Untersuchungen zum Einfluss der Rho-Aktivität auf die Expression SaeRS-abhängiger Virulenzfaktoren in Staphylococcus aureus

Staphylococcus aureus ist ein Gram-positives pathogenes Bakterium, welches bei ca. 30 % der gesunden Bevölkerung zur kommensalen Flora der Nasenschleimhaut gehört. Jedoch zählt S. aureus auch zu den häufigsten Erregern bakterieller Infektionen beim Menschen. Aus diesem Grund wurden S. aureus-Stämme in zahlreichen Studien untersucht, um die Pathophysiologie und Virulenz der Bakterien sowie die zugrundeliegenden Regulationsmechanismen zu verstehen. Die Expression von Virulenzfaktoren wird direkt oder indirekt durch verschiedene Regulatoren beeinflusst. Zu diesen zählen beispielsweise das Quorum-Sensing-System Agr, der alternative Sigma-Faktor SigB und das Zweikomponentensystem SaeRS. Bei der Regulation der Genexpression spielt neben Mechanismen, die die Transkriptionsinitiation beeinflussen, auch die Transkriptions-termination eine Rolle. Bei Bakterien unterscheidet man zwischen der Rho-unabhängigen und der Rho-abhängigen Transkriptionstermination. In bisherigen Studien wurde die Rolle des Transkriptionsterminationsfaktors Rho in Escherichia coli, Bacillus subtilis und Mycobacterium tuberculosis untersucht. Hierzu zählt unter anderem das Silencing von horizontal erworbenen Genen, die Verhinderung von DNA-Doppelstrangbrüchen und die Unterdrückung der persistierenden Antisense-Transkripten. Besonders die erhöhte Antisense-Transkription konnte auch in einer Tiling Array-Studie des S. aureus Wildtypstammes HG001 und einer isogenen Δrho-Mutante ST1258 festgestellt werden. In dieser Transkriptom-Analyse wurden die S. aureus-Stämme in RPMI- und TSB-Medium in der exponentiellen und stationären Wachstumsphase untersucht. Es konnten insgesamt 416 chromosomale Regionen identifiziert werden, deren Transkriptmenge in einer der vier Bedingungen in der Δrho-Mutante im Vergleich zum Wildtyp wenigstens 4-fach erhöht waren. Von diesen Regionen ließen sich nur 11 % annotierten Genen zuordnen, während eine massive Erhöhung der Menge solcher Transkripte festgestellt wurde, die vom Gegenstrang kodierender Gene stammen. Ausgehend von diesen Befunden wurde in dieser Studie das zelluläre und extrazelluläre Proteom des S. aureus Wildtyps HG001 und der Δrho-Mutante ST1258 verglichen, um die Auswirkungen der Abwesenheit von Rho auf das Proteom zu untersuchen. Dabei lag die Mehrheit der relativ quantifizierten Proteine in erhöhten Mengen in der Δrho-Mutante im Vergleich zum Wildtyp vor. Viele dieser Proteine konnten dem SaeRS-Zweikomponentensystem von S. aureus zugeordnet werden. In der Proteomanalyse konnten 34 von 39 Proteinen, die durch SaeR reguliert werden, quantifiziert werden. Von diesen wiesen 29 erhöhte Proteinmengen in der Δrho-Mutante auf. Durch das Sae-System werden Gene reguliert, von denen die meisten für Virulenzfaktoren, wie Adhäsine, Toxine und immune evasion-Proteine, kodieren. Die Daten der Proteomanalyse zeigen, dass in S. aureus-Zellen, denen die Aktivität von Rho fehlt, das Sae-System aktiviert wird und dadurch die Induktion des SaeR-Regulons zu beobachten ist. Die Relevanz dieser Ergebnisse wurde durch ein in vivo-Infektionsexperiment untersucht. In einem Bakteriämie-Modell führte die Inaktivierung von Rho zu einer signifikant erhöhten Virulenz von S. aureus, welche sich in einer signifikant reduzierten Überlebensrate der Mäuse äußerte. Zwischen dem Wildtyp und dem Komplementationsstamm konnte kein signifikanter Unterschied in der Überlebensrate der infizierten Mäuse gezeigt werden. Es ist bekannt, dass SaeRS-abhängige Virulenzfaktoren auch für die Invasion in Epithel- und Endothelzellen entscheidend sind. Anhand der Zahl der internalisierten S. aureus-Bakterien nach Infektion von humanen Lungenepithelzellen konnten in dieser Arbeit keine Unterschiede zwischen dem Wildtyp HG001 und ∆rho-Mutante ST1258 im zeitlichen Verlauf festgestellt werden. Dabei konnten weder Unterschiede im Überleben in 16HBE14o- Zellen noch in der Internalisierungsrate in A549-Zellen zwischen den beiden Stämmen gezeigt werden. Das Antibiotikum Bicyclomycin ist ein spezifischer Inhibitor des Transkriptionsterminationsfaktors Rho und wird in Studien zur Rho-abhängigen Transkriptionstermination in Gram-negativen Bakterien eingesetzt, da in diesen Rho ein essentielles Protein ist. In Transkriptom- und Proteomanalysen konnten vergleichbare Effekte durch die Behandlung des Wildtyps mit Bicyclomycin wie in der ∆rho-Mutante hervorgerufen werden. Die Antisense-Transkription und die Expression SaeRS-abhängigen Gene waren im Wildtyp nach Gabe von Bicyclomycin deutlich erhöht. Es konnte gezeigt werden, dass die Aktivierung des Sae-Systems unter Rho-defizienten Bedingungen direkt mit der Transkriptionsterminationsaktivität von Rho verbunden ist und eine neue Verbindung zwischen antibiotischer Wirkung und schädlicher Virulenzgenexpression in S. aureus herstellt werden konnte. In anderen Studien konnten Effekte von Antibiotika auf die Expression von Virulenzfaktoren in S. aureus gezeigt werden, jedoch wurden in diesen Studien die Effekte von Anti-Staphylokokken-Wirkstoffen untersucht. Im Gegensatz dazu ist im Fall von Bicyclomycin ein Antibiotikum verwendet worden, das gegen Gram-negative Bakterien wirksam ist und dennoch die Expression von Virulenzfaktoren in S. aureus beeinflusst. Diese Untersuchungen haben damit auch klinische Relevanz, nicht nur für Patienten, die an gemischten Infektionen mit verschiedenen Bakterienarten leiden, sondern auch für Patienten mit einer Gram-negativen bakteriellen Infektion, die jedoch Träger von S. aureus sind.The Gram-positive bacterium Staphylococcus aureus causes various diseases ranging from skin and soft tissue to life-threatening infections. The goal of the present study was a detailed comparative analysis of S. aureus HG001 and its isogenic rho deletion mutant. Proteome analysis revealed significant differences in cellular and extracellular protein profiles between the HG001 wild-type and rho mutant, most notably increased amounts of SaeRS-dependent virulence factors in the Rho deficient strain. These higher levels of virulence factors such as adhesins, toxins and immune evasion proteins in the rho mutant resulted in higher virulence in a murine bacteremia model. Inhibition of Rho activity by bicyclomycin, a specific inhibitor of Rho activity, also induced the expression of SaeRS-dependent virulence factor genes, both at the mRNA and protein levels, to the same extent as observed in the rho deletion mutant. Taken together, these findings indicate that activation of the Sae system in the absence of Rho is directly linked to Rho’s transcription termination activity and establish a new link between antibiotic action and virulence gene expression in S. aureus

Aboriginal stone-walled intertidal fishtrap morphology, function and chronology investigated with high-resolution close-range Unmanned Aerial Vehicle photogrammetry

Stone-walled intertidal fishtraps surround the Australian coastline and are among the largest structures built by Indigenous Australians. Globally, fishtraps are considered important elements in food production, domestication, territoriality and ceremonial landscapes, yet the level of detail in documentation is highly varied and scholarly fishtrap knowledge sparse. Comparative analysis is currently restricted by a lack of detail and reproducibility in recording, hindering analysis of morphology, function and chronology. In this study we employ high-resolution close-range Unmanned Aerial Vehicle (UAV) photogrammetry and a suite of spatial information analytical techniques to investigate the Kaiadilt Aboriginal stone-walled intertidal fishtraps of Sweers Island, southern Gulf of Carpentaria, Australia. Tidal inundation modelling is undertaken to assess (1) fishtrap working range, (2) individual and simultaneous trap function, (3) seasonal functionality and (4) chronology based on function relative to sea-level history. Thirteen fishtraps were identified in the study area, ranging from 38m to 287m in length. Flow accumulation indicates that shape and placement of fishtraps reflects underlying topography. Inundation modelling shows that all fishtraps operate most efficiently at present mean-sea level (PMSL), indicating construction in the last 3500 years. Quantitative recording techniques, analytical procedures and terminology developed in this study provide an opportunity to improve approaches to recording large-scale stone features and standardise documentation of stone-walled intertidal fishtrap sites

EDC-mediated oligonucleotide immobilization on a long period grating optical biosensor

We present the development and simplification of label-free fiber optic biosensors based on immobilization of oligonucleotides on dual-peak long period gratings (dLPGs). This improvement is the result of a simplification of biofunctionalization methodology. A one-step 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated reaction has been developed for the straightforward immobilization of unmodified oligonucleotides on the glass fiber surface along the grating region, leading to covalent attachment of a 5´-phosphorylated probe oligonucleotide to the amino-derivatized fiber grating surface. Immobilization is achieved via a 5´phosphate-specific linkage, leaving the remainder of the oligonucleotide accessible for binding reactions. The dLPG has been tested in different external media to demonstrate its inherent ultrahigh sensitivity to the surrounding-medium refractive index (RI) achieving 50- fold improvement in RI sensitivity over the previously-published LPG sensor in media with RI’s relevant to biological assays. After functionalization, the dLPG biosensor was used to monitor the hybridization of complementary oligonucleotides showing a detectable oligonucleotide concentration of 4 nM. The proposed one-step EDC reaction approach can be further extended to develop fiber optic biosensors for disease analysis and medical diagnosis with the advances of label-free, real-time, multiplex, high sensitivity and specificity

Improving Clinical ECG-based Atrial Fibrosis Quantification With Neural Networks Through in silico P waves From an Extensive Virtual Patient Cohort

Fibrotic atrial cardiomyopathy is characterized by a replacement of healthy atrial tissue with diffuse patches exhibiting slow electrical conduction properties and altered myocardial tissue structure, which provides a substrate for the maintenance of reentrant activity during atrial fibrillation (AF). Therefore, an early detection of atrial fibrosis could be a valuable risk marker for new-onset AF episodes to select asymptomatic subjects for screening, allowing for timely intervention and optimizing therapy planning. We examined the potential of estimating the fibrotic tissue volume fraction in the atria based on P waves of the 12-lead ECG recorded in sinus rhythm in a quantitative and noninvasive way. Our dataset comprised 68,282 P waves from healthy subjects and 42,227 P waves from AF patients with low voltage areas in the atria, as well as 642,400 simulated P waves of a virtual cohort derived from statistical shape models with different extents of the left atrial myocardium replaced by fibrosis. The root mean squared error for estimating the left atrial fibrotic volume fraction on a clinical test set with a neural network trained on features extracted from simulated and clinical P waves was 16.57 %. Our study shows that the 12-lead ECG contains valuable information on atrial tissue structure. As such it could potentially be employed as an inexpensive and widely available tool to support AF risk stratification in clinical practic

Sensory adaptation to chemical cues by vomeronasal sensory neurons

Sensory adaptation is a source of experience-dependent feedback that impacts responses to environmental cues. In the mammalian main olfactory system (MOS), adaptation influences sensory coding at its earliest processing stages. Sensory adaptation in the accessory olfactory system (AOS) remains incompletely explored, leaving many aspects of the phenomenon unclear. We investigated sensory adaptation in vomeronasal sensory neurons (VSNs) using a combination of in situ Ca2+ imaging and electrophysiology. Parallel studies revealed prominent short-term sensory adaptation in VSNs upon repeated stimulation with mouse urine and monomolecular bile acid ligands at interstimulus intervals (ISIs) less than 30 s. In such conditions, Ca2+ signals and spike rates were often reduced by more than 50%, leading to dramatically reduced chemosensory sensitivity. Short-term adaptation was reversible over the course of minutes. Population Ca2+ imaging experiments revealed the presence of a slower form of VSN adaptation that accumulated over dozens of stimulus presentations delivered over tens of minutes. Most VSNs showed strong adaptation, but in a substantial VSN subpopulation adaptation was diminished or absent. Investigation of same-and opposite-sex urine responses in male and female VSNs revealed that adaptation to same-sex cues occurred at ISIs up to 180 s, conditions that did not induce adaptation to opposite-sex cues. This result suggests that VSN sensory adaptation can be modulated by sensory experience. These studies comprehensively establish the presence of VSN sensory adaptation and provide a foundation for future inquiries into the molecular and cellular mechanisms of this phenomenon and its impact on mammalian behavior

Label-free oligonucleotide biosensor based on dual-peak long period fiber grating

We report the simplification and development of biofunctionalization methodology based on one-step 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-mediated reaction. The dual-peak long period grating (dLPG) has been demonstrated its inherent ultrahigh sensitivity to refractive index (RI), achieving 50-fold improvement in RI sensitivity over a standard LPG sensor used in low RI range. With the simple and efficient immobilization of unmodified oligonucleotides on sensor surface, dLPG-based biosensor has been used to monitor the hybridization of complementary oligonucleotides showing a detectable oligonucleotide concentration of 4 nM with the advantages of label-free, real-time, and ultrahigh sensitivity

Thin chalcogenide capillaries as efficient waveguides in the mid-IR - THz spectral range

We present chalcogenide glass As2Se3 capillaries as efficient waveguides in the mid-IR and THz spectral ranges. The capillaries are fabricated using a double crucible glass drawing technique. The wall thickness of the glass capillary is properly designed and controlled during drawing, and we are able to produce capillaries with different wall thickness, starting from 12 \mum and up to 130 \mum. Such capillaries show low loss properties in the whole target wavelength region. In the mid-IR range guidance is governed by Fresnel reflection and antiguidance mechanisms (ARROWs), while in the THz spectral range thin walls capillaries guide via total internal reflection

Polymeric microspheres as protein transduction reagents

Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere-protein linkage was stable for ≥90 h at 37°C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake