Mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from Bacillus cereus

Bacillus cereus is increasingly recognized as an opportunistic pathogen causing local and systemic infections. The causative strains typically produce three pore-forming enterotoxins. This study focusses on the tripartite non-hemolytic enterotoxin (Nhe). Until today, studies have tried to elucidate the structure, complex formation and cell binding mechanisms of the tripartite Nhe toxin. Here, we demonstrate the synthesis of the functional tripartite Nhe toxin using eukaryotic cell-free systems. Single subunits, combinations of two Nhe subunits as well as the complete tripartite toxin were tested. Functional activity was determined by hemolytic activity on sheep blood agar plates, planar lipid bilayer measurements as well as cell viability assessment using the MTT assay. Our results demonstrate that cell-free protein synthesis based on translationally active eukaryotic lysates is a platform technology for the fast and efficient synthesis of functionally active, multicomponent toxins

Nachweis, Charakterisierung und Tenazität von Spezies der Bacillus cereus- Gruppe in Kräutern und Gewürzen

Table of contents iii List of abbreviations v List of figures and tables vii 1 Introduction 1 1.1 Spices and herbs 1 1.1.1 Definition and market overview 1 1.1.2 Production and decontamination 2 1.1.3 Contamination of spices and herbs 3 1.1.4 Microbiological quality standards 5 1.1.5 Antimicrobial effects 7 1.2 The Bacillus cereus group 8 1.2.1 Taxonomy and species differentiation within the Bacillus cereus group 8 1.2.2 Pathogenesis of Bacillus cereus 13 1.2.3 Detection of Bacillus cereus in food 15 1.2.4 Occurrence and survival of Bacillus cereus in food 17 1.3 Aims of the study 20 2 Publications 21 2.1 List of publications and own contribution 21 2.2 Publication 1 23 2.3 Publication 2 33 2.4 Publication 3 42 2.5 Publication 4 52 3 General discussion 62 3.1 Background 62 3.2 Multiplex real-time PCR to detect Bacillus cereus group species in spices and herbs 63 3.2.1 DNA-extraction from spores in condiment matrices 63 3.2.2 Quantitative multiplex real-time PCR 64 3.3 Characterization of Bacillus cereus group isolates from spices and herbs 65 3.3.1 Bacillus cereus group species in spices and herbs 66 3.3.2 Toxinogenic potential of Bacillus cereus group species isolated from spices and herbs 68 3.4 Tenacity of Bacillus cereus and Bacillus thuringiensis spores in spices and herbs 71 3.5 Significance of spiking techniques on the results of tenacity studies 74 4 Summary 76 5 Zusammenfassung 78 6 References 80 7 Danksagung 94 8 Eidesstattliche Erklärung 95The Gram positive spore forming bacterium B. cereus can cause foodborne diseases with diarrhoeal and emetic symptoms. Spices and herbs have frequently been found to contain spores of B. cereus group species which has been linked to several foodborne disease outbreaks. The spice market is growing and a considerable part of condiments is used in the industrial food production and in the catering sector. Thus, spices and herbs can act as a vehicle to transfer B. cereus spores to various foodstuffs that might enable germination and growth under temperature abuse conditions. The informative value of standard microbiological methods applied to spices and herbs is limited as they can be biased by a high microbial background flora and possible antimicrobial effects. Moreover, they neither provide information on the exact B. cereus group species nor on the toxinogenic potential. Hence, data on these parameters are scarce for presumptive B. cereus isolated from spices and herbs. To facilitate data gathering culture independent detection and characterization methods should be developed. In this work a multiplex real- time PCR for the quantification of spores of B. cereus group species with simultaneous discrimination of B. pseudomycoides, cry1-positive B. thuringiensis as well as B. weihenstephanensis/B. mycoides was applied to artificially contaminated spices and herbs. For this, an internal amplification control was included in the PCR-method and different DNA- extraction procedures were evaluated. The best results with pure spore suspensions were achieved with the Qiagen DNeasy Blood & Tissue Kit coupled to quantitative real-time PCR (LOD ~10E2 cfu/ml). Yet, applied to spices and herbs kit based DNA-extraction yielded poor LODs of ≥10E5 cfu/g, whereas the open-formula CTAB-DNA-extraction achieved LODs of 10E2 to 10E3 cfu/g. In order to further investigate the pathogenic potential of B. cereus group species naturally present in spices and herbs, we analysed presumptive B. cereus strains isolated from six different condiments with presumptive B. cereus concentrations of 10E1 to 10E3 cfu/g. In a total of 59 isolates, 44 were classified as B. cereus, five as B. thuringiensis, four as B. toyonensis-like, two as B. pseudomycoides/B. mycoides one as B. weihenstephanensis, and three as undefined B. cereus group species. Most isolates carried the enterotoxin genes nheA and hblD and were also able to produce the respective toxins (Nhe and Hbl). Half of the isolates additionally harboured the cytK-2 gene. One isolate possessed the ces gene and was able to produce cereulide. For some isolates MLST analyses revealed disagreements between phylogenetic relationship and the classification as B. weihenstephanensis and B. mycoides based on previously described real-time PCR species markers (motB and bpm sequences). Hence, the suitability of these markers for species differentiation should be further investigated. For the purpose to investigate the long-term survival of B. cereus and B. thuringiensis in spices and herbs, tenacity studies were conducted using artificially contaminated condiments with spores of each species. After 50 weeks of storage in the dark no significant spore reduction could be observed for either strain. Beforehand, an appropriate spiking technique for spices and herbs was investigated and the suitability of the applied cultural detection method was evaluated. Using sand as a carrier proved to be a suitable method for dry-spiking. The examination of the cultural detection method indicated that the enumeration of vegetative cells in suspensions of spices and herbs can be biased due to substances with inhibitory effect on the bacteria. In contrast, spores remained unaffected unless stored for prolonged times in condiment suspension. This finding might be relevant for cultural enrichment.Das Gram positive sporenbildende Bakterium B. cereus kann lebensmittelbedingte Erkrankungen verursachen, die von Symptomen wie Durchfall, Übelkeit und Erbrechen geprägt sind. In Gewürzen und Kräutern wurden bisher häufig Spezies der B. cereus-Gruppe nachgewiesen, wobei deren Vorkommen bereits mit lebensmittelbedingten Krankheitsausbrüchen in Verbindung gebracht wurde. Der Gewürzmarkt wächst und ein beträchtlicher Teil der Gewürzprodukte wird in der industriellen Lebensmittelproduktion und im Gastronomiebereich eingesetzt. So können Gewürze und Kräuter als Vehikel fungieren, um B. cereus Sporen auf verschiedene Lebensmittel zu übertragen, in welchen sie bei Nicht-einhalten der optimalen Lagertemperaturen keimen und wachsen können. Der Aussagewert von kulturellen mikrobiologischen Methoden, die an Gewürzen und Kräutern angewendet werden, ist begrenzt, da sie durch eine hohe mikrobielle Begleitflora und mögliche antimikrobielle Effekte beeinflusst werden. Darüber hinaus liefern sie weder Informationen über die exakte B. cereus- Gruppenspezies noch über die Toxinbildungs-fähigkeit. Daher fehlen Daten über diese Parameter für präsumtive B. cereus aus Gewürzen und Kräutern. Um solche Daten einfacher erfassen zu können, sind kulturunabhängige Detektions- und Charakterisierungsmethoden erstrebenswert. In dieser Arbeit wurde eine Multiplex Real-Time PCR eingesetzt, um Sporen von Mitgliedern der B. cereus- Gruppe in künstlich kontaminierten Kräutern und Gewürzen zu quantifizieren und gleichzeitig B. pseudomycoides, cry1-positive B. thuringiensis sowie B. weihenstephanensis/B. mycoides von B. cereus zu unterscheiden. Dazu wurde eine interne Amplifikationskontrolle in die PCR-Methode integriert und verschiedene DNA-Extraktionsverfahren untersucht. Die besten Ergebnisse mit reinen Sporen- Suspensionen wurden mit dem Qiagen DNeasy Blood & Tissue Kit gekoppelt mit quantitativer Real-Time PCR erzielt (Detektionslimit ~10E2 KbE/ml). Für die mikrobielle DNA-Extraktion aus Gewürzen und Kräutern erwies sich die Kit- basierte DNA-Extraktion jedoch als ungeeignet (Nachweisgrenzen von ≥10E5 KbE/g), wohingegen mit der CTAB-DNA-Extraktion Nachweisgrenzen von 10E2 bis 10E3 KbE/g erreicht wurden. Um das pathogene Potenzial von präsumtiven B. cereus aus Kräutern und Gewürzen zu untersuchen, wurden Stämme aus verschiedenen Gewürzprodukten, mit präsumtiven B. cereus-Gehalten von 10E1 - 10E3 KbE/g, isoliert und näher charakterisiert. Von insgesamt 59 Isolaten wurden 44 als B. cereus, fünf als B. thuringiensis, vier als B. toyonensis- ähnlich, zwei als B. pseudomycoides/B. mycoides, eins als B. weihenstephanensis und drei als undefinierte B. cereus-Gruppenspezies klassifiziert. Fast alle Isolate trugen die Enterotoxin-Gene nheA und hblD und konnten auch die entsprechenden Toxine (Nhe und Hbl) produzieren. Die Hälfte der Stämme enthielt zusätzlich das cytK-2-Gen. Ein Stamm besaß das ces-Gen und konnte Cereulid produzieren. Für einige Isolate zeigten die MLST-Analysen Widersprüche zwischen phylogenetischer Einordnung und der Klassifikation als B. weihenstephanensis und B. mycoides, basierend auf zuvor beschriebenen Real- Time PCR-Speziesmarkern (motB und bpm Sequenzen). Die Eignung dieser Marker zur Speziesabgrenzung sollte daher in weiteren Studien genauer untersucht werden. Mit dem Ziel das Langzeitüberleben von B. cereus und B. thuringiensis in Gewürzen und Kräutern zu untersuchen, wurden Tenazitätsstudien mit Sporen dieser Spezies in verschiedenen Gewürzprodukten durchgeführt. Nach 50-wöchiger Lagerung konnte bei keiner Spezies eine signifikante Sporenreduktion beobachtet werden. Im Vorfeld wurde eine zweckmäßige Spiking-Technik für Gewürze etabliert und die Eignung der angewandten kulturellen Detektionsmethode überprüft. Die Verwendung von Sand als Träger erwies sich als eine geeignete Methode zum Trocken-Spiken. Bei der Überprüfung der kulturellen Nachweismethode stellte sich heraus, dass die Quantifizierung von vegetativen Zellen in Gewürzen und Kräutern aufgrund von antimikrobiellen Substanzen in der Erstverdünnung beeinflusst sein kann. Im Gegensatz dazu zeigte sich kein Effekt auf den Nachweis von Sporen, wenn sie nicht für längere Zeiträume der Gewürzsuspension ausgesetzt waren, wie dies beispielsweise bei Anreicherungen der Fall wäre

Decontamination of High-risk Animal and Zoonotic Pathogens

Program obilježavanja Noći muzeja 31. siječnja 2020. na Sveučilištu u Zagrebu

The Pore-Forming Hemolysin BL Enterotoxin from <i>Bacillus cereus</i>: Subunit Interactions in Cell-Free Systems

The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by Bacillus cereus. Previous studies have described the formation of the Hbl complex and aimed to identify the toxin’s mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L1, L2 and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl’s ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L1-L2 showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions

Temperature-dependent growth characteristics of Bacillus thuringiensis in a ratatouille food model

In contrast to Bacillus cereus, the role of Bacillus thuringiensis in foodborne illness has been controversially discussed. As B. thuringiensis-based biopesticides containing a mixture of crystal toxins and viable spores are widely used, a current European Food Safety Authority opinion underlines the need for additional data to enable risk assessment. However, it is currently poorly understood if B. thuringiensis is able to multiply in food, which is crucial to sound risk assessment. Therefore, the aim of this study was to investigate growth of selected B. thuringiensis strains from food and insecticides in a ratatouille food model. To this end, the growth parameters of three B. thuringiensis strains were determined: insecticide strain ABTS-351 (CH_119, B. thuringiensis serovar kurstaki), insecticide strain ABTS-1857 (CH_121, B. thuringiensis serovar aizawai), and CH_48 (wild-type B. thuringiensis isolated from rosemary), producing extremely high levels of enterotoxins. After an initial drop in colony counts, we observed a statistically significant growth for the tested B. thuringiensis strains between 6 and 24 h at 22, 30, and 37°C, conditions mimicking prolonged holding times. We were also able to show that the enterotoxin overproducer CH_48 can grow up to 108 CFU/g in the ratatouille matrix within 24 h at 37°C. The two midlevel enterotoxin formers ABTS-351 (CH_119) and ABTS-1857 (CH_121) isolated from biopesticides exhibited growth between 6 and 24 h, with one of the strains growing to 107 CFU/g. To our knowledge, this is the first study providing evidence of B. thuringiensis growth in a food model with intact competitive flora. Our findings suggest strain-specific variation and stress the complexity of assessing the risk related to B. thuringiensis in food, indicating that some strains can represent a risk to consumer health when vegetable-based foods are stored under conditions of prolonged temperature abuse