RESUMEN
The most commonly used biopesticides to control agricultural, forest and insect vectors of human diseases are derived from the bacterium Bacillus thuringiensis, which begins to produce Cry and Cyt insecticidal proteins during the onset of the sporulation phase. Some B. thuringiensis strains also produce S-layer proteins that are toxic to certain pests. S-layer proteins are the most abundant proteins in bacteria and archaea. This proteins' key trait to design high performace processes for mass production is their continuous expression during the vegetative phase, unlike Cry and Cyt, which are restricted to the sporulation phase. In this work, a S-layer protein expressed by the GP543 strain of B. thuringiensis that is toxic to the cattle tick Rhipicephalus microplus was mass produced using the batch culture fermentation technique. In addition, the spore-protein complex showed a mortality rate of 75% with a dose of 300 µg·mL-1 on adult females of R. microplus after fourteen days. The lethal concentration 50 was 69.7 µg·mL-1. The treatment also caused a decrease of 13% in the weight of the mass of oviposited eggs with 200 µg·mL-1 of the spore-protein complex and inhibition of the hatching of eggs from 80 to 92%. Therefore, this could be a good option for controlling this parasite. The advantages of S-layer protein synthesis are focused on the production of a new generation of proteins in pest control. This is the first report on the mass production of an S-layer protein that is responsible for toxicity.
Asunto(s)
Bacillus thuringiensis/química , Técnicas Bacteriológicas/métodos , Agentes de Control Biológico/aislamiento & purificación , Microbiología Industrial/métodos , Glicoproteínas de Membrana/aislamiento & purificación , Rhipicephalus/efectos de los fármacos , Animales , Anticuerpos Antibacterianos/biosíntesis , Bacillus thuringiensis/efectos de los fármacos , Bacillus thuringiensis/crecimiento & desarrollo , Bacillus thuringiensis/metabolismo , Agentes de Control Biológico/toxicidad , Biomasa , Reactores Biológicos , Bovinos , Medios de Cultivo/farmacología , Femenino , Fermentación , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/toxicidad , Oviposición/efectos de los fármacos , Óvulo/efectos de los fármacos , Conejos , Esporas BacterianasRESUMEN
Lysinibacillus sphaericus strains belonging the antigenic group H5a5b produce spores with larvicidal activity against larvae of Culex mosquitoes. C7, a new isolated strain, which presents similar biochemical characteristics and Bin toxins in their spores as the reference strain 2362, was, however, more active against larvae of Culex mosquitoes. The contribution of the surface layer protein (S-layer) to this behaviour was envisaged since this envelope protein has been implicated in the pathogenicity of several bacilli, and we had previously reported its association to spores. Microscopic observation by immunofluorescence detection with anti S-layer antibody in the spores confirms their attachment. S-layers and BinA and BinB toxins formed high molecular weight multimers in spores as shown by SDS-PAGE and western blot detection. Purified S-layer from both L. sphaericus C7 and 2362 strain cultures was by itself toxic against Culex sp larvae, however, that from C7 strain was also toxic against Aedes aegypti. Synergistic effect between purified S-layer and spore-crystal preparations was observed against Culex sp. and Aedes aegypti larvae. This effect was more evident with the C7 strain. In silico analyses of the S-layer sequence suggest the presence of chitin-binding and hemolytic domains. Both biochemical characteristics were detected for both S-layers strains that must justify their contribution to pathogenicity.
Asunto(s)
Aedes/efectos de los fármacos , Bacillaceae/química , Culex/efectos de los fármacos , Glicoproteínas de Membrana/toxicidad , Secuencia de Aminoácidos , Animales , Quitina/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/farmacología , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Esporas Bacterianas/químicaRESUMEN
Cysteine-rich secretory proteins (CRiSPs) are widespread in reptile venoms, but most have functions that remain unknown. In the present study we describe the purification and characterization of a CRiSP (patagonin) from the venom of the rear-fanged snake Philodryas patagoniensis, and demonstrate its biological activity. Patagonin is a single-chain protein, exhibiting a molecular mass of 24,858.6 Da, whose NH(2)-terminal and MS/MS-derived sequences are nearly identical to other snake venom CRiSPs. The purified protein hydrolyzed neither azocasein nor fibrinogen, and it could induce no edema, hemorrhage or inhibition of platelet adhesion and aggregation. In addition, patagonin did not inhibit contractions of rat aortic smooth muscle induced by high K(+). However, it caused muscular damage to murine gastrocnemius muscle, an action that has not been previously described for any snake venom CRiSPs. Thus, patagonin will be important for studies of the structure-function and evolutionary relationships of this family of proteins that are widely distributed among snake venoms.