Snake genes of venom toxins and b-defensins

Abstract

Snakes are one of the most successful group of animals in the world living in a variety of habitats, from desert to ocean and rivers, on the trees and on the floor. They have many feeding strategies and may present venom glands that are rich in bioactive molecules. In general, these molecules can grouped into structural families such as phospholipase A2, metalloproteases, serine proteases and B-defensins. This project will focus on two families, the serine proteases responsible for thrombin-like activity and the B-defensins that has antimicrobial activity. The serine proteases are present in Bothrops and Crotalus venoms; however, some species such as B. erythromelas did not exhibit this activity thrombin-like in a pool of venom tested. Therefore, we will test venoms individually to coagulant activities, such as minimum clotting dose in plasma and fibrinogen; in addition, these snakes will be tested for the presence of the serine protease gene by PCR using genomic DNA obtained from the blood of individuals. In this way, we can make a comparative study of genes in snakes that have thrombin like activity with those that have not and analyze if this lack of activity was due to mutation or deletion of the serine protease gene. We will compare the serine protease genes of B. erythromelas to B. jararaca, because it is a snake which venom is coagulant and has thrombin like activity. The B-defensins are components of the innate immune system and are the first components to be recruited at the onset of an infection. They are present in all vertebrates and seem to have a common origin. Besides the antimicrobial activity, they have functions in the immunomodulation and reproduction. Our group described several B-defensins in Brazilian pitvipers. We intend to increase the amounts of data including in this phylogenetic study, the B-defensin from colubrid snakes using PCR for gene amplification from purified genomic DNA. In this work we intent to analyze B-defensin sequences from Thamnodynastes strigatus and Phalotris mertensi. In B. jararaca, three different groups of sequences were described amplified from genome by PCR or found in the transcriptome of venom gland. In addition, we will analyze the expression of these sequences in several tissues such as liver, brain, lung, skin, gut, testicle, and ovary by real time PCR. We want to characterize the organization of B-defensin genes in the B. jararaca genome as well. As the B-defensin are antimicrobial peptides, they will be tested against bacteria and fungi using a qualitative method, modified microbroth dilution assay, in which a serial dilution of peptides is tested against fixed quantities of microorganisms. This assay will be used to test peptides derived from snake B-defensins in order to extend the spectrum of activity or improve the antimicrobial activity. Previous studies have indicated that some snake B-defensins have antibiotic activity against the Gram-negative bacteria Staphylococcus aureus, Citrobacter freundii, and Escherichia coli (ATCC 25922) as well against the Gram-positive Micrococcus luteus, with minimum inhibitory concentrations ranging 2-128 mg/mL. This search is important at a time when few products with antimicrobial activity come on the market despite the resistance of microorganisms against various medications in use. In this way, we intend to continue our way in understanding the polymorphism of snake venoms and knowing the evolutionary story of B-defensin genes. (AU)