Untersuchung antibiotischer Wirkmechanismen mit Röntgenkleinwinkelstreuung

Die weltweit steigende Zahl der Infektionen durch multiresistente Krankheitserreger ist eine bedeutende Herausforderung für unser modernes Gesundheitssystem. Zu ihrer effektiven Bekämpfung werden Antibiotika mit neuen Wirkmechanismen benötigt. Im Entwicklungsprozess eines neuen Antibiotikums können zwar viele antimikrobielle Substanzen erzeugt werden, die Identifikation neuer Wirkmotive stellt jedoch eine zeit- und kostenintensive Hürde dar. In dieser Arbeit wird die Anwendung von Röntgenkleinwinkelstreuung (SAXS) mit Synchrotronstrahlung als Hochdurchsatzverfahren zur Klassifizierung antibiotischer Wirkmechanismen präsentiert. Hierzu wurden Escherichia coli mit einem Satz klinisch relevanter Antibiotika und mit einem kurzen kationischen Peptid mit unbekanntem Wirkmechanismus behandelt, und mit SAXS untersucht. Mithilfe einer Hauptkomponentenanalyse konnten die Antibiotika anhand ihrer morphologischen Änderungen klassifiziert werden. Die hierfür relevanten Zellstrukturen konnten durch Korrelation mit Transmissionselektronenmikroskopie und zonenplattenbasierter Röntgenmikroskopie (TXM) identifiziert werden. Ein einfaches Modell erlaubte es, aus Kleinwinkelstreukurven ganzer E. coli den Volumenanteil und die Anzahl wichtiger Zellkomponenten (DNS und Ribosomen) zu extrahieren. In der Röntgenstreukammer HORST wurde kryo-SAXS im weichen Röntgenbereich etabliert, um kryogene biologische Proben, zusammen mit der existierenden kryo-TXM Einheit, korrelativ in Real- und Fourierraum zu untersuchen

Water Window Ptychographic Imaging with Characterized Coherent X-rays

We report on a ptychographical coherent diffractive imaging experiment in the water window with focused soft X-rays at $500~\mathrm{eV}$. An X-ray beam with high degree of coherence was selected for ptychography at the P04 beamline of the PETRA III synchrotron radiation source. We measured the beam coherence with the newly developed non-redundant array method. A pinhole $2.6~\mathrm{\mu m}$ in size selected the coherent part of the beam and was used for ptychographic measurements of a lithographically manufactured test sample and fossil diatom. The achieved resolution was $53~\mathrm{nm}$ for the test sample and only limited by the size of the detector. The diatom was imaged at a resolution better than $90~\mathrm{nm}$.Comment: 22 pages. 7 figure

BioSAXS measurements reveal that two antimicrobial peptides induce similar molecular changes in gram-negative and gram-positive bacteria

Two highly active short broad-spectrum AMPs (14D and 69D) with unknown mode of action have been investigated in regards to their effect against the Gramnegative bacteria Escherichia coli and the Gram-positive bacteria methicillinresistant Staphylococcus aureus (MRSA). Minimal inhibitory concentration (MIC) measurements using a cell density of 108 cfu/ml resulted in values between 16 and 32 μg/ml. Time-kill experiments using 108 cfu/ml revealed complete killing, except for 69D in combination with MRSA, where bacterial load was reduced a million times. Small-angle X-ray scattering of biological samples (BioSAXS) at 108 cfu/ml was applied to investigate the ultrastructural changes in E. coli and MRSA in response to these two broad-spectrum AMPs. In addition, electron microscopy (EM) was performed to visualize the treated and non-treated bacteria. As expected, the scattering curves generated using BioSAXS show the ultrastructure of the Grampositive and Gram-negative bacteria to be very different (BioSAXS is not susceptible to the outer shape). After treatment with either peptide, the scattering curves of E.coli and MRSA cells are much more alike. Whereas in EM, it is notoriously difficult to observe changes for spherical Gram-positives; the BioSAXS results are superior and reveal strongly similar effects for both peptides induced in Gram-positive as well as Gram-negative bacteria. Given the high-throughput possibility and robust statistics, BioSAXS can support and speed up mode of action research in AMPs and other antimicrobial compounds, making a contribution toward the development of urgently needed drugs against resistant bacteria

Ptychographic Imaging of Fossil Diatom Structures with Soft X-Rays

Imaging of biological samples in the water window offer high chemical contrast between Oxygen and Carbon. To avoid resolution limitations due to lens based microscopy techniques the coherent diffractive imaging (CDI) method is favourable for high resolution imaging. In order to measure extended samples the ptychographic coherent diffractive imaging technique (PCDI) is applied. Images of a test pattern and a fossil diatom are reconstructed with the extended ptychographic iterative engine (ePIE) at 55 nm and 286 nm resolution respectively

Quantitative ptychographic bio-imaging in the water window

Coherent X-ray ptychography is a tool for highly dose efficient lensless nano-imaging of biological samples. We have used partially coherent soft X-ray synchrotron radiation to obtain a quantitative image of a laterally extended, dried, and unstained fibroblast cell by ptychography. We used data with and without a beam stop that allowed us to measure coherent diffraction with a high-dynamic range of 1.7·10$^6$. As a quantitative result, we obtained the refractive index values for two regions of the cell with respect to a reference area. Due to the photon energy in the water window we obtained an extremely high contrast of 53% at 71 nm half-period resolution. The dose applied in our experiment was 9.5·10$^4$ Gy and is well below the radiation damage threshold. The concept for dynamic range improvement for low dynamic range detectors with a beam stop opens the path for high resolution nano-imaging of a variety of samples including cryo-preserved, hydrated and unstained biological cells

Micro x-ray fluorescence analysis of trace element distribution in frozen hydrated HeLa cells at the P06 beamline at Petra III

X-ray fluorescence analysis enables the study of trace element distributions in biological specimens. When this analysis is done under cryogenic conditions, cells are cryofixed as closely as possible to their natural physiological state, and the corresponding intracellular elemental densities can be analyzed. Details about the experimental setup used for analysis at the P06 beamline at Petra III, DESY and the used cryo-transfer system are described in this work. The system was applied to analyze the elemental distribution in single HeLa cells, a cell line frequently used in a wide range of biological applications. Cells adhered to silicon nitride substrates were cryoprotected within an amorphous ice matrix. Using a continuous scanning scheme and a KB x-ray focus, the distribution of elements in the cells was studied. We were able to image the intracellular potassium and zinc levels in HeLa cells as two key elements relevant for the physiology of cells

Rational Designed Hybrid Peptides Show up to a 6-Fold Increase in Antimicrobial Activity and Demonstrate Different Ultrastructural Changes as the Parental Peptides Measured by BioSAXS

Antimicrobial peptides (AMPs) are a promising class of compounds being developed against multi-drug resistant bacteria. Hybridization has been reported to increase antimicrobial activity. Here, two proline-rich peptides (consP1: VRKPPYLPRPRPRPL-CONH2 and Bac5-v291: RWRRPIRRRPIRPPFWR-CONH2) were combined with two arginine-isoleucine-rich peptides (optP1: KIILRIRWR-CONH2 and optP7: KRRVRWIIW-CONH2). Proline-rich antimicrobial peptides (PrAMPs) are known to inhibit the bacterial ribosome, shown also for Bac5-v291, whereas it is hypothesized a “dirty drug” model for the arginine-isoleucine-rich peptides. That hypothesis was underpinned by transmission electron microscopy and biological small-angle X-ray scattering (BioSAXS). The strength of BioSAXS is the power to detect ultrastructural changes in millions of cells in a short time (seconds) in a high-throughput manner. This information can be used to classify antimicrobial compounds into groups according to the ultrastructural changes they inflict on bacteria and how the bacteria react towards that assault. Based on previous studies, this correlates very well with different modes of action. Due to the novelty of this approach direct identification of the target of the antimicrobial compound is not yet fully established, more research is needed. More research is needed to address this limitation. The hybrid peptides showed a stronger antimicrobial activity compared to the proline-rich peptides, except when compared to Bac5-v291 against E. coli. The increase in activity compared to the arginine-isoleucine-rich peptides was up to 6-fold, however, it was not a general increase but was dependent on the combination of peptides and bacteria. BioSAXS experiments revealed that proline-rich peptides and arginine-isoleucine-rich peptides induce very different ultrastructural changes in E. coli, whereas a hybrid peptide (hyP7B5GK) shows changes, different to both parental peptides and the untreated control. These different ultrastructural changes indicated that the mode of action of the parental peptides might be different from each other as well as from the hybrid peptide hyP7B5GK. All peptides showed very low haemolytic activity, some of them showed a 100-fold or larger therapeutic window, demonstrating the potential for further drug development

Micro x-ray fluorescence analysis of trace element distribution in frozen hydrated HeLa cells at the P06 beamline at Petra III

X-ray fluorescence analysis enables the study of trace element distributions in biological specimens. When this analysis is done under cryogenic conditions, cells are cryofixed as closely as possible to their natural physiological state, and the corresponding intracellular elemental densities can be analyzed. Details about the experimental setup used for analysis at the P06 beamline at Petra III, DESY and the used cryo-transfer system are described in this work. The system was applied to analyze the elemental distribution in single HeLa cells, a cell line frequently used in a wide range of biological applications. Cells adhered to silicon nitride substrates were cryoprotected within an amorphous ice matrix. Using a continuous scanning scheme and a KB x-ray focus, the distribution of elements in the cells was studied. We were able to image the intracellular potassium and zinc levels in HeLa cells as two key elements relevant for the physiology of cells