Team:USP-EEL-Brazil/Description
Ophidian accidents are categorized as neglected tropical diseases by the World Health Organization (WHO), which means that despite having a high incidence rate, these occurrences do not receive the proper attention. Among the cases in Brazil, one of the serpents which causes the most accidents is the Bothrops jararaca, which lives in most of Latin America. The effect of this serpent bite includes myonecrosis, a phenomenon which includes the degradation of cells, especially muscle ones, due to the external destruction of cellular membranes, causing the tissues affected by the venom to be more susceptible to microbial infection.
The main goal of the project is to produce a protein capable of inhibiting one of the many toxins present in B. jararaca venom, specifically an enzyme that is primarily responsible for the myonecrosis effect: phospholipase A2. The inhibition will be performed using the model bacterium Escherichia coli and using recombinant gene technology to express the 𝛾PLI protein (𝛾 phospholipase inhibitor), the natural inhibitor found in the blood serum of venomous snakes such as the genus Bothrops and Crotalus. Thus, with the inhibition of phospholipase A2, we intend to control more effectively one of the most harmful effects that require urgent medical treatment. Additionally, producing this protein through synthetic biology can reduce the use of animals to obtain the anti venom serum, also avoiding occasional deaths of animals during the traditional process of obtaining the serum.
According to the World Health Organization (WHO), it is estimated that annually the number of people bitten by snakes can reach 5.4 million, among these the number of deaths ranges between 81 and 138 thousand. In addition, up to 400,000 victims become disabled due to the action of the toxin in the bloodstream (WHO, 2019).
WHO includes accidents with venomous animals in its list of neglected tropical diseases, including snakes that produce venom. Taking into consideration affected people who are from regions without adequate infrastructure (WHO, 2019), this is a worrisome factor since the venom weakens the victim, and may be a hindrance to seeking timely care.
Among the most important snakes, two families predominate in Brazil: Viperidae, in which the Bothrops genus stands out, and Elapidae, in which the Crotalus genus belongs. Botropical accidents can happen in several areas, such as lakeshores, coastlines, humid environments, agricultural sites, savannahs, and open areas. Crotalus accidents usually occur in rural, open, arid and cerrado areas (Ministério da Saúde, 1991).
In Brazil, the highest rate of accidents with snakes comes from those that belong to the Bothrops genus. Only in 2019, there were around 20,897 cases notified with Bothrops, of this total, 17,644 were cured, 96 resulted in death by the grievance notified, 7 resulted in death from another cause, and 3,262 have no record on the evolution of the case. However, in the same year, only 2,610 cases with Crotalus were reported. Data from DATASUS.
Still, there were 238,899 accidents in which there was no identification of the snake type, showing that, due to the amount of species already identified and the proportion already established, the number of botropical accidents may be even higher than those recorded.
Snake venom, considered the most complex mixture in nature by some authors, is composed of 90 to 95% proteins. The metalloproteases and phospholipases A2 (PLA2) are the main component enzymes of the venom, and they are also the most damaging to the affected cells. In botropic venom there are also toxins called hemorrhagins, which are metalloproteases capable of breaking the integrity of the vascular endothelium and inhibiting platelet aggregation, thus causing hemorrhagic activity (PICELLI, 2013).
PLA2 proteins cause strong edematogenic activity, which is characterized by increased microvascular permeability, leading to edema formation and inflammation. These enzymes are also the main responsible for muscle necrosis (myonecrosis), causing an infection, due to the presence of bacteria located in the snake's mouth, which evolves quickly and may cause shock and death (PICELLI, 2013).
Snakes have natural PLA2 inhibitors, called Phospholipase Inhibitors (PLI), which are found in snake plasma. Based on amino acid sequence homology, PLIs are divided into 3 classes, ⍺-PLI, β-PLI, and 𝛾-PLI. Despite the existence of inhibitor variations, the project is focused on the production of 𝛾PLI via synthetic biology, as it has a broad spectrum of action, being able to inhibit most of the PLA2 classes found in venoms while the other classes cannot.
Swith the production of the 𝛾PLI inhibitor via synthetic biology, it is hoped that this project can facilitate access to anti-hyphidic treatment and also that this work will be a step towards the production of sera from fully synthetic genes, and with this there will be no need to use animals for serum production, thus preserving the health and welfare of these animals.
The snake that is most related to accidents is B. jararaca, a viper endemic to cerrados and forests in South and Central America.
This snake has a natural pattern on its scales, whose color varies according to the region and subspecies, which makes it easier to camouflage on dry land and leaves. It has an almost exclusively nocturnal habit, feeding on amphibians and small mammals, which allows B. jararaca to reach up to 1.6 meters in length. B. jararaca is terrestrial, but the younger specimens can be found in trees (arboreal). They are active most of the year and, like many other animals, have slightly larger females than males. This reptile has viviparous reproduction, the young are born between 2 and 3 months of gestation, usually 12 to 16 individuals, during rainy seasons (ALVES, 2000).
Like other specimens of the genus Bothrops sp., the venom of this animal has high amounts of the enzyme PLA2, responsible for the myonecrosis effect in its victims. This fact, together with the endemic region of the snake and its being the cause of several ophidic accidents, led to the choice of using the gene responsible for the production of γPLI in Bothrops Jararaca.
The choice of this inhibitor is not an unprecedented bet of the team, there are in the bibliography several successful tests in laboratory. In 2018, Serino-Silva et al. demonstrated, including experimentally, the action of γPLI on paw edema (laboratory test in rats) after PLA2 injection. The results showed significant reduction of myonecrosis effects.
The various studies about the venom of B. jararaca point out a high similarity between gamma-type PLA2 with respect to other snakes of the genus, pointing to a possible inhibitory potential in the venom of other snake species of this same genus (SERINO-SILVA et al., 2018).
Due to the high similarity between the PLA2 of Bothrops snakes, once the inhibitor of this toxin is synthesized, the final product may also be useful in combating other snake venoms of this genus (ESTEVÃO-COSTA et al., 2017).
For our research, we chose to prioritize local snake species in order to make the study more feasible. In Brazil, the highest rate of snake accidents is related to those belonging to the Bothrops genus. According to DATASUS data, in 2019, according to screening done on 21,009 cases of Bothrops bites, 10,687 were considered mild cases, 7,971 were considered moderate cases, 1,468 were considered severe cases, and 883 cases were not recorded.
In the same year, only 2,610 cases with Crotalus were reported. Still, more than 238 thousand cases in which there was no identification of the snake type were reported, showing that due to the amount of species already identified and the proportion already established, the number of botropical accidents is even higher than the one registered. Therefore, the Bothrops were chosen as the animals of interest.
Phospholipases are a superfamily of proteins that cleave phospholipids into lysophospholipids and fatty acids (SANTOS-FILHO, 2009). These enzymes act mainly on cell membranes and generally have optimal catalytic activity in the presence of calcium cations (SILVA, 2017).
Depending on the classification of phospholipases, they can have a variety of functions in different organisms. They can act, for example, as digestive enzymes, play an important role in membrane maintenance and remodeling, and also in the regulation of cellular mechanisms, such as the synthesis of bioactive lipid molecules (WAITE, 1987).
Being a large family of proteins, their variations are currently classified into 5 groups: the so-called secreted PLA2s (sPLA2), among them the PLA2s found in snake venom, the cytosolic PLA2s (cPLA2), the Ca2+ independent PLA2s (iPLA2), the acetylhydrolases, platelet-activating factors (PAF-AH) and the lysosomal PLA2s (SANTOS FILHO, 2009).
Among these 5 major groups, we will focus only on secreted phospholipases A2, which are classified into 15 subgroups, among which we highlight subgroup 2, which makes up the PLA2 targets of our study. This subgroup is mostly composed of phospholipases that, according to the amino acid at position 49, are classified as ASP-49, responsible for high catalytic activity due to its affinity for calcium cation, and Lys-49, which has low catalytic activity, but extreme importance in inducing myonecrosis (MAGALHÃES, 2017).
Botropic venom has tissue effect (at the site of inoculation) and systemic effects. Tissue injury is one of the main problems, being characterized by edema, intense pain and inflammation caused mainly by cytokines, besides coagulation and hemorrhage, caused by components called hemorrhages, inhibitors of platelet aggregation, causing the rupture of the vascular endothelium, and may evolve to severe necrosis.
After poisoning, the person may experience complications such as blistering, abscess formation, gangrene, compartment syndrome, acute renal failure, and shock (FERNANDES, 2017).
The myotoxic phospholipases A2 disrupt the sarcolemma, the connective tissue surrounding the muscle fiber, causing calcium ion influx and muscle necrosis. Tissue edema is multifactorial, by the action of hemorrhagic metalloproteases, and also catalytically active phospholipases A2 that play the role of producers of inflammatory mediators of localized edema, inducing mast cell exocytosis, releasing histamines, and producing bioactive lipids such as prostaglandins (COSTA, 2017).
Phospholipases A2, besides causing necrosis of the muscle tissue where the inoculation occurred, end up damaging, in more severe cases, the kidneys and cause various side effects to the body. After the tissue death of the affected muscles, the cytoplasmic content of the cells is released into the bloodstream, increasing the concentration of toxins called myoglobins, which are extremely harmful to the kidneys.
When the myoglobins reach the renal tubules, they cause acute tubular necrosis (ATN) and, consequently, acute renal failure (ARF), which causes several compromising effects to the organism (CASTRO, 2006).
Snakes are naturally immune to their own venoms, and this also applies when it comes specifically to phospholipases A2. This is because of a natural protection consisting of inhibitors. These inhibitors have a molecular mass of 75 to 180 kDa and are formed by 3 to 6 subunits (some are glycosylated), equal or not, each with a mass of 20 to 50 kDa, linked in a non-covalent way.
Based on amino acid sequence homology, PLI's are divided into 3 classes, each with their different characteristics and spectrum of actions:
The 𝛾PLIs are divided into two subgroups, I and II, according to their amino acid sequence, biochemical characteristics, and inhibition profile. The 𝛾PLI I have a heteromeric composition with two subgroups A and B with less than 33% identity with each other, and inhibit group I, II and III PLA2s. The 𝛾PLI II, on the other hand, inhibit group II PLA2s, and consist of a single subunit (PICELLI et al., 2003).
An important fact is that 𝛾PLI-I has a broad spectrum of action, being able to inhibit most of the PLA2 classes found in venoms, which makes it of high interest for the development of the project, while 𝛾PLI-II is able to inhibit only PLA2-II.
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