Structural analysis of Clostridium botulinum neurotoxin type D as a platform for the development of targeted secretion inhibitors.

The botulinum neurotoxin type D is one of seven highly potent toxins produced by Clostridium botulinum which inhibit neurotransmission at cholinergic nerve terminals. A functional fragment derived from the toxin, LHn, consisting of the catalytic and translocation domains, has been heralded as a platform for the development of targeted secretion inhibitors. These secretion inhibitors are aimed at retargeting the toxin towards a specific cell type to inhibit vesicular secretion. Here we report crystal structures of LHn from serotype D at 2.3 Å, and that of SXN101959 at 3.1 Å resolution. SXN101959, a derivative that combines LHn from serotype D with a fragment of the growth hormone releasing hormone, has previously revealed promising results in inhibiting growth hormone release in pituitary somatotrophs. These structures offer for the first time insights into the translocation domain interaction with the catalytic domain in serotype D. Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B, and D. Taken together, these results demonstrate the robustness of the 'LHn fold' across serotypes and its use in engineering additional polypeptide components with added functionality. Our study demonstrates the suitability of botulinum neurotoxin, and serotype D in particular, as a basis for engineering novel secretion inhibitors.

The C terminus of the catalytic domain of type A botulinum neurotoxin may facilitate product release from the active site.

Botulinum neurotoxins are the most toxic of all compounds. The toxicity is related to a poor zinc endopeptidase activity located in a 50-kDa domain known as light chain (Lc) of the toxin. The C-terminal tail of Lc is not visible in any of the currently available x-ray structures, and it has no known function but undergoes autocatalytic truncations during purification and storage. By synthesizing C-terminal peptides of various lengths, in this study, we have shown that these peptides competitively inhibit the normal catalytic activity of Lc of serotype A (LcA) and have defined the length of the mature LcA to consist of the first 444 residues. Two catalytically inactive mutants also inhibited LcA activity. Our results suggested that the C terminus of LcA might interact at or near its own active site. By using synthetic C-terminal peptides from LcB, LcC1, LcD, LcE, and LcF and their respective substrate peptides, we have shown that the inhibition of activity is specific only for LcA. Although a potent inhibitor with a Ki of 4.5 µm, the largest of our LcA C-terminal peptides stimulated LcA activity when added at near-stoichiometric concentration to three versions of LcA differing in their C-terminal lengths. The result suggested a product removal role of the LcA C terminus. This suggestion is supported by a weak but specific interaction determined by isothermal titration calorimetry between an LcA C-terminal peptide and N-terminal product from a peptide substrate of LcA. Our results also underscore the importance of using a mature LcA as an inhibitor screening target.

Phenotypic characterization of Clostridium botulinum strains isolated from infant botulism cases in Argentina / Caracterización fenotípica de cepas de Clostridium botulinum aisladas de casos de botulismo del lactante en Argentina

Infant botulism is the most common form of human botulism; however, its transmission has not been completely explained yet. Some of the most recognized potential sources of Clostridium botulinum spores are the soil, dust, honey and medicinal herbs. In Argentina, 456 cases of infant botulism were reported between 1982 and 2007. C. botulinum type A was identified in 455 of these cases whereas type B was identified in just one case. However, in Argentina, types A, B, E, F, G, and Af have been isolated from environmental sources. It is not clearly known if strains isolated from infant botulism cases have different characteristics from strains isolated from other sources. During this study, 46 C. botulinum strains isolated from infant botulism cases and from environmental sources were typified according to phenotypic characteristics. Biochemical tests, antimicrobial activity, and haemagglutinin-negative botulinum neurotoxin production showed uniformity among all these strains. Despite the variability observed in the botulinum neurotoxin's binding to cellular receptors, no correlation was found between these patterns and the source of the botulinum neurotoxin. However, an apparent geographical clustering was observed, since strains isolated from Argentina had similar characteristics to those isolated from Italy and Japan, but different to those isolated from the United States.

El botulismo del lactante es la forma más común del botulismo humano; sin embargo, su forma de transmisión no ha sido totalmente explicada. El suelo, el polvo ambiental, la miel y algunas hierbas medicinales son potenciales fuentes de esporas de Clostridium botulinum. Entre 1982 y 2007 se informaron en Argentina 456 casos de botulismo del lactante, 455 casos debidos al serotipo A y uno al serotipo B. Sin embargo, los serotipos A, B, E, F, G y Af han sido aislados de suelos y otras fuentes en Argentina. No se conoce si las cepas aisladas de casos de botulismo del lactante poseen características diferentes de las cepas aisladas de otras fuentes. Durante este estudio se caracterizaron 46 cepas de C. botulinum. Las pruebas bioquímicas y de sensibilidad a los antimicrobianos y la producción de neurotoxina botulínica hemaglutinina-negativa mostraron uniformidad entre estas cepas. A pesar de la variabilidad observada respecto de la unión de la neurotoxina a receptores celulares, no se observó una correlación entre estos patrones de unión y la fuente de aislamiento. Sin embargo, se observó una aparente agrupación geográfica, ya que las cepas aisladas en Argentina tuvieron características similares a las observadas en las cepas aisladas en Italia y Japón, pero diferentes de las que se registraron en las cepas aisladas en los Estados Unidos.

Molecular characterization of the protease from Clostridium botulinum serotype C responsible for nicking in botulinum neurotoxin complex.

A protease was purified from the culture medium of Clostridium botulinum serotype C strain Stockholm (C-St). The purified protease belonged to the cysteine protease family based on assays for enzyme inhibitors, activators and kinetic parameters. The protease formed a binary complex consisting of 41- and 17-kDa proteins held together non-covalently. The DNA sequence encoding the protease gene was shown to be a single open reading frame of 1593 nucleotides, predicting 530 amino acid residues including a signal peptide. The N-terminal region of the native enzyme underwent further proteolytic modification after processing by a signal peptidase. The protease introduced intermolecular cleavage into an intact single chain botulinum neurotoxin (BoNT) at a specific site. Homology modeling and docking simulation of C-St BoNT and C-St protease demonstrated that the specific nicking-site of the BoNT appears to fit into the deep pocket in the active site of the protease.

Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855-1296 with antitoxin antibodies from three host species.

Botulism due to food poisoning is caused mainly by protein toxins, botulinum neurotoxins (BoNTs), produced by Clostridium botluinum in seven known immunological serotypes. These are the most potent toxins and poisons known. BoNT effects blockade of neuromuscular transmission by preventing neurotransmitter release. Human botulism is most frequently caused by types A, B, and E. Recent studies have shown that immunization with a 43-kDa C-terminal fragment (Hc, residues 860-1296) of BoNT/A affords excellent protection against BoNT/A poisoning. We raised antibodies (Abs) against BoNT/A in horse, and against pentavalent toxoid (BoNTs A, B, C, D, E) in human volunteers and outbred mice. Thirty-one 19-residue peptides that started at residue 855, overlapped consecutively by 5 residues, and encompassed the entire length of the Hc of BoNT/A were synthesized and used for mapping the Ab-binding regions recognized by the anti-BoNT/A antisera. Horse Abs against BoBT/A were bound by peptides 855-873, 939-957, 1079-1097/1093-1111 overlap, 1191-1209/1205-1223 overlap, 1261-1279 and 1275-1296. In addition, peptides 883-901, 911-929, 995-1013, 1023-1041/1037-1055 overlap, 1121-1139, and 1149-1167 gave low, but significant and reproducible, binding. With human antisera, high amounts of Abs were bound by peptides 869-887, 925-943, 981-999, 995-1013, 1051-1069, and 1177-1195. In addition, lower amounts of Abs were bound by peptides 911-929, 939-957, 967-985, and the overlaps 1121-1139/1135-1153 and 1247-1265/1261-1279/1275-1296. With outbred mouse antisera, high amounts of Abs were bound by peptides 869-887, 1051-1069, and 1177-1195, while peptides 939-957, 995-1013, 1093-1111, and 1275-1296 bound lower amounts of Abs. The results indicate that horse antiserum against BoNT/A or human and mouse (outbred) antisera against the toxoid recognized similar regions on BoNT/A, but exhibited some boundary frame shifts and differences in immunodominance of these regions among the antisera. Selected synthetic epitopes will be used as immunogens to stimulate active or passive (by Ab transfer) immunity against toxin poisoning.

Diagnóstico bacteriológico de un caso de botulismo infantil y primer aislamiento de C. botulinum en Chile / Bacteriological diagnosis of an infant botulism case and first isolation of C. botulinum in Chile

En noviembre de 1994 se recibió, en el Laboratorio de Anaerobicos del Instituto de Salud Pública de Chile, una muestra de deposición de una lactante de dos meses de edad con el diagnóstico presuntivo de botulismo infantil. La muestra fus sembrada en los medios de cultivo correspondientes y en atmósfera anaerobia hasta obtener un cultivo puro. Este fue sometido a diferentes pruebas bioquímicas, las cuales fueron coincidentes con Clostridium botulinum grupo I. POsteriormente se procedió a confirmar el diagnóstico realizando el test de patogenicidad y neutralización en ratones CF-1. En base a los resultados observados se concluyó que la cepa aislada correspondía a clostridium botulinum grupo I tipo A, lo cual fue confirmado por el Centers for Disease Control de Atlanta. Este fue el primer aislamiento de C. botulinum a partir de una muestra clínica en Chile

Clostridium botulinum: cronologia das descobertas, caracterizaçäo, manifestaçöes clínicas, diagnóstico e controle / Clostridium botulinum: chronological discovery, characteristics, clinical manifestations, diagnosis and control

Nesta revisäo bibliográfica säo apresentados, em ordem cronológica, as principais descobertas sobre o Clostridium botulinum, suas principais características, as manifestaçöes clínicas e o diagnóstico do botulismo, bem como o isolamento da bactéria. Também säo feitos comentários sobre os principais fatores no controle do desenvolvimento de Clostridium botulinum em alimentos

Characterization, self-assembly and reattachment of S layer from Clostridium botulinum type E saroma.

S layer of Clostridium botulinum type E Saroma and its subunits were isolated and characterized for their chemical and morphological properties. The S layer was composed of a number of subunits with apparent molecular weights ranging from about 10 to 150 kDa. The isolated S layer subunits possess the ability to assemble into recrystallized flat sheets in the absence of any supporting layer and to reattach to the cell wall from which they have been removed. Immunoblot analysis using an antiserum against whole cells of the organism showed that 60 kDa and 90 kDa subunits of the S layer were major somatic antigens of the organism. Immunogold-labeling using monospecific antiserum raised to the individual 60 and 90 kDa proteins revealed that both subunits were exposed evenly over the entire cell surface. The amino acid compositions of both subunits showed that aspartate and glutamate were predominant whereas cystine and methionine were poor. The amino acid composition and acidic property of the two subunits of the S layer agree well with the results obtained from the S layers of other bacterial species as well as other pathogenic clostridia.

Clostridium botulinum tipos D y A en materiales de necropsia de bovinos afectados por el mal de Aguapey / Clostridium botulinum type D and A in necropsy materials of bovines affected by Aguapey disease

Se realizaron exámenes bacteriológicos en materiales de necropsia de cinco animales muertos con diagnóstico clínico de botulismo bovino, enfermedad enzoótica regional llamada Mal de Aguapey (Provincia de Corrientes). Se identificó C. botulinum tipo D o su toxina en todos los animales, alternativamente en contenidos de rumen, yeyuno, íleon y ciego, y en muestras obtenidas de bazo, riñon e hígado. C. botulinum tipo A fue identificado, respectivamente, en hígado y riñón de dos animales. En los cultivos de cien muestras de diferentes tipos de suelo tomados en el área enzoótica, se identificó toxina butulínica sólo del tipo A en tres y en ninguna tipo D. Estos resultados amplían y confirman los hallazgos previos realizados sobre la etiología botulínica del Mal de Aguapey

Characterization of proteins, insoluble at low temperature, produced by Clostridium botulinum type C and C. subterminale. Their antigenic relationship with a similar protein synthesized by C. botulinum type G.

The production of a protein insoluble at low temperature ("cryoprotein"), by cultures of Clostridium botulinum type G has been shown to be a metabolic characteristic also shared by C. botulinum type C and by C. subterminale. These new cryoproteins have been purified and some of their chemical and immunological properties studied. It was found that both proteins were chemically very similar among themselves and to the cryoprotein isolated from C. botulinum type G. All these proteins are formed by a single polypeptide chain of approximately Mr = 180,000, with closely related amino acid compositions, isoelectric points and do not contain either free cysteine or disulfide bridges. Homologous and heterologous radioimmunoassays established the existence of an antigenic similitude among the cryoproteins from C. botulinum type G and C. subterminale thus becoming the first purified antigens which relate both bacterial species. If the production of cryoproteins can be shown to be a generalized phenomenon within the genus Clostridium these substances would provide an important tool to examine immunological and genetical relatedness between strains in this bacterial group.

Botulism: a pyrolysis-gas-liquid chromatographic study.

Forty samples of dried Clostridia bacteria were subjected to pyrolysis-gas-liquid chromatography (PGLC). Examination of the key fingerprint peaks enabled the analyst to differentiate the samples into their respective antigenic groups. Peaks occurring at the high boiling end of profile could be used to distinguish proteolytic from non-proteolytic strains of C-botulinum. PGLC has proven to be a highly reproducible as well as a rapid specific method for differentiating and identifying samples of Clostridium botulinum.

Characterization of clostridia by gas chromatography differentiation of species by trimethylsilyl derivatives of whole-cell hydrolysates.

Trimethylsilyl (TMS) derivatives prepared from whole-cell hydrolysates of 36 strains, representing 10 species of Clostridium were examined by gas-liquid chromatography (GLC). The TMS profile of each species contained a group of peaks which characterized the species. Variation among strains within a species was much lower than variation between species. Some of the closely related clostridia could be differentiated by comparing their TMS profiles. Strains of Clostridium botulinum were distinguished from C. sporogenes on the basis of the ratio of two GLC peaks which corresponded to arabinose and glucose. A peak with a retention time identical to that of mannose was present in all C. bifermentans strains but was absent in those of C. sordellii.

Differentiation of Clostridium botulinum types A, B, and E by pyrolysis-gas-liquid chromatography.

Vegetative cells and spores of 10 strains of Clostridium botulinum representing types A, B, and E were grown in Trypticase-peptone-sucrose-yeast extract (TPSY) medium. Five type E strains were also grown in Multipeptone-sucrose-Nutramino acids (MSN) medium. Lyophilized samples were subjected to pyrolysis-gas-liquid chromatography (PGLC) analysis, and the resulting pyrograms were examined for variations in elution patterns between spores and vegetative cells of types A, B, and E grown in the TPSY medium and spores and vegetative cells of type E grown in the TPSY medium and spores and vegetative cells of type E grown in TPSY and MSN media. Growth and toxin production of all 10 strains of C. botulinum were investigated by using a modified dialysis sac culture technique. The dialysate supernatant fluid (DSF) obtained after centrifugation of the 5-day-old cultures from the dialysate was also subjected to PGLC analysis. Control samples consisting of (i) noninoculated DSF, (ii) noninoculated DSF plus partially purified toxin, and (iii) 1.0 mg of partially purified toxin were also analyzed by PGLC. Differences between pyrograms of cultures were suitable for positive identification at the type level but not at the strain level. Pyrograms permitting differentiation were also obtained between spores and vegetative cells as well as between the same cultures grown in different media. The dialysis sac technique was useful in detecting growth but not toxin production of C. botulinum.