Functional detection of Shiga toxin-producing E. coli based on innovative Förster Resonance Energy Transfer substrates

Shigatoxin-produzierende E. coli (STEC) sind bedeutende humanpathogene Erreger, die für Erkrankungen mit (blutigen) Durchfällen bis hin zum lebensbedrohlichen hämolytisch-urämischen Syndrom verantwortlich sind. In Deutschland werden dem RKI jährlich etwa 1.900 STEC-Infektionen und 70 HUS-Fälle gemeldet, wobei meist Kinder unter fünf Jahren betroffen sind. Die Übertragung von STEC erfolgt sowohl über den Kontakt zu Tieren als auch über kontaminierte Lebensmittel. Da STEC große Ausbrüche verursachen können, ist ihr rechtzeitiger und qualifizierter Nachweis einschließlich einer Isolatgewinnung von großer Bedeutung, der jedoch schwierig und arbeitsintensiv ist. STEC sind durch eine Vielzahl an Virulenzfaktoren, zu denen beispielsweise Adhäsions- und Kolonisationsfak-toren, Pathogenitätsinseln und Toxine gehören, sowie verschiedenen Stoffwechsel- und Oberflächen-merkmalen (O- und H-Antigene) geprägt. Da das gemeinsame Merkmal und der Hauptvirulenzfaktor der vielfältigen STEC das Shigatoxin (Stx) ist, wurde im Rahmen dieser Arbeit eine Nachweismethode für Stx entwickelt. Der Nachweis fokussiert sich speziell auf die katalytische RNA-N-Glykosylase-Aktivität von Stx, die auf den Sarcin-Ricin-Loop (SRL) der 28S ribosomalen RNA abzielt. Synthetische ssDNA-Substrate, die auf dem SRL basieren, wurden designt und mit einem Fluorophor- und Quencher-Paar verknüpft. Diese erzeugen nach der Spaltung durch Stx ein Fluoreszenzsignal auf der Grundlage des Förster-Resonanz-Energie-Transfers (FRET). Die enzymatische Stx-Aktivität der Bakterienpräparate wurde mit einem Realtime-Cycler erfasst und mit insgesamt 65 STEC-Stämmen verschiedener Serotypen und unterschiedlicher Stx-Subtypen sowie 11 Shigella-Stämmen analysiert. Die Sensitivität des Assays sowie die Spezifität wurden anhand von 29 Stämmen anderer darmpatho-gener Erreger wie Salmonella untersucht. Optimale Ergebnisse wurden mit bakteriellen Kulturüberständen oder einzelnen Kolonien für das Substrat StxSense4 nach 30 bis 60 Minuten Inkubation erreicht. Die Nachweisgrenze von 10 29 ng/mL lag im Bereich etablierter Nachweismethoden wie dem Verozell-Zytotoxizitätstest. Verschiedene Stx1-Subtypen (stx1a, stx1c und stx1d) und Stx2-Subtypen (stx2a-stx2g) sowie diverse STEC-Serotypen und Shigella wurden mit dem entwickelten Test nachgewiesen. 5 µL Kulturüberstand reichten für den Nachweis von enzymatisch aktivem Stx2 aus, während einzelne Kolonien den Nachweis sowohl von Stx1 als auch von Stx2 ermöglichten. Bei anderen darmpathogenen Erregern, die kein stx enthalten, wurde keine Fluoreszenz oberhalb der Basislinienwerte festgestellt. Der in dieser Arbeit entwickelte Test ermöglicht einen schnellen und einfachen Nachweis von STEC durch die Echtzeitanzeige der Stx-Aktivität. Daher kann er die Bewertung von STEC-Risiken, Therapie-entscheidungen und die Erkennung von Ausbrüchen und Infektionsquellen verbessern sowie die Forschung nach antimikrobiellen Mitteln vereinfachen.Shigatoxin-producing E. coli (STEC) are important human pathogens causing diseases ranging from diarrhea to severe hemolytic uremic syndrome. In Germany, about 1900 STEC infections and 70 HUS cases are reported to the RKI annually and children under the age of five are mostly affected. As STEC are transmitted via animals, food, and water, and may produce large outbreaks, their timely and qualified detection including isolate recovery is of high importance, but challenging and labor-intense. Thus, the availability of an easy-to-perform rapid test would be a tremendous advance. STEC are a group of pathogens with high variation in marker genes such as adhesion and colonization factors, pathogenicity island and toxins, as well as metabolic and surface characteristics (O and H antigens). Since the common feature and major virulence factor of otherwise multifaceted STEC is the Shiga toxin (Stx), this study aimed to develop a detection method for Stx, specifically for its catalytic RNA-N-glycosidase activity targeting the Sarcin Ricin Loop (SRL) of 28S ribosomal RNA. To this end, synthetic ssDNA substrates mimicking the SRL were designed and linked to a fluorophore and quencher pair, which conferred a fluorescence signal after cleavage by Stx, based on the Förster Resonance Energy Transfer (FRET). Stx enzymatic activity of the bacterial preparations was tested using a Realtime-Cycler for fluorescence readout. In a proof-of-principle study, the Stx activity of 65 STEC strains of distinct serogroups harboring different Stx subtypes and 11 Shigella strains was analyzed. The sensitivity of the assay was determined, and specificity was examined using 29 strains of other intestinal pathogens without Stx, such as Salmonella. Optimal results using bacterial culture supernatants or single colonies were achieved for substrate StxSense4 following 30 to 60 minutes of incubation. The detection limit of 10 29 ng/mL was within the range of established detection methods, such as the Vero cell cytotoxicity assay. Importantly, different Stx1 subtypes (stx1a, stx1c, and stx1d) and Stx2 subtypes (stx2a–stx2g), diverse STEC serotypes, and Shigella were detected. 5 µL of culture supernatants were sufficient for the detection of enzymatically active Stx2, while single colonies allowed the detection of both Stx1 and Stx2. For other enterobacteria not comprising stx, no fluorescence above baseline levels was detected. In conclusion, the here-developed assay offers rapid and facile detection of STEC based on a Realtime readout for Stx activity. Therefore, it may improve STEC risk evaluation, therapy decisions, outbreak, and source detection, and simplify research for antimicrobials

Impact of Von Willebrand Factor on Bacterial Pathogenesis.

Von Willebrand factor (VWF) is a mechano-sensitive protein with crucial functions in normal hemostasis, which are strongly dependant on the shear-stress mediated defolding and multimerization of VWF in the blood stream. Apart from bleeding disorders, higher plasma levels of VWF are often associated with a higher risk of cardiovascular diseases. Herein, the disease symptoms are attributed to the inflammatory response of the activated endothelium and share high similarities to the reaction of the host vasculature to systemic infections caused by pathogenic bacteria such as Staphylococcus aureus and Streptococcus pneumoniae. The bacteria recruit circulating VWF, and by binding to immobilized VWF on activated endothelial cells in blood flow, they interfere with the physiological functions of VWF, including platelet recruitment and coagulation. Several bacterial VWF binding proteins have been identified and further characterized by biochemical analyses. Moreover, the development of a combination of sophisticated cell culture systems simulating shear stress levels of the blood flow with microscopic visualization also provided valuable insights into the interaction mechanism between bacteria and VWF-strings. In vivo studies using mouse models of bacterial infection and zebrafish larvae provided evidence that the interaction between bacteria and VWF promotes bacterial attachment, coagulation, and thrombus formation, and thereby contributes to the pathophysiology of severe infectious diseases such as infective endocarditis and bacterial sepsis. This mini-review summarizes the current knowledge of the interaction between bacteria and the mechano-responsive VWF, and corresponding pathophysiological disease symptoms

Streptococcus pneumoniae Affects Endothelial Cell Migration in Microfluidic Circulation

Bloodstream infections caused by Streptococcus pneumoniae induce strong inflammatory and procoagulant cellular responses and affect the endothelial barrier of the vascular system. Bacterial virulence determinants, such as the cytotoxic pore-forming pneumolysin, increase the endothelial barrier permeability by inducing cell apoptosis and cell damage. As life-threatening consequences, disseminated intravascular coagulation followed by consumption coagulopathy and low blood pressure is described. With the aim to decipher the role of pneumolysin in endothelial damage and leakage of the vascular barrier in more detail, we established a chamber-separation cell migration assay (CSMA) used to illustrate endothelial wound healing upon bacterial infections. We used chambered inlets for cell cultivation, which, after removal, provide a cell-free area of 500 μm in diameter as a defined gap in primary endothelial cell layers. During the process of wound healing, the size of the cell-free area is decreasing due to cell migration and proliferation, which we quantitatively determined by microscopic live cell monitoring. In addition, differential immunofluorescence staining combined with confocal microscopy was used to morphologically characterize the effect of bacterial attachment on cell migration and the velocity of gap closure. In all assays, the presence of wild-type pneumococci significantly inhibited endothelial gap closure. Remarkably, even in the presence of pneumolysin-deficient pneumococci, cell migration was significantly retarded. Moreover, the inhibitory effect of pneumococci on the proportion of cell proliferation versus cell migration within the process of endothelial gap closure was assessed by implementation of a fluorescence-conjugated nucleoside analogon. We further combined the endothelial CSMA with a microfluidic pump system, which for the first time enabled the microscopic visualization and monitoring of endothelial gap closure in the presence of circulating bacteria at defined vascular shear stress values for up to 48 h. In accordance with our CSMA results under static conditions, the gap remained cell free in the presence of circulating pneumococci in flow. Hence, our combined endothelial cultivation technique represents a complex in vitro system, which mimics the vascular physiology as close as possible by providing essential parameters of the blood flow to gain new insights into the effect of pneumococcal infection on endothelial barrier integrity in flow

Polyhalonitrobutadienes as Versatile Building Blocks for the Biotargeted Synthesis of Substituted N-Heterocyclic Compounds.

Substituted nitrogen heterocycles are structural key units in many important pharmaceuticals. A new synthetic approach towards heterocyclic compounds displaying antibacterial activity against Staphylococcus aureus or cytotoxic activity has been developed. The selective synthesis of a series of 64 new N-heterocycles from the three nitrobutadienes 2-nitroperchloro-1,3-butadiene, 4-bromotetrachloro-2-nitro-1,3-butadiene and (Z)-1,1,4-trichloro-2,4-dinitrobuta-1,3-diene proved feasible. Their reactions with N-, O- and S-nucleophiles provide rapid access to push-pull substituted benzoxazolines, benzimidazolines, imidazolidines, thiazolidinones, pyrazoles, pyrimidines, pyridopyrimidines, benzoquinolines, isothiazoles, dihydroisoxazoles, and thiophenes with unique substitution patterns. Antibacterial activities of 64 synthesized compounds were examined. Additionally, seven compounds (thiazolidinone, nitropyrimidine, indole, pyridopyrimidine, and thiophene derivatives) exhibited a significant cytotoxicity with IC50-values from 1.05 to 20.1 µM. In conclusion, it was demonstrated that polyhalonitrobutadienes have an interesting potential as structural backbones for a variety of highly functionalized, pharmaceutically active heterocycles

Streptococcus pneumoniae Affects Endothelial Cell Migration in Microfluidic Circulation

Bloodstream infections caused by Streptococcus pneumoniae induce strong inflammatory and procoagulant cellular responses and affect the endothelial barrier of the vascular system. Bacterial virulence determinants, such as the cytotoxic pore-forming pneumolysin, increase the endothelial barrier permeability by inducing cell apoptosis and cell damage. As life-threatening consequences, disseminated intravascular coagulation followed by consumption coagulopathy and low blood pressure is described. With the aim to decipher the role of pneumolysin in endothelial damage and leakage of the vascular barrier in more detail, we established a chamber-separation cell migration assay (CSMA) used to illustrate endothelial wound healing upon bacterial infections. We used chambered inlets for cell cultivation, which, after removal, provide a cell-free area of 500 μm in diameter as a defined gap in primary endothelial cell layers. During the process of wound healing, the size of the cell-free area is decreasing due to cell migration and proliferation, which we quantitatively determined by microscopic live cell monitoring. In addition, differential immunofluorescence staining combined with confocal microscopy was used to morphologically characterize the effect of bacterial attachment on cell migration and the velocity of gap closure. In all assays, the presence of wild-type pneumococci significantly inhibited endothelial gap closure. Remarkably, even in the presence of pneumolysin-deficient pneumococci, cell migration was significantly retarded. Moreover, the inhibitory effect of pneumococci on the proportion of cell proliferation versus cell migration within the process of endothelial gap closure was assessed by implementation of a fluorescence-conjugated nucleoside analogon. We further combined the endothelial CSMA with a microfluidic pump system, which for the first time enabled the microscopic visualization and monitoring of endothelial gap closure in the presence of circulating bacteria at defined vascular shear stress values for up to 48 h. In accordance with our CSMA results under static conditions, the gap remained cell free in the presence of circulating pneumococci in flow. Hence, our combined endothelial cultivation technique represents a complex in vitro system, which mimics the vascular physiology as close as possible by providing essential parameters of the blood flow to gain new insights into the effect of pneumococcal infection on endothelial barrier integrity in flow

Identification of antibiotics that diminish disease in a murine model of enterohemorrhagic infection.

Infections with enterohemorrhagic Escherichia coli (EHEC) cause disease ranging from mild diarrhea to hemolytic uremic syndrome (HUS) and are the most common cause of renal failure in children in high income countries. The severity of the disease derives from the release of Shiga toxins (Stx). The use of antibiotics to treat EHEC infections is generally avoided as it can result in increased stx expression. Here, we systematically tested different classes of antibiotics and found that their influence on stx expression and release varies significantly. We assessed a selection of these antibiotics in vivo using the Citrobacter rodentium φstx2dact mouse model and show that stx2d-inducing antibiotics resulted in weight loss and kidney damage despite clearing the infection. However, several non-Stx-inducing antibiotics cleared bacterial infection without causing Stx-mediated pathology. Our results suggest that these antibiotics could be useful in the treatment of EHEC-infected human patients and decrease the risk of HUS development