Substratbindung und -freigabe während des Katalysezyklus eines biotinspezifischen ECF-Transporters

ECF (Energy-Coupling Factor)-Transporter sind prokaryotische Aufnahmesysteme für Mikronährstoffe, die eine spezielle Gruppe von Transportern mit ATP-Bindekassette (ABC) darstellen. Sie beinhalten zwei asymmetrische Membranproteine, von denen eins (S) für die spezifische Bindung und Translokation des Substrates und das andere (T) für die Kopplung mit den ATPasen (A1,A2) zuständig ist. Bei ECF-Transportern der Subklasse I bilden diese Komponenten eine Einheit, während bei Vertretern der Subklasse II ein AAT-Modul mit wechselnden S-Einheiten interagiert. In der vorliegenden Arbeit wurde der Transportmechanismus, der eine Drehung der kompletten S-Einheit in der Membran beinhaltet, anhand des Biotintransporters BioMNY erstmals experimentell validiert. Durch Rekonstitution in Lipid-Nanodiscs, chemische Quervernetzung, fluoreszenz- und ESR-spektroskopische Techniken sowie einen Bindungstest mit radioaktivem Biotin wurde gezeigt, dass (i) die ATP-Bindung an die ATPasen zu einer Aufrichtung der S-Einheit (BioY) führt, (ii) diese Bewegung die Substratbeladung ermöglicht und (iii) BioY dabei ununterbrochen mit der T-Einheit (BioN) interagiert. Dies stellt einen Gegensatz zu Systemen der Subklasse II dar, für die ein ATP-abhängiger Austausch von S-Einheiten im Transportzyklus gezeigt worden war. Darüber hinaus wurde ein Escherichia coli-Stamm konstruiert, der durch Blockierung seines hochaffinen Biotintransporters und des -synthesewegs auf Spuren von Biotin nicht wachsen kann. Dieser Stamm ermöglichte einen eindeutigen Nachweis der Transportaktivität einiger solitärer BioY-Proteine. Aufgrund der einheitlichen Topologie von S-Einheiten ist ein Kippen auch für solitäre BioY-Varianten wahrscheinlich. Auch die metallspezifischen S-Einheiten CbiM und NikM besitzen ohne AAT-Modul eine basale Co2+- bzw. Ni2+-Transportaktivität. Ein ESR-spektroskopischer Kobaltnachweises zeigte, dass die aus nur zwei Membranhelices bestehende CbiN-Einheit für die Metallbeladung von CbiM essentiell ist.ECF (Energy-Coupling Factor) transporters are a subgroup of ABC transporters that mediate uptake of micronutrients into prokaryotic cells. In contrast to canonical ABC importers, ECF transporters comprise two unrelated membrane proteins, one of which is responsible for specific and high affinity substrate binding (S) and the other one constitutes the coupling component (T) between S and the cytosolic ABC-ATPases (A1,A2). Subclass I transporters consist of four dedicated components whereas in subclass II transporters, a central AAT-module may interact with various S units. The biotin specific subclass I ECF transporter BioMNY was used to experimentally verify the hitherto hypothetic transport mechanism, which involves a rotation of the S unit within the membrane. With a series of experiments including reconstitution of BioMNY into lipid nanodiscs, site-specific cross-linking, a substrate binding assay with radioactive biotin and both fluorescence and EPR spectroscopic techniques, the ATP-dependent rotation of BioY (S) as a prerequisite for substrate binding and release was shown for the first time for an ECF transporter. Unlike subclass II transporters, for which an ATP-dependent release of the S unit was proposed, BioY interacts continuously with BioN (T) during the transport cycle. In a second focus of the work, an Escherichia coli reporter strain for biotin transporters was constructed. Due to inactivation of both biotin synthesis and the intrinsic high affinity biotin transporter, this strain was not capable of growing on trace amounts of biotin. With the use of this strain, transport activity of recombinantly produced solitary BioY proteins that naturally lack other ECF components was evidenced. Transport activity in the absence of AAT modules is also a feature of the Co2+ and Ni2+ specific S components CbiM and NikM. An EPR spectroscopic Co2+ detection assay helped underscoring the essential role of the small membrane protein CbiN for Co2+ loading of CbiM

Detection, differentiation, and identification of botulinum neurotoxin serotypes C, CD, D, and DC by highly specific immunoassays and mass spectrometry

Botulinum neurotoxin (BoNT) serotypes C and D and their mosaic variants CD and DC cause severe cases of botulism in animal husbandry and wildlife. Epidemiological data on the exact serotype or toxin variant causing outbreaks are rarely available, mainly because of their high sequence identity and the lack of fast and specific screening tools to detect and differentiate the four similar toxins. To fill this gap, we developed four highly specific sandwich enzyme-linked immunosorbent assays (ELISAs) able to detect and differentiate botulinum neurotoxins type BoNT/C, D, CD, and DC based on four distinct combinations of specific monoclonal antibodies targeting both conserved and divergent subdomains of the four toxins. Here, highly sensitive detection with detection limits between 2 and 24 pg mL−1 was achieved. The ELISAs were extensively validated and results were compared with data obtained by quantitative real-time PCR using a panel of Clostridium botulinum strains, real sample materials from veterinary botulism outbreaks, and non-BoNT-producing Clostridia. Additionally, in order to verify the results obtained by ELISA screening, the new monoclonal antibodies were used for BoNT enrichment and subsequent detection (i) on a functional level by endopeptidase mass spectrometry (Endopep-MS) assays and (ii) on a protein sequence level by LC-MS/MS spectrometry. Based on all technical information gathered in the validation study, the four differentiating ELISAs turned out to be highly reliable screening tools for the rapid analysis of veterinary botulism cases and should aid future field investigations of botulism outbreaks and the acquisition of epidemiological data

Detection, differentiation, and identification of botulinum neurotoxin serotypes C, CD, D, and DC by highly specific immunoassays and mass spectrometry

Botulinum neurotoxin (BoNT) serotypes C and D and their mosaic variants CD and DC cause severe cases of botulism in animal husbandry and wildlife. Epidemiological data on the exact serotype or toxin variant causing outbreaks are rarely available, mainly because of their high sequence identity and the lack of fast and specific screening tools to detect and differentiate the four similar toxins. To fill this gap, we developed four highly specific sandwich enzyme-linked immunosorbent assays (ELISAs) able to detect and differentiate botulinum neurotoxins type BoNT/C, D, CD, and DC based on four distinct combinations of specific monoclonal antibodies targeting both conserved and divergent subdomains of the four toxins. Here, highly sensitive detection with detection limits between 2 and 24 pg mL−1 was achieved. The ELISAs were extensively validated and results were compared with data obtained by quantitative real-time PCR using a panel of Clostridium botulinum strains, real sample materials from veterinary botulism outbreaks, and non-BoNT-producing Clostridia. Additionally, in order to verify the results obtained by ELISA screening, the new monoclonal antibodies were used for BoNT enrichment and subsequent detection (i) on a functional level by endopeptidase mass spectrometry (Endopep-MS) assays and (ii) on a protein sequence level by LC-MS/MS spectrometry. Based on all technical information gathered in the validation study, the four differentiating ELISAs turned out to be highly reliable screening tools for the rapid analysis of veterinary botulism cases and should aid future field investigations of botulism outbreaks and the acquisition of epidemiological data