Botulinum neurotoxin A complex recognizes host carbohydrates through its hemagglutinin component.

Botulinum neurotoxins (BoNTs) are potent bacterial toxins. The high oral toxicity of BoNTs is largely attributed to the progenitor toxin complex (PTC), which is assembled from BoNT and nontoxic neurotoxin-associated proteins (NAPs) that are produced together with BoNT in bacteria. Here, we performed ex vivo studies to examine binding of the highly homogeneous recombinant NAPs to mouse small intestine. We also carried out the first comprehensive glycan array screening with the hemagglutinin (HA) component of NAPs. Our data confirmed that intestinal binding of the PTC is partly mediated by the HA moiety through multivalent interactions between HA and host carbohydrates. The specific HA-carbohydrate recognition could be inhibited by receptor-mimicking saccharides

A Dynamic Leadership Approach in Different Situations and Contexts

This paper is one of the first to provide empirical evidence supporting the conceptualization of a dynamic leadership approach in which employee maturity and organizational dynamism help leaders make the right choice of leadership styles to improve employee job satisfaction. As business environments change and employee maturity differs in this dynamic business era, and as there is a chaos characterized by numerous leadership styles in the leadership literature, adopting a dynamic leadership approach has become important to identify the most effective leadership styles suitable for employees. A quantitative study using conditional process analysis was conducted to examine a dynamic leadership approach that draws on servant/transactional/transformational leadership to influence employee job satisfaction in stable and turbulent environments. Results indicated that leaders could employ transactional/transformational/servant leadership styles to further improve employee job satisfaction and should adopt servant leadership when the organizational environment is stable or weakly dynamic and should adopt transformational leadership when the environment is moderately dynamic. Results also show that highly mature employees mediate the relationship between transformational/servant leadership and job satisfaction. Findings supported the development of a new dynamic leadership approach in which leaders can skillfully change their leadership style over time by navigating through multiple leadership styles as appropriate depending on the organizational dynamism and employee maturity levels

A Genomic and Proteomic Investigation Into the <i>Clostridium botulinum</i> Neurotoxin Complex

Clostridium botulinum and some strains of C. baratii and C. butyricum produce one of the most potent toxins known to man, botulinum neurotoxin, and are responsible for the disease botulism. This severe neuroparalytic disease is the result of botulinum neurotoxin negotiating a complex path to the cholinergic nerve endings. There, it interferes with the release of excitatory neurotransmitters resulting in flaccid paralysis and if untreated, death. The neurotoxin, itself a multi-faceted protein, does not act alone but is produced as part of a large, hetero-multimeric complex with the associated non-toxic proteins. This complex, known to protect the toxin from acids and proteases in the gut, has recently been suggested to play a more active role in toxicity. Here, the proteins from the lesser-studied toxin complex type (the OrfX type) are shown for the first time to share sequence similarity and synteny with clusters of proteins that are co-localised with various putative toxin genes in diverse other species. The extracellular supernatant proteome of C. butyricum is characterised and mined for potential novel virulence factors, with metabolic cost of extracellular protein being highlighted as a potential marker of virulence associated extracellular proteins. The supernatant of 22 clinical strains of C. butyricum were investigated for the presence of the toxin complex; all toxin complex components were identified in the majority of strains indicating the importance of these proteins in the causation of botulism. A relationship between OrfX-encoding strains and infant botulism was also uncovered in clinical strains from the UK. Hypotheses to explain this association are explored. Finally, the transcriptome of C. butyricum was investigated using RNA-sequencing. This uncovered a complex and diverse picture of transcription in C. butyricum and raised questions as to the role of the alternative sigma factor BotR in the regulation of bont

Tables of toxicity of botulinum and tetanus neurotoxins

Tetanus and botulinum neurotoxins are the most poisonous substances known, so much so as to be considered for a possible terrorist use. At the same time, botulinum neurotoxin type A1 is successfully used to treat a variety of human syndromes characterized by hyperactive cholinergic nerve terminals. The extreme toxicity of these neurotoxins is due to their neurospecificity and to their metalloprotease activity, which results in the deadly paralysis of tetanus and botulism. Recently, many novel botulinum neurotoxins and some botulinum-like toxins have been discovered. This large number of toxins differs in terms of toxicity and biological activity, providing a potential goldmine for novel therapeutics and for new molecular tools to dissect vesicular trafficking, fusion, and exocytosis. The scattered data on toxicity present in the literature require a systematic organization to be usable by scientists and clinicians. We have assembled here the data available in the literature on the toxicity of these toxins in different animal species. The internal comparison of these data provides insights on the biological activity of these toxins

Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

In the framework of the EU project EQuATox a first international proficiency test (PT) on the detection and quantification of BoNT was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. A variety of methods was applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo / in vitro approaches (mouse bioassay, hemidiaphrama assay, Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently seen as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need of certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphrama assay delivered quantitative results superior to the mouse bioassay.JRC.D.2-Standards for Innovation and sustainable Developmen

Botolinum toxins: their structure, properties, and genetics

Clostridium botulinum is Gram positive, spore-forming anaerobic bacteria, which can produce botulinum neurotoxins (BoNTs). The toxins block the release of neurotransmitter, acetylcholine, at peripheral cholinergic nerve terminal and cause flaccid paralysis of muscle in human and animals, a condition known as botulism. BoNTs are classified into seven different serotypes (designated as BoNT/ A-BoNT/G), in which serotype A, B, E, and F cause botulism in human. BoNTs are comprised of one domain of light chain (L-chain) at N-terminus and two domains of heavy chains (H-chain) at C-terminus. The function of L-chain is to cleave SNARE (soluble N-ethylmaleimide-sensitive factor attachmentprotein receptors) proteins that involve in the exocytosis of neurotransmitter whereas H-chain is responsible for binding of toxin with nerve terminal and translocating of L-chain into cytosol from synaptic vesicle. The BoNTs are usually produced as complexes called progenitor toxin complex (PTC). They bind together with neurotoxin-associated proteins (NAPs), which are haemagglutinin (HA) and non-toxin non-haemagglutinin (NTNH). The NAPs can protect BoNTs from gastrointestinal environment and facilitate the absorption andtranslocation of neurotoxin into main circulation. The genes encoding BoNTs and NAPs are arranged as gene cluster, which are organized in two operons: ha and orfX operons. Mostly, they are located on the chromosome, large plasmid, or bacteriophage at the specific location and can be transferred horizontallyto other clostridia strains

Clostridium botulinumin geeniaktiivisuus ja neurotoksiinigeenien aktiivisuuteen vaikuttavat tekijät

Clostridium botulinum on elintarvikevälitteinen patogeeni, joka tuottaa voimakkainta tunnettua myrkkyä botuliinitoksiinia. Botuliini on hermomyrkky, joka aiheuttaa botulismia sekä ihmisille että eläimille estämällä välittäjäaineen vapautumisen hermo-lihasliitoksessa. Hengityslihaksistoon edetessään botulismi on hoitamattomana mahdollisesti kuolemaan johtava sairaus. C. botulinum kannan ATCC 3502 botuliinitoksiinikompleksi koostuu toksiiniosasta (BoNT/A1), nonhemagglutiniiniosasta (NTNH) ja kolmesta hemagglutiniiniosasta (HA-17, HA-33 ja HA-70). Toksiinikompleksin osia koodaavat geenit sijaitsevat samassa lokuksessa ja muodostavat kaksi erillistä operonia. Operonien välissä sijaitsee geeni (botR), joka koodaa toksiinituotantoa säätelevää BotR-sigmatekijää. C. botulinum tuottaa itiöitä, jotka ovat ympäristössä hyvin kestäviä. Itiöitymisen pääsäätelijänä toimii Spo0A, joka aktivoituessaan aktivoi edelleen sigmatekijäkaskadin. Tässä säätelykaskadissa emosolun puolella itiöitymistä ohjaavia rakennegeenejä säätelevät SigE ja SigK, ja esi-itiön puolella SigF ja SigG. Tutkimus koostui kahdesta eri osasta. Tutkimuksen metodologisessa osassa I oli tarkoituksena arvioida pienistä bakteeripopulaatioista saatavaa RNA-määrää ja sen laatua. RNA:n tutkimusmenetelmiin vaaditaan tyypillisesti suuria määriä hyvälaatuista RNA:ta, ja menetelmästä riippuen tarvittava määrä vaihtelee 200 nanogrammasta 5:een mikrogrammaan. Tarkoituksena oli selvittää, kuinka paljon bakteerisoluja tarvitaan RNA-sekvensointiin ja RT-qPCR-menetelmään. Tutkimuksessa käytetyt bakteerisolupelletit säilytettiin -70°C:ssa ja niistä eristettiin RNA. Tutkimuksen biologisessa osassa II oli tarkoituksena vertailla kahden eri C. botulinum ATCC 3502 -kannan isolaatin transkriptioprofiilia toksiinituotantoon ja itiöitymiseen liittyvien geenien osalta. Isolaattien transkriptioprofiilin avulla saatiin tietoa siitä, mitkä geenit ilmentyvät solussa tietyllä ajanhetkellä. ATCC 3502 -kannan isolaatti tox+ tuottaa botuliinia, kun taas kannasta laboratorio-olosuhteissa spontaanisti mutatoitunut isolaatti tox- tuottaa botuliinia huomattavasti vähemmän. Neurotoksiinituotantokyvyn lisäksi myös isolaattien itiöitymiskyvyn tiedetään eroavan toisistaan. Tox--isolaatin itiöitymiskyky on aikaisempien tutkimusten perusteella heikentynyt. Tox+- ja tox--isolaattien aiempi genomianalyysi toi esiin tärkeitä eroavaisuuksia niiden genotyyppien ja fenotyyppien suhteen. Tox--isolaatin vaihtoehtoista sigmatekijää BotR koodaavasta geenistä (botR) löydettiin geenin lukukehyksen muuttava insertiomutaatio. Molemmat isolaatit (tox+ ja tox-) kasvatettiin identtisissä olosuhteissa (anaerobinen TPGY-liemikasvatusalusta, 37 °C), ja bakteereiden eri kasvuvaiheista eristettiin solunäytteet. Bakteerisoluista eristettiin RNA, jonka perusteella valmistettua cDNA:ta käytettiin templaattina kvantitatiivisessa reaaliaikaisessa RT-qPCR-analyysissa. RT-qPCR-menetelmän avulla tutkittiin kohdegeenien (botA, ha33, spo0A, sigF, sigE ja sigG) ilmentymistä bakteerin eri kasvuvaiheissa. Referenssigeeninä käytettiin 16S rRNA:ta. Geenien esiintymiselle muodostettiin suhdeluvut, joiden perusteella isolaattien transkriptioprofiileja verrattiin toisiinsa. Tutkimuksen osion II tulokset osoittavat, että botuliinin rakennegeenien (botA ja ha33) ilmentyminen tox+-isolaatilla oli suurimmillaan 30–65-kertaisesti (p<0,05) suurempaa kuin tox--isolaatilla. Tuloksen avulla voidaan selittää aikaisemmissa tutkimuksissa havaittu ero isolaattien neurotoksiinituotannossa. spo0A-geenin ilmentyminen oli enimmillään noin 30-kertaisesti suurempaa tox+-isolaatilla kuin tox--isolaatilla, mutta ero ei ollut näin suuri eikä tilastollisesti merkitsevä solupopulaatioiden kaikissa kasvuvaiheissa. Itiöitymisen sigmatekijöitä koodaavien geenien (sigE, sigF ja sigG) kohdalla erot isolaattien välillä olivat suurempia kuin spo0A-geenin kohdalla. Geenien ilmentyminen oli yleisesti 20–30-kertainen tox+-isolaatilla tox--isolaattiin nähden. Saadun tuloksen avulla voidaan selittää isolaatin tox- heikentynyt kyky itiöityä. Toksiinituotannolla ja itiöitymisellä on siten todennäköisesti yhteisiä säätelymekanismeja ja/tai -signaaleja. Mutaatio BotR sigmatekijässä todennäköisesti vaikuttaa myös itiöitymisen säätelyyn. On todennäköistä, että C. botulinumin itiöitymisen säätelykaskadi osallistuu myös toksiinituotannon säätelyyn. Tutkimus antaa tärkeää lisätietoa C. botulinumin itiöitymisen ja toksiinituotannon säätelystä saman kannan eri tavoin käyttäytyvillä solupopulaatioilla. Tutkimuksen osiosta I saatujen tulosten perusteella bakteerisolujen ja niistä eristettävän RNA-määrän välille saatiin standardisuora. Saatua tietoa voidaan hyödyntää RNA-sekvensoinnin ja RT-qPCR:n tutkimuskäytössä. Saatujen tulosten avulla voidaan laskea halutun RNA-massan perusteella soluviljelmään tarvittava bakteerisolumäärä, tai vastaavasti kuinka suuri määrä RNA:ta on mahdollista saada tietyn kokoisesta bakteerisolupopulaatiosta. Tutkimuksen osiosta II saatujen tulosten perusteella C. botulinumin itiöitymisprosessi ja toksiinituotanto linkittyvät siis toisiinsa useaa eri reittiä pitkin, ja vaikuttavat näin toinen toisensa säätelyyn. Tärkeänä tekijänä niiden välillä useimmilla C. botulinum –kannoilla toimii vaihtoehtoinen sigmatekijä BotR, joka toksiinituotannon säätelyn lisäksi linkittyy myös itiöitymisprosessin säätelyyn mitä todennäköisimmin orpokinaasien välityksellä säätelemällä Spo0A:n fosforyloitumista

A historical and proteomic analysis of botulinum neurotoxin type/G

<p>Abstract</p> <p>Background</p> <p><it>Clostridium botulinum </it>is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an <it>in silico </it>and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity <it>in vitro</it>. In addition, we review previous literature to provide a complete description of the BoNT/G complex.</p> <p>Results</p> <p>An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT) and three NAPs--nontoxic-nonhemagglutinin (NTNH) and two hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison.</p> <p>Conclusions</p> <p>The <it>in silico </it>protein sequence comparisons established that the/G complex is phenetically related to the other six serotypes of <it>C. botulinum</it>. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and NAPs, along with the activity of the commercial/G complex. The use of data-independent MS^Edata analysis, coupled to label-free quantification software, suggested that the weight ratio BoNT:NAPs is 1:3, whereas the molar ratio of BoNT:NTNH:HA70:HA17 is 1:1:2:1, within the BoNT/G progenitor toxin.</p