The Potassium Channel Blocker β-Bungarotoxin from the Krait Bungarus multicinctus Venom Manifests Antiprotozoal Activity

Protozoal infections are a world-wide problem. The toxicity and somewhat low effectiveness of the existing drugs require the search for new ways of protozoa suppression. Snake venom contains structurally diverse components manifesting antiprotozoal activity; for example, those in cobra venom are cytotoxins. In this work, we aimed to characterize a novel antiprotozoal component(s) in the Bungarus multicinctus krait venom using the ciliate Tetrahymena pyriformis as a model organism. To determine the toxicity of the substances under study, surviving ciliates were registered automatically by an original BioLaT-3.2 instrument. The krait venom was separated by three-step liquid chromatography and the toxicity of the obtained fractions against T. pyriformis was analyzed. As a result, 21 kDa protein toxic to Tetrahymena was isolated and its amino acid sequence was determined by MALDI TOF MS and high-resolution mass spectrometry. It was found that antiprotozoal activity was manifested by β-bungarotoxin (β-Bgt) differing from the known toxins by two amino acid residues. Inactivation of β-Bgt phospholipolytic activity with p-bromophenacyl bromide did not change its antiprotozoal activity. Thus, this is the first demonstration of the antiprotozoal activity of β-Bgt, which is shown to be independent of its phospholipolytic activity.

[1]  V. Jimenez,et al.  Down the membrane hole: Ion channels in protozoan parasites , 2022, PLoS pathogens.

[2]  M. Lourenzoni,et al.  Antileishmanial activity, cytotoxicity and cellular response of amphotericin B in combination with crotamine derived from Crotalus durissus terrificus venom using in vitro and in silico approaches. , 2022, Toxicon : official journal of the International Society on Toxinology.

[3]  Hoang Ngoc Anh,et al.  Comparative Study of the Effect of Snake Venoms on the Growth of Ciliates Tetrahymena pyriformis: Identification of Venoms with High Antiprotozoal Activity , 2022, Doklady. Biochemistry and biophysics.

[4]  V. Tsetlin,et al.  Antiviral Effects of Animal Toxins: Is There a Way to Drugs? , 2022, International journal of molecular sciences.

[5]  J. Van Houten,et al.  Ion channels of cilia: Paramecium as a model , 2022, The Journal of eukaryotic microbiology.

[6]  D. He,et al.  Antiprotozoal Effect of Snake Venoms and Their Fractions: A Systematic Review , 2021, Pathogens.

[7]  A. H. Laustsen,et al.  Black-necked spitting cobra (Naja nigricollis) phospholipases A2 may cause Trypanosoma brucei death by blocking endocytosis through the flagellar pocket , 2021, Scientific Reports.

[8]  F. Gonçalves,et al.  Venom of Viperidae: A Perspective of its Antibacterial and Antitumor Potential. , 2021, Current drug targets.

[9]  N. Moretti,et al.  Panacea within a Pandora's box: the antiparasitic effects of phospholipases A2 (PLA2s) from snake venoms. , 2021, Trends in parasitology.

[10]  Marco M. Domingues,et al.  Mechanistic Insights into the Leishmanicidal and Bactericidal Activities of Batroxicidin, a Cathelicidin-Related Peptide from a South American Viper (Bothrops atrox). , 2021, Journal of natural products.

[11]  T. R. Costa,et al.  A comparative study on the leishmanicidal activity of the L-amino acid oxidases BjussuLAAO-II and BmooLAAO-II isolated from Brazilian Bothrops snake venoms. , 2020, International journal of biological macromolecules.

[12]  I. Sharifi,et al.  The Effect of Naja naja oxiana Snake Venom Against Leishmania tropica Confirmed by Advanced Assays , 2020, Acta Parasitologica.

[13]  V. Rodrigues,et al.  Insights into the antiviral activity of phospholipases A2 (PLA2s) from snake venoms , 2020, International Journal of Biological Macromolecules.

[14]  T. Andreeva,et al.  Screening Snake Venoms for Toxicity to Tetrahymena Pyriformis Revealed Anti-Protozoan Activity of Cobra Cytotoxins , 2020, Toxins.

[15]  P. Volf,et al.  Suicidal Leishmania , 2020, Pathogens.

[16]  D. Shahbazzadeh,et al.  The in vitro study of anti-leishmanial effect of Naja naja oxiana snake venom on Leishmania major. , 2020, Infectious disorders drug targets.

[17]  N. Santos,et al.  Snake Venom Cathelicidins as Natural Antimicrobial Peptides , 2019, Front. Pharmacol..

[18]  A. Soares,et al.  Identification of a peptide derived from a Bothrops moojeni metalloprotease with in vitro inhibitory action on the Plasmodium falciparum purine nucleoside phosphorylase enzyme (PfPNP). , 2019, Biochimie.

[19]  Araceli Castillo-Romero,et al.  Identification of a novel potassium channel (GiK) as a potential drug target in Giardia lamblia: Computational descriptions of binding sites , 2019, PeerJ.

[20]  M. T. dos Santos Correia,et al.  In vitro effect of Bothrops leucurus lectin (BLL) against Leishmania amazonensis and Leishmania braziliensis infection. , 2018, International journal of biological macromolecules.

[21]  R. Wheeler,et al.  Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections , 2018, bioRxiv.

[22]  Bernhard Y. Renard,et al.  Evaluating de novo sequencing in proteomics: already an accurate alternative to database‐driven peptide identification? , 2018, Briefings Bioinform..

[23]  J. R. Almeida,et al.  Harnessing snake venom phospholipases A2 to novel approaches for overcoming antibiotic resistance , 2018, Drug development research.

[24]  Z. Harrat,et al.  Isolation and characterization of an anti‐leishmanial disintegrin from Cerastes cerastes venom , 2018, Journal of biochemical and molecular toxicology.

[25]  A. Soares,et al.  ASP49‐phospholipase A2‐loaded liposomes as experimental therapy in cutaneous leishmaniasis model , 2018, International immunopharmacology.

[26]  Jef Rozenski,et al.  Astemizole analogues with reduced hERG inhibition as potent antimalarial compounds. , 2017, Bioorganic & medicinal chemistry.

[27]  G. Rádis-Baptista,et al.  Antichagasic effect of crotalicidin, a cathelicidin-like vipericidin, found in Crotalus durissus terrificus rattlesnake's venom gland , 2017, Parasitology.

[28]  J. Guitian,et al.  Past and Ongoing Tsetse and Animal Trypanosomiasis Control Operations in Five African Countries: A Systematic Review , 2016, PLoS neglected tropical diseases.

[29]  M. Soliman,et al.  In vitro antischistosomal activity of venom from the Egyptian snake Cerastes cerastes. , 2016, Revista da Sociedade Brasileira de Medicina Tropical.

[30]  M. Serebryakova,et al.  Peptides from puff adder Bitis arietans venom, novel inhibitors of nicotinic acetylcholine receptors. , 2016, Toxicon : official journal of the International Society on Toxinology.

[31]  A. Nematollahi,et al.  Histopathological study on parasites in freshwater ornamental fishes in Iran , 2016, Journal of Parasitic Diseases.

[32]  B. Reading,et al.  A Diverse Family of Host-Defense Peptides (Piscidins) Exhibit Specialized Anti-Bacterial and Anti-Protozoal Activities in Fishes , 2016, PloS one.

[33]  M. Hayashi,et al.  Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom , 2016, Peptides.

[34]  T. Pons,et al.  The Kunitz-Type Protein ShPI-1 Inhibits Serine Proteases and Voltage-Gated Potassium Channels , 2016, Toxins.

[35]  Y. Utkin,et al.  Quantitative proteomic analysis of Vietnamese krait venoms: Neurotoxins are the major components in Bungarus multicinctus and phospholipases A2 in Bungarus fasciatus. , 2015, Toxicon : official journal of the International Society on Toxinology.

[36]  M. Engstler,et al.  Flagellar motility in eukaryotic human parasites. , 2015, Seminars in cell & developmental biology.

[37]  S. Ghisla,et al.  Evaluating the microbicidal, antiparasitic and antitumor effects of CR-LAAO from Calloselasma rhodostoma venom. , 2015, International journal of biological macromolecules.

[38]  T. Chakraborti,et al.  Effect of different serine protease inhibitors in validating the 115 kDa Leishmania donovani secretory serine protease as chemotherapeutic target. , 2015, Indian journal of biochemistry & biophysics.

[39]  T. Souto-Padrón,et al.  Crovirin, a Snake Venom Cysteine-Rich Secretory Protein (CRISP) with Promising Activity against Trypanosomes and Leishmania , 2014, PLoS neglected tropical diseases.

[40]  L. Kozlov,et al.  A Universal Method for Measuring Functional Activity of Complement in Humans, Laboratory, Domestic, and Agricultural Animals, Amphibians, and Birds , 2014, Bulletin of Experimental Biology and Medicine.

[41]  R. Stábeli,et al.  Purification and Biochemical Characterization of Three Myotoxins from Bothrops mattogrossensis Snake Venom with Toxicity against Leishmania and Tumor Cells , 2014, BioMed research international.

[42]  R. Menna-Barreto,et al.  Effects of a marine serine protease inhibitor on viability and morphology of Trypanosoma cruzi, the agent of Chagas disease. , 2013, Acta tropica.

[43]  A. Gomes,et al.  In vivo and in vitro antileishmanial activity of Bungarus caeruleus snake venom through alteration of immunomodulatory activity. , 2013, Experimental parasitology.

[44]  M. Nonato,et al.  Isolation and biochemical, functional and structural characterization of a novel L-amino acid oxidase from Lachesis muta snake venom. , 2012, Toxicon : official journal of the International Society on Toxinology.

[45]  J. Gutiérrez,et al.  In Vitro Antiplasmodial Activity of Phospholipases A2 and a Phospholipase Homologue Isolated from the Venom of the Snake Bothrops asper , 2012, Toxins.

[46]  J. C. Alarcón,et al.  Antiplasmodial effect of the venom of Crotalus durissus cumanensis, crotoxin complex and Crotoxin B. , 2012, Acta tropica.

[47]  A. Bacic,et al.  Ciliate pellicular proteome identifies novel protein families with characteristic repeat motifs that are common to alveolates. , 2011, Molecular biology and evolution.

[48]  A. Bhattacharyya,et al.  Purification and biochemical characterization of a serine proteinase inhibitor from Derris trifoliata Lour. seeds: insight into structural and antimalarial features. , 2009, Phytochemistry.

[49]  C. Vieira,et al.  Toxoplasma gondii: effects of neuwiedase, a metalloproteinase from Bothrops neuwiedi snake venom, on the invasion and replication of human fibroblasts in vitro. , 2008, Experimental parasitology.

[50]  N. Portman,et al.  Swimming with protists: perception, motility and flagellum assembly , 2008, Nature Reviews Microbiology.

[51]  A. Schwab,et al.  Potassium channels keep mobile cells on the go. , 2008, Physiology.

[52]  Stephan Herminghaus,et al.  Hydrodynamic Flow-Mediated Protein Sorting on the Cell Surface of Trypanosomes , 2007, Cell.

[53]  Y. Utkin,et al.  Toxicity of venoms from vipers of Pelias group to crickets Gryllus assimilis and its relation to snake entomophagy. , 2007, Toxicon : official journal of the International Society on Toxinology.

[54]  Ming Li,et al.  PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. , 2003, Rapid communications in mass spectrometry : RCM.

[55]  D. Suckau,et al.  Screening for disulfide bonds in proteins by MALDI in-source decay and LIFT-TOF/TOF-MS. , 2002, Analytical chemistry.

[56]  E Piccinni,et al.  Tetrahymena pyriformis: a tool for toxicological studies. A review. , 1999, Chemosphere.

[57]  L. Schechter.  The potassium channel blockers 4-aminopyridine and tetraethylammonium increase the spontaneous basal release of [3H]5-hydroxytryptamine in rat hippocampal slices. , 1997, The Journal of pharmacology and experimental therapeutics.

[58]  Y. Chen,et al.  Resolution of isotoxins in the β-bungarotoxin family , 1995 .

[59]  S. W. Chen,et al.  The non-phospholipase A2 subunit of beta-bungarotoxin plays an important role in the phospholipase A2-independent neurotoxic effect: characterization of three isotoxins with a common phospholipase A2 subunit. , 1994, The Biochemical journal.

[60]  C. Benishin.  Potassium channel blockade by the B subunit of beta-bungarotoxin. , 1990, Molecular pharmacology.

[61]  C. Bon,et al.  A sensitive and continuous fluorometric assay for phospholipase A2 using pyrene-labeled phospholipids in the presence of serum albumin. , 1989, Analytical biochemistry.

[62]  A. Harvey,et al.  Potassium channel blocking actions of beta-bungarotoxin and related toxins on mouse and frog motor nerve terminals. , 1988, British journal of pharmacology.

[63]  M. Sokabe,et al.  A cation channel for K+ and Ca2+ from Tetrahymena cilia in planar lipid bilayers. , 1988, Cell structure and function.

[64]  K. Kondo,et al.  Amino acid sequence of beta 2-bungarotoxin from Bungarus multicinctus venom. The amino acid substitutions in the B chains. , 1982, Journal of biochemistry.

[65]  K. Kondo,et al.  Characterization of phospholipase A activity of beta1-bungarotoxin from Bungarus multicinctus venom. II. Identification of the histidine residue of beta1-bungarotoxin modified by p-bromophenacyl bromide. , 1978, Journal of biochemistry.

[66]  K. Jeevaratnam,et al.  Anti‐malarial drugs: Mechanisms underlying their proarrhythmic effects , 2022, British journal of pharmacology.

[67]  T. Souto-Padrón,et al.  Venoms as Sources of Novel Anti-Parasitic Agents , 2015 .

[68]  Y. Chen,et al.  Resolution of isotoxins in the beta-bungarotoxin family. , 1995, Journal of Chromatography A.

[69]  A. Balber.  The pellicle and the membrane of the flagellum, flagellar adhesion zone, and flagellar pocket: functionally discrete surface domains of the bloodstream form of African trypanosomes. , 1990, Critical reviews in immunology.

[70]  P. Stanfield.  Tetraethylammonium ions and the potassium permeability of excitable cells. , 1983, Reviews of physiology, biochemistry and pharmacology.