLDs) present in the venom. These enzymes have a strongly conserved sequence, 273ATXXDNPW280, in the ...C-terminal region (SMD-tail) that make contact with β-sheets of the TIM barrel, in which the amino acids Asp277 and Trp280 establish the energetically strongest contacts. The SMD-tail is conserved in PLDs from different species but absent in the non-toxic PLD ancestral glycerophosphodiester phosphodiesterases (GDPDs). This work aims to understand the role of the C-terminal region in the structural stability and/or function of phospholipases D. Through site-directed mutagenesis of the rLiD1 protein (recombinant Loxosceles intermedia dermonecrotic protein 1), we produced two mutants: rLiD1D277A and rLiD1W280A (both with sphingomyelinase activity), in which Asp277 and Trp280 were replaced by alanine. rLiD1D277A showed similar sphingomyelinase activity but at least 2 times more dermonecrotic activity than rLiD1 (wild-type protein). Conversely, while the rLiD1W280A displayed a slight increase in sphingomyelinase activity, its biological activity was similar or lower compared to rLiD1, potentially due to its decreased thermostability and formation of amyloid aggregates. In conclusion, these new findings provide evidence that SMD-tail mutants impact the structure and function of these proteins and point out that residues outside the active site can even increase the function of these enzymes.
•Spider PLD's C-terminal mutation, Asp277 to Ala, increases dermonecrosis in vivo.•The Asp277 to Ala mutation does not affect enzymatic activity in vitro.•Spider PLD's C-terminal mutation, Trp280 to Ala, lowers enzyme thermostability.•Trp280 to Ala mutation slightly increases enzyme activity in vitro.•Mutations in PLD's conserved C-terminal amino acids affect its structure and function.
The spider's genus Loxosceles (also known as “brown spiders”) is one of the few ones of medical importance in Brazil, being Loxosceles anomala a species of common occurrence in the Southeast region. ...This species is usually smaller in size than the other members of the Loxosceles group. A single human accident involving L. anomala was reported to date and the clinical picture shared similar characteristics with accidents caused by other Loxosceles species. Despite the potential relevance of L. anomalafor loxocelism in Minas Gerais state, its venom activity has never been characterized. In this work, we provide a preliminary characterization of L. anomala venom, considering its most relevant enzymatic activities and its venom immunorecognition by current therapeutic antivenoms. The results showed that L. anomala venom is immunorecognised by therapeutic antivenoms and by anti-phospholipase D antibodies. Its venom also shows enzymatic activities (sphingomyelinase activity, fibrinogenolytic) described for other Loxosceles venoms. This work contributes to a better knowledge on the venom content and activities of synanthropic Loxosceles species that have the potential of causing relevant human accidents.
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•Loxosceles anomala is a synanthropic spider original from Minas Gerais State, Brazil.•L. anomala venom is recognized by anti-loxoscelic, anti-arachnidic and anti-phospholipase-D antibodies.•L. anomala venom presented sphingomyelinase-D and fibrinogenolytic activities.•Due to its life habits and venom activities, L. anomala spiders can potentially cause significant human accidents.
Loxosceles
spp. (brown spiders) bites are responsible for the development of a syndrome consisting mainly of dermonecrotic lesions, and also systemic effects. Rabbits are one of the main experimental ...models used for better understanding the systemic and local effects of
Loxosceles
venom. The aim of this study is to evaluate the toxic and protective effects of rabbits immunized with
Loxosceles
spp. venom. Male New Zealand rabbits were allocated as a control group (CG;
n
= 5) that received adjuvant (Montanide) and phosphate-buffer saline (PBS), or as venom group (VG;
n
= 5) that received 21 μg of
Loxosceles
venom using Montanide as adjuvant. After five immunization cycles, a trial with 7 μg of
Loxosceles intermedia
(
L
.
intermedia
) venom was performed, and dermonecrotic lesions were measured. The rabbits were then euthanized, and their organs were collected for histopathology analysis. Rabbits that had undergone
Loxosceles
venom immunization protocol showed minor clinical disturbances during the experimental period. The used immunization protocol protected the rabbits against the toxic effect of the
Loxosceles
venom because they showed minor clinical disturbances during the experimental period.
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.
We present the case of a 32-year-old male patient hospitalized during the COVID-19 pandemic because of a Brown spider bite on his lower lip. The Brown spider accident occurred in southern Brazil; at ...hospital admission, the patient presented on his lip: edema, pustules, necrotic regions, and ulcerations. The patient complained of lower back pain, fever and dyspnea. Laboratory tests showed monocytosis, leukocytosis, neutrophilia, increased D-dimer levels, C-reactive protein, glutamate-pyruvate transaminase, delta bilirubin, creatine phosphokinase, procalcitonin, and fibrinogen. The patient was hospitalized and a multi-professional team carried out the treatment. The medical team diagnosed loxoscelism with moderate changes. The dentist treated the oral cavity. The patient began to develop nausea, vomiting, and desaturation episodes during hospitalization. A computed tomography of the chest was performed, which showed signs of viral infection. The RT-PCR test for COVID-19 was positive. The systemic conditions worsened (renal dysfunction, systemic inflammatory response, pulmonary complications). This condition may have resulted from the association of the two diseases (loxoscelism and COVID-19), leading to the patient's death. This case illustrates the difficulties and risks in treating patients with venomous animal accidents during the pandemic, and the importance of a multi-professional team in treating such cases.
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•Describes a local and systemic reaction to a Brown spider bite on the lower lip.•The combined systemic effect of Brown spider venom and SARS-CoV-2 infection.•Special attention in treating patients after Brown spider bites.•Loxoscelism -a condition that increases and exacerbates inflammatory response.
Spiders of
genus are widely distributed and their venoms contain phospholipases D (PLDs), which degrade phospholipids and trigger inflammatory responses, dermonecrosis, hematological changes, and ...renal injuries. Biochemical, functional, and structural properties of three recombinant PLDs from
,
and
, the principal species clinically relevant in South America, were analyzed. Sera against
and
PLDs strongly cross-reacted with other PLDs, but sera against
PLD mostly reacted with homologous molecules, suggesting underlying structural and functional differences. PLDs presented a similar secondary structure profile but distinct melting temperatures. Different methods demonstrated that all PLDs cleave sphingomyelin and lysophosphatidylcholine, but
and
PLDs excelled.
PLD showed greater "in vitro" hemolytic activity.
and
PLDs were more lethal in assays with mice and crickets. Molecular dynamics simulations correlated their biochemical activities with differences in sequences and conformations of specific surface loops, which play roles in protein stability and in modulating interactions with the membrane. Despite the high similarity, PLDs from
and
venoms are more active than
PLD, requiring special attention from physicians when these two species prevail in endemic regions.
The genus
Loxosceles
comprises 140 species widely distributed around the world. These spiders are nocturnal, sedentary and remarkably nonaggressive, although they cause accidents in humans with wide ...degrees of severity, generating signs and symptoms that define the clinical condition known as loxoscelism. Its local signs and symptoms were first reported in 1872, and over the years, a large medical literature has been accumulated; unfortunately, it is not always trustworthy. Assessing the reliability of such information, we reviewed 120 case reports of loxoscelism published in 84 articles over the past 20 years. This search allowed us to gather information on the clinical aspects, diagnosis and treatment of loxoscelism, showing that the severity of these accidents has multiple degrees and that it is influenced by many factors. Thus, coupled with epidemiological and species occurrence information, this study can be a useful tool for the clinical practice of loxoscelism. It may support and provide a multidisciplinary view that should be taken into consideration when establishing the therapeutic approach in cases of
Loxosceles
envenomation.
The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the clinical manifestations following spider bites include skin necrosis with gravitational lesion ...spreading and occasional systemic manifestations, such as intravascular hemolysis, thrombocytopenia and acute renal failure. Brown spider venoms are complex mixtures of toxins especially enriched in three molecular families: the phospholipases D, astacin-like metalloproteases and Inhibitor Cystine Knot (ICK) peptides. Other toxins with low level of expression also present in the venom include the serine proteases, serine protease inhibitors, hyaluronidases, allergen factors and translationally controlled tumor protein (TCTP). The mechanisms by which the Loxosceles venoms act and exert their noxious effects are not fully understood. Except for the brown spider venom phospholipase D, which causes dermonecrosis, hemolysis, thrombocytopenia and renal failure, the pathological activities of the other venom toxins remain unclear. The objective of the present review is to provide insights into the brown spider venoms and loxoscelism based on recent results. These insights include the biology of brown spiders, the clinical features of loxoscelism and the diagnosis and therapy of brown spider bites. Regarding the brown spider venom, this review includes a description of the novel toxins revealed by molecular biology and proteomics techniques, the data regarding three-dimensional toxin structures, and the mechanism of action of these molecules. Finally, the biotechnological applications of the venom components, especially for those toxins reported as recombinant molecules, and the challenges for future study are discussed.
•Data from the last 13 years of brown spider venom and loxoscelism.•Overview of the biology of brown spiders and the loxoscelism.•Molecular biology and proteomic analysis are shown.•Molecular mechanisms of the main toxins are discussed.•The biotechnological use of toxins and future directions are approached.
Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause ...hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.
•Identification of key residues involved in the catalytic activity of PLD from Loxosceles sp.•Role of Tyrosine 228 in enzymatic activity and substrate binding.•Correlation between biological and catalytic activity.
Antivenom production against Loxosceles venom relies on horses being immunized and bled for plasma harvest. One horse can partake in several cycles of antivenom production, which will require years ...of constant venom and adjuvant inoculation and bleeding. The actual impact on the health of horses that participate in several antivenom-producing cycles is unknown. Therefore, this study aimed to evaluate the general health status of horses that underwent at least six cycles of loxoscelic antivenom production. Seven crossbred horses that had partaken in six to eight complete antivenom-producing cycles were used and established as the immunized group (IG). Under the same handling and general management, eleven horses were established as the control group (CG). The horses were evaluated regarding their general clinical status and had their blood sampled, and an ECG recorded. The IG presented lower RBC and PCV, despite keeping values within inferior limits for the species. Renal function was not impaired, and liver-related enzymes were higher than those in the CG, probably due to liver exertion from immunoglobulin synthesis. ECG showed some abnormalities in the IG, such as atrioventricular block and a wandering atrial pacemaker, corroborated by an increase in CK-MB. The cardiovascular abnormalities were mainly found in the horses that participated in several antivenom-producing cycles. The overall results indicate that these horses had some impairment of their general health status. Once available, some alternative, less toxic antigens should replace the venom for immunization of horses used for antivenom production.
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