Preferential Entry of Botulinum Neurotoxin A Hc Domain through Intestinal Crypt Cells and Targeting to Cholinergic Neurons of the Mouse Intestine

Botulism, characterized by flaccid paralysis, commonly results from botulinum neurotoxin (BoNT) absorption across the epithelial barrier from the digestive tract and then dissemination through the blood circulation to target autonomic and motor nerve terminals. The trafficking pathway of BoNT/A passage through the intestinal barrier is not yet fully understood. We report that intralumenal administration of purified BoNT/A into mouse ileum segment impaired spontaneous muscle contractions and abolished the smooth muscle contractions evoked by electric field stimulation. Entry of BoNT/A into the mouse upper small intestine was monitored with fluorescent HcA (half C-terminal domain of heavy chain) which interacts with cell surface receptor(s). We show that HcA preferentially recognizes a subset of neuroendocrine intestinal crypt cells, which probably represent the entry site of the toxin through the intestinal barrier, then targets specific neurons in the submucosa and later (90–120 min) in the musculosa. HcA mainly binds to certain cholinergic neurons of both submucosal and myenteric plexuses, but also recognizes, although to a lower extent, other neuronal cells including glutamatergic and serotoninergic neurons in the submucosa. Intestinal cholinergic neuron targeting by HcA could account for the inhibition of intestinal peristaltism and secretion observed in botulism, but the consequences of the targeting to non-cholinergic neurons remains to be determined

Passage de la neurotoxine botulique à travers la barrière intestinale

paralysis, is produced by anaerobic bacteria of the Clostridium genus. BoNTs are classified into 7 types (A to G) and form different complexes by association with non-toxic proteins, including the non-toxic non-hemagglutinin component (NTNH) and hemagglutinins (HAs). En coding genes are clustered in the botulinum locus and organized in two divergent operons, ntnh-bont/A and ha34-ha17-ha70 for type A, whose expressions are positively regulated by the alternative sigma factor BotR. A synchronous expression peak of all the botulinum locus genes is measured by real-time RT-PCR at the transition between exponential and stationary growth phases, concomitantly to toxin accumulation in culture supernatant. In an intestinal epithelium model, purified BoNT/A is transcytosed after binding, via the Hc/A domain, to apical receptors including gangliosides and putative SV2-related proteins. Binding affinity and transport efficiency into intestinal cells are higher with crypt-type than with enterocytetype cell lines. Injected into the lumen of ligatured mouse ileal loops, BoNT/A inhibits smooth muscle contractions and fluorescent Hc/A migrates across mucosa, through some crypt cells, to submucosa and musculosa where it targets certain nerve endings, mostly cholinergic. Hc /A is internalized via different pathways into neuronal cells (dynamin- and clathrin-dependent) and intestinal cells (dynamin- and Cdc42-dependent, and clathrinindependent).flasque, est produite par des bactéries anaérobies du genre Clostridium. Les BoNTs, classifiées en 7 types (A à G), forment divers complexes avec des protéines non toxiques, dont le composant non toxique non hémagglutinant (NTNH) et les hémagglutinines (HAs). Les gènes sont organisés, au sein du locus botulique, selon deux opérons divergents, ntnhbont/ A et ha34-ha17-ha70 pour le type A, dont l'expression est positivement régulée par le facteur sigma alternatif BotR. Un pic d'expression synchrone de tous les gènes du locus de type A est mesuré par RT-PCR en temps réel lors de la transition entre les phases exponentielle et stationnaire de croissance, en parallèle avec l'augmentation du titre en toxine du surnageant de culture. Dans un modèle d'épithélium intestinal, BoNT/A purifiée est transcytosée après liaison via le domaine Hc/A à des récepteurs apicaux comprenant des gangliosides et des protéines potentiellement apparentées à SV2. L'intensité de liaison et l'efficacité de transport de la toxine sont supérieures dans les cellules intestinales de type crypte plutôt qu'entérocyte. Injectés dans la lumière d'une anse iléale ligaturée, BoNT/A inhibe les contractions des muscles lisses et le domaine Hc/A fluorescent progresse de la muqueuse, via certaines cellules des cryptes, vers la sous-muqueuse et la musculeuse où il cible certaines terminaisons nerveuses, majoritairement cholinergiques. Hc/A entre par des voies distinctes dans les cellules neuronales (voie clathrine dépendante de la dynamine) et les cellules intestinales (voie non-clathrine, dépendante de Cdc42 et de la dynamine)

Differential entry of Botulinum Neurotoxin A into neuronal and intestinal cells: an ultrastructural approach

International audienceRecombinant C-terminus (Hc) of botulinum neurotoxin A (BoNT/A) was used to monitor toxin entry into NG108-15 neuronal cells and m-ICcl2 intestinal cells by both fluorescence and electronic microscopy. BoNT/A Hc entered neuronal cells via a clathrin pathway and intestinal cells mainly via non-clathrin vesicles. Upon incubation at 4°C, BoNT/A Hc gold particles were mainly distributed to neuronal cell periphery and to a lower extent to m-ICcl2 cells. After incubation at 37°C, BoNT/A Hc mostly transited in coated vesicles in NG108-15 cells and in uncoated vesicles of m-ICcl2 cells, which support the transcytotic passage of the toxin

Performances des méthodes diagnostiques de la fièvre Q chez les ruminants domestiques : état de l’art et intérêt de l’activité de référence du LNR

International audienceLa fièvre Q est une maladie zoonotique causée par la bactérie Coxiella burnetii, la plupart des cas cliniques humains recensés sont liés à une exposition à des ruminants domestiques excréteurs de la bactérie. Parmi les méthodes diagnostiques des infections par C. burnetii en élevages de ruminants, les méthodes PCR et ELISA sont recommandées respectivement pour le diagnostic direct et indirect par l’EFSA et l’OIE. Cet article fait l’état des lieux des connaissances sur les performances analytiques et diagnostiques de ces méthodes. Il présente également les moyens mis en œuvre par le LNR fièvre Q pour contribuer à standardiser ces méthodes et à valider leurs performances (PCR) ou les évaluer (ELISA), et ainsi garantir comparabilité et fiabilité des résultats fournis par un réseau de laboratoires. Les travaux mis en œuvre par les équipes de recherche (bactériologie, épidémiologie, statistiques) et le LNR ont permis de préciser l’interprétation des résultats diagnostiques de la fièvre Q chez les ruminants domestiques. Les recommandations d’usage de ces tests, dans le contexte de l’imputation d’une série abortive à C. burnetii dans les élevages de ruminants (protocole national du dispositif OSCAR), sont discutées à la lumière des nouvelles connaissances. Poursuivre ces travaux permettra d’appuyer davantage la démarche diagnostique des vétérinaires praticiens, la surveillance nationale et internationale et les échanges

Evaluation of Clostridium botulinum A5 neurotoxin actions in vivo and ex vivo at the mouse skeletal neuromuscular junction

International audienceClostridium botulinum neurotoxins (BoNTs), the most potent toxins known, are the cause of a worldwide lifethreatening disease in humans and animals known as botulism. This disease usually manifests as descending symmetrical flaccid paralysis of skeletal muscles together with autonomic dysfunction. BoNTs include a large family of zinc metalloproteases that can be immunologically distinguished by homologous antitoxins into seven primary serotypes, designated A to G. Additionally, for BoNT/A, at least 8 subtypes (/A1-/A8) have been identified from gene sequence analysis. In France, several cases of human botulism due to BoNT/A5, /A6 or /A7 have been reported, though BoNT/A1 and /A2 are the prevalent forms of type-A botulism. The characterization of the C. botulinum strains involved in two cases of BoNT/A5-poisoning revealed that they possess the gene encoding for BoNT/A5 identical to the one previously reported. In the present study, we produced the BoNT/A5 crude complex (3 x 107 LD50/mg) from the C. botulinum A5 strain 126.07 [1], and studied in vivo and ex vivo, in murine models, the skeletal neuromuscular block caused by BoNT/A5, at different times after a single local toxin injection into the hind-limbs. The results show that the duration and degree of paralysis depended on the dose of BoNT/A5 and on the mouse strain studied. The transgenic Thy-1-YFP-16 black C57BL6 mice [2] were more sensitive to the action of the toxin than Swiss mice, as revealed by the digit abduction score (DAS) assay and by compound muscle action potential (CMAP) recordings from the same mouse in vivo, at different times after toxin injection. functional and morphological ex vivo studies on muscles locally injected with BoNT/A5 in vivo reveal (a) the presence of axonal outgrowths (nodal and nerve terminal sprouts), (b) an extension of nicotinic acetylcholine receptor clusters, (c) a reduction of both muscle weight and muscle fiber cross sectional area, and (d) the prolonged atrophy of muscle fibers. Pre- and post-synaptic remodeling was completely abolished by an immune-purified rabbit polyclonal antibody directed against the BoNT/A1-Heavy chain (HcA1), when injected together with BoNT/A5. In conclusion, our results show that the actions of BoNT/A5 have many similarities to those of BoNT/A1 previously reported, although no direct comparison was performed in the present study. BoNT/A5 differed from BoNT/A1 by 5 identified amino acids in the Light chain domain (Lc) and by 32 amino acids in the Heavy chain domain (Hc). The fact that the antibody directed to the HcA1 completely prevented the lethal effect of BoNT/A5, as well as the block of the CMAP in vivo, and the morphological changes induced by the neurotoxin suggest that the antibody blocks the entry of BoNT/A5 into motor nerve terminals of the neuromuscular junction

Visualization of HcA in neuroendocrine cells from mouse intestinal crypts.

<p>Fluorescent HcA (0.5 µg) was injected into the lumen of a mouse ileum (<b>A, B</b>) or duodenum (<b>C</b>) loop, and after 30 min incubation the intestinal loop was washed and prepared for immunostaining with chromogranin A or serotonin antibodies. (<b>A</b>) Two cells stained with chromogranine A antibodies in an intestinal crypt (dotted circle) were co-labeled with HcA (green). Magnification of one cell (square) shows a uniform punctuate distribution of chromogranin A staining, whereas HcA was preferentially localized at the basal pole. Phase contrast shows that the chromogranin A-immunoreactive cell contained no large granules, in contrast to Paneth cell (black cross). (<b>B</b>) Co-labeling of a cell from an intestinal crypt (dotted circle) with HcA (green) and serotonin antibodies (red). Magnification of the cell (square) shows a basal distribution of both HcA and serotonin. Note that a cell extension was also labeled with serotonin antibodies but not by HcA (arrowhead), and that the serotonin-immunoreactive cell contained no large granules as in Paneth cells (black cross) (scale bars = 10 µm). (<b>C</b>) Co-labeling of HcA (red) with serotonin (green) in a duodenum crypt cell. Neurofilament staining (blue) was observed at the crypt periphery.</p