Heterologous expression of four recombinant toxins from Panamanian scorpions of the genus Tityus and Centruroides for production of antivenom.

BACKGROUND: The development of more effective antivenoms remains a necessity for countries where scorpionism is a public health problem. Also, the regionalization of antivenoms may be important for some countries with special scorpionism characteristics. OBJECTIVE: Production of antibodies capable of neutralizing the lethal effect of the venom of three scorpion species from Panama. METHODS: The primary structures of two neurotoxins from T. pachyurus, one from T. cerroazul and another from C. bicolor were elucidated using N-terminal amino acid degradation and Sanger gene cloned sequencing. The obtained mRNA transcripts were cloned and expressed using E. coli vectors. Different bacterial expression conditions were tested and the best culture conditions for each expressed protein is reported. The expressed scorpion toxins were purified by chromatographic methods and used as immunogens in rabbits. RESULTS: The antibodies produced under the reported immunization scheme show better neutralization (ED50) than other reported commercial antivenoms used to neutralize similar species scorpion venoms under similar LD50 conditions. CONCLUSION: The information reported here shows the proof of concept for selecting recombinant immunogens with the ability to produce antibodies for neutralizing the lethal effects of the most important medical species of scorpions in Panama.

Short-chain consensus alpha-neurotoxin: a synthetic 60-mer peptide with generic traits and enhanced immunogenic properties.

The three-fingered toxin family and more precisely short-chain α-neurotoxins (also known as Type I α-neurotoxins) are crucial in defining the elapid envenomation process, but paradoxically, they are barely neutralized by current elapid snake antivenoms. This work has been focused on the primary structural identity among Type I neurotoxins in order to create a consensus short-chain α-neurotoxin with conserved characteristics. A multiple sequence alignment considering the twelve most toxic short-chain α-neurotoxins reported from the venoms of the elapid genera Acanthophis, Oxyuranus, Walterinnesia, Naja, Dendroaspis and Micrurus led us to propose a short-chain consensus α-neurotoxin, here named ScNtx. The synthetic ScNtx gene was de novo constructed and cloned into the expression vector pQE30 containing a 6His-Tag and an FXa proteolytic cleavage region. Escherichia coli Origami cells transfected with the pQE30/ScNtx vector expressed the recombinant consensus neurotoxin in a soluble form with a yield of 1.5 mg/L of culture medium. The 60-amino acid residue ScNtx contains canonical structural motifs similar to α-neurotoxins from African elapids and its LD50 of 3.8 µg/mice is similar to the most toxic short-chain α-neurotoxins reported from elapid venoms. Furthermore, ScNtx was also able to antagonize muscular, but not neuronal, nicotinic acetylcholine receptors (nAChR). Rabbits immunized with ScNtx were able to immune-recognize short-chain α-neurotoxins within whole elapid venoms. Type I neurotoxins are difficult to isolate and purify from natural sources; therefore, the heterologous expression of molecules such ScNtx, bearing crucial motifs and key amino acids, is a step forward to create common immunogens for developing cost-effective antivenoms with a wider spectrum of efficacy, quality and strong therapeutic value.

Venoms of Centruroides and Tityus species from Panama and their main toxic fractions.

The scorpionism in Panama is notorious for the confluence and coexistence of buthid scorpions from the genera Centruroides and Tityus. This communication describes an overview of the larger representative toxic venom fractions from eight dangerous buthid scorpion species of Panama: Centruroides (C. granosus, C. bicolor, C. limbatus and C. panamensis) and Tityus (T. (A.) asthenes, T. (A.) festae, T. (T.) cerroazul and T. (A.) pachyurus). Their venoms were separated by HPLC and the corresponding sub-fractions were tested for lethality effects on mice and insects. Many fractions toxic to either mice or insects, or both, were found and have had their molecular masses determined by mass spectrometry analysis. The great majority of the lethal components had a molecular mass close to 7000 Da, assumed to be peptides that recognize Na+-channels, responsible for the toxicity symptoms observed in other buthids scorpion venoms. A toxic peptide isolated from the venom of T. pachyurus was sequenced by Edman degradation, allowing the synthesis of nucleotide probe for cloning the correspondent gene. The mature toxin based on the cDNA sequencing has the C-terminal residue amidated, contains 62 amino acid packed by 4 disulfide linkages, with molecular mass of 7099.1 Da. This same toxic peptide seems to be present in scorpions of the species T. pachyurus collected in 5 different regions of Panama, although the overall HPLC profile is quite different. The most diverse neurotoxic venom components from the genus Centruroides were found in the species C. panamensis, whereas T. cerroazul was the one from the genus Tityus. The most common neurotoxins were observed in the venoms of T. festae, T. asthenes and T. pachyurus with closely related molecular masses of 7099.1 and 7332 Da. The information reported here is considered very important for future generation of a neutralizing antivenom against scorpions from Panama. Furthermore, it will contribute to the growing interest in using bioactive toxins from scorpions for drug discovery purposes.

Péptidos con actividad antimicrobiana producidos por microorfanismos nativos / Peptides with antimicrobial activity produced by isolated native microorganisms

Al igual que las proteínas, los péptidos antimicrobianos (PAM) son moléculas versátiles sintetizadas por microorganismos siguiendo rutas enzimáticas, y con interesantes propiedades alimentarias y farmacéuticas, entre ellas la capacidad antibiótica sobre patógenos. La búsqueda convencional de biomoléculas provenientes de microorganismos consiste en analizar químicamente su extracto crudo, lo cual es engorroso y prolongado. El método de aislamiento usado en esta investigación permitió localizar microorganismos con capacidad para producir PAM por interacción de cargas entre moléculas generadas en el metabolismo microbiano y un colorante cargado presente en el medio de cultivo. Se aislaron 20 muestras de suelos de varios pisos térmicos en medios selectivos. Se purificaron 35 cepas con base en la interacción con un colorante básico y se identificaron microorganismos del género Streptomyces sp. y Bacillus sp. Se obtuvieron proteínas de los extractos crudos de fermentaciones, identificando péptidos y aminoácidos por cromatografía de capa fina y electroforesis. Aquellos extractos con alto contenido proteico se evaluaron por bioautografía, y se encontraron 2 extractos de 35 con actividad inhibitoria sobre E. coli ATCC 8739 (halos de 8 mm). Se demuestra la efectividad del método para aislar y purificar microorganismos productores de péptidos cargados y que poseen actividad biológica e industrial de gran interés.

Like proteins, antimicrobial peptides (AMP) are versatile molecules synthesized by microorganisms using enzymatic pathways with no genetic code instruction. AMP have interesting properties in the food and pharmaceutical industries, like their antimicrobial ability against pathogens. Looking for biomolecules from microorganisms requires hard and time consuming chemical analysis of each microorganism extract. The microorganism isolation method proposed in this research allowed us to find antimicrobial peptides produced by bacteria, through interaction between a charged dye mixed with selective agar and metabolites produced by microorganisms. Twenty soil samples from different zones were isolated in selective media; thirty five strains were purified based on interaction between basic dye and charged molecules from bacteria. Streptomyces sp. y Bacillus sp. both genera were identified. Protein extracts were obtained from the isolated microorganisms cultivated in liquid media; peptides and amino acids were identified by thin layer chromatography and electrophoresis. Those extracts with high protein level were used to evaluate bioautography. Two extracts from 35 showed inhibitory activity against E. coli ATCC 8739 (8 mm halo). Method effectiveness for the isolation and the purifying of microorganisms able to produce charged molecules, of industrial interest is demonstrated.