Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase.

GumK is a membrane-associated glucuronosyltransferase of Xanthomonas campestris that is involved in xanthan gum biosynthesis. GumK belongs to the inverting GT-B superfamily and catalyzes the transfer of a glucuronic acid (GlcA) residue from uridine diphosphate (UDP)-GlcA (UDP-GlcA) to a lipid-PP-trisaccharide embedded in the membrane of the bacteria. The structure of GumK was previously described in its apo- and UDP-bound forms, with no significant conformational differences being observed. Here, we study the behavior of GumK toward its donor substrate UDP-GlcA. Turbidity measurements revealed that the interaction of GumK with UDP-GlcA produces aggregation of protein molecules under specific conditions. Moreover, limited proteolysis assays demonstrated protection of enzymatic digestion when UDP-GlcA is present, and this protection is promoted by substrate binding. Circular dichroism spectroscopy also revealed changes in the GumK tertiary structure after UDP-GlcA addition. According to the obtained emission fluorescence results, we suggest the possibility of exposure of hydrophobic residues upon UDP-GlcA binding. We present in silico-built models of GumK complexed with UDP-GlcA as well as its analogs UDP-glucose and UDP-galacturonic acid. Through molecular dynamics simulations, we also show that a relative movement between the domains appears to be specific and to be triggered by UDP-GlcA. The results presented here strongly suggest that GumK undergoes a conformational change upon donor substrate binding, likely bringing the two Rossmann fold domains closer together and triggering a change in the N-terminal domain, with consequent generation of the acceptor substrate binding site.

Biophysical characterization of the outer membrane polysaccharide export protein and the polysaccharide co-polymerase protein from Xanthomonas campestris.

This study investigated the structural and biophysical characteristics of GumB and GumC, two Xanthomonas campestris membrane proteins that are involved in xanthan biosynthesis. Xanthan is an exopolysaccharide that is thought to be a virulence factor that contributes to bacterial in planta growth. It also is one of the most important industrial biopolymers. The first steps of xanthan biosynthesis are well understood, but the polymerization and export mechanisms remain unclear. For this reason, the key proteins must be characterized to better understand these processes. Here we characterized, by biochemical and biophysical techniques, GumB, the outer membrane polysaccharide export protein, and GumC, the polysaccharide co-polymerase protein of the xanthan biosynthesis system. Our results suggested that recombinant GumB is a tetrameric protein in solution. On the other hand, we observed that both native and recombinant GumC present oligomeric conformation consistent with dimers and higher-order oligomers. The transmembrane segments of GumC are required for GumC expression and/or stability. These initial results provide a starting point for additional studies that will clarify the roles of GumB and GumC in the xanthan polymerization and export processes and further elucidate their functions and mechanisms of action.

Linden flower (Tilia spp.) as potential vehicle of Clostridium botulinum spores in the transmission of infant botulism / El té de tilo como vehículo potencial de esporas de Clostridium botulinum en la transmisión del botulismo infantil

Infant botulism is an intestinal toxemia caused principally by Clostridium botulinum. Since the infection occurs in the intestinal tract, numerous food products have been investigated for the presence of C. botulinum and its neurotoxins. In many countries, people use linden flower (Tilia spp) tea as a household remedy and give it to infants as a sedative. Therefore, to help provide a clear picture of this disease transmission, we investigated the presence of botulinum spores in linden flowers. In this study, we analyzed 100 samples of unwrapped linden flowers and 100 samples of linden flowers in tea bags to determine the prevalence and spore-load of C. botulinum. Results were analyzed by the Fisher test. We detected a prevalence of 3% of botulinum spores in the unwrapped linden flowers analyzed and a spore load of 30 spores per 100 grams. None of the industrialized linden flowers analyzed were contaminated with botulinum spores. C. botulinum type A was identified in two samples and type B in one sample. Linden flowers must be considered a potential vehicle of C. botulinum, and the ingestion of linden flower tea can represent a risk factor for infant botulism.

El botulismo del lactante es una toxiinfección causada, principalmente, por Clostridium botulinum. Debido a que esta infección ocurre en el tracto intestinal, la presencia de esta bacteria y sus neurotoxinas ha sido investigada en numerosos alimentos. En muchos países se utiliza el té de tilo (Tilia spp.) como sedante natural, el que se administra incluso a los lactantes. A fin de contribuir al esclarecimiento de la transmisión de esta enfermedad, se investigó la prevalencia y la carga de esporas botulínicas en esta hierba. Se analizaron 100 muestras de tilo comercializado a granel y 100 muestras de tilo industralizado en “saquitos”. Los resultados de prevalencia fueron analizados por el test de Fisher y la carga de esporas por la técnica del número más probable. Se halló una prevalencia de esporas de C. botulinum del 3% en el tilo comercializado a granel, con una carga de 30 esporas/100 g de hierba. En tanto, ninguna de las muestras en saquitos acusó la presencia del patógeno. Se identificaron tres cepas de C. botulinum, dos tipo A y una tipo B. En virtud de estos resultados, el tilo podría considerarse un potencial vehículo de esporas de C. botulinum y la administración de sus infusiones a menores y lactantes, un riesgo para la transmisión de la enfermedad.

Phenotypic characterization of Clostridium botulinum strains isolated from infant botulism cases in 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.

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.

Linden flower (Tilia spp.) as potential vehicle of Clostridium botulinum spores in the transmission of infant botulism.

Infant botulism is an intestinal toxemia caused principally by Clostridium botulinum. Since the infection occurs in the intestinal tract, numerous food products have been investigated for the presence of C. botulinum and its neurotoxins. In many countries, people use linden flower (Tilia spp) tea as a household remedy and give it to infants as a sedative. Therefore, to help provide a clear picture of this disease transmission, we investigated the presence of botulinum spores in linden flowers. In this study, we analyzed 100 samples of unwrapped linden flowers and 100 samples of linden flowers in tea bags to determine the prevalence and spore-load of C. botulinum. Results were analyzed by the Fisher test. We detected a prevalence of 3% of botulinum spores in the unwrapped linden flowers analyzed and a spore load of 30 spores per 100 grams. None of the industrialized linden flowers analyzed were contaminated with botulinum spores. C. botulinum type A was identified in two samples and type B in one sample. Linden flowers must be considered a potential vehicle of C. botulinum, and the ingestion of linden flower tea can represent a risk factor for infant botulism.

[Presence of botulinum-producing clostridia in the soils of Entre Ríos]. / Presencia de clostridios productores de toxina botulínica en suelos de Entre Ríos.

Botulism, well known for centuries, continues to preoccupy the Public Health authorities, food and agricultural industries. In its oldest form it results from ingestion of food containing botulinum toxin. To correctly evaluate the incidence of botulism it is fundamental to know the distribution of botulinum toxin-producing clostridia (Clostridium botulinum, C. baratii, C. butyricum and C. argentinense) in nature, specially in the soil, its main reservoir. In our country, this study has been carried out in a partial way. With the aim to contribute to such knowledge, 240 soil samples from Entre Ríos province were examined for C. botulinum and 35 (14.6%) samples resulted positive.

Presencia de clostridios productores de toxina botulínica en suelos de Entre Ríos / Presence of botulinum toxin-producing clostridia in the soil of Entre Ríos

El botulismo, reconocido desde hace siglos, continúa preocupando a las autoridades de salud pública y a las industrias alimentaria y agropecuaria. En su forma más antigua, resulta de la ingestión de alimentos conteniendo toxina botulínica. Para evaluar correctamente el riesgo de la incidencia del botulismo, es fundamental el conocimiento de la distribución de clostridios productores de toxina botulínica (Clostridium botulinum, C.baratii, C.butyricum y C.argentinense) en el medio natural, sobre todo en el suelo, su principal reservorio. En nuestro país este estudio se ha realizado en forma parcial. Con el objetivo de contribuir con dicho conocimiento se analizaron 240 muestras provenientes de la provincia de Entre Ríos, de las cuales 35 (17,6 por ciento ) resultaron positivas.

Presencia de clostridios productores de toxina botulínica en suelos de Entre Ríos / Presence of botulinum toxin-producing clostridia in the soil of Entre Ríos

El botulismo, reconocido desde hace siglos, continúa preocupando a las autoridades de salud pública y a las industrias alimentaria y agropecuaria. En su forma más antigua, resulta de la ingestión de alimentos conteniendo toxina botulínica. Para evaluar correctamente el riesgo de la incidencia del botulismo, es fundamental el conocimiento de la distribución de clostridios productores de toxina botulínica (Clostridium botulinum, C.baratii, C.butyricum y C.argentinense) en el medio natural, sobre todo en el suelo, su principal reservorio. En nuestro país este estudio se ha realizado en forma parcial. Con el objetivo de contribuir con dicho conocimiento se analizaron 240 muestras provenientes de la provincia de Entre Ríos, de las cuales 35 (17,6 por ciento ) resultaron positivas. (AU)

Presencia de clostridios productores de toxina botulínica en suelos de Entre Ríos. / [Presence of botulinum-producing clostridia in the soils of Entre Ríos]

Botulism, well known for centuries, continues to preoccupy the Public Health authorities, food and agricultural industries. In its oldest form it results from ingestion of food containing botulinum toxin. To correctly evaluate the incidence of botulism it is fundamental to know the distribution of botulinum toxin-producing clostridia (Clostridium botulinum, C. baratii, C. butyricum and C. argentinense) in nature, specially in the soil, its main reservoir. In our country, this study has been carried out in a partial way. With the aim to contribute to such knowledge, 240 soil samples from Entre Ríos province were examined for C. botulinum and 35 (14.6

) samples resulted positive.