Can BioSAXS detect ultrastructural changes of antifungal compounds in Candida albicans?-an exploratory study.

The opportunistic yeast Candida albicans is the most common cause of candidiasis. With only four classes of antifungal drugs on the market, resistance is becoming a problem in the treatment of fungal infections, especially in immunocompromised patients. The development of novel antifungal drugs with different modes of action is urgent. In 2016, we developed a groundbreaking new medium-throughput method to distinguish the effects of antibacterial agents. Using small-angle X-ray scattering for biological samples (BioSAXS), it is now possible to screen hundreds of new antibacterial compounds and select those with the highest probability for a novel mode of action. However, yeast (eukaryotic) cells are highly structured compared to bacteria. The fundamental question to answer was if the ultrastructural changes induced by the action of an antifungal drug can be detected even when most structures in the cell stay unchanged. In this exploratory work, BioSAXS was used to measure the ultrastructural changes of C. albicans that were directly or indirectly induced by antifungal compounds. For this, the well-characterized antifungal drug Flucytosine was used. BioSAXS measurements were performed on the synchrotron P12 BioSAXS beamline, EMBL (DESY, Hamburg) on treated and untreated yeast C. albicans. BioSAXS curves were analysed using principal component analysis (PCA). The PCA showed that Flucytosine-treated and untreated yeast were separated. Based on that success further measurements were performed on five antifungal peptides {1. Cecropin A-melittin hybrid [CA (1-7) M (2-9)], KWKLFKKIGAVLKVL; 2. Lasioglossin LL-III, VNWKKILGKIIKVVK; 3. Mastoparan M, INLKAIAALAKKLL; 4. Bmkn2, FIGAIARLLSKIFGKR; and 5. optP7, KRRVRWIIW}. The ultrastructural changes of C. albicans indicate that the peptides may have different modes of action compared to Flucytosine as well as to each other, except for the Cecropin A-melittin hybrid [CA (1-7) M (2-9)] and optP7, showing very similar effects on C. albicans. This very first study demonstrates that BioSAXS shows promise to be used for antifungal drug development. However, this first study has limitations and further experiments are necessary to establish this application.

BioSAXS-an emerging method to accelerate, enrich and de-risk antimicrobial drug development.

Antimicrobial resistance is a worldwide threat to modern health care. Low-profit margin and high risk of cross-resistance resulted in a loss of interest in big pharma, contributing to the increasing threat. Strategies to address the problem are starting to emerge. Novel antimicrobial compounds with novel modes of action are especially valued because they have a lower risk of cross-resistance. Up to now determining the mode of action has been very time and resource consuming and will be performed once drug candidates were already progressed in preclinical development. BioSAXS is emerging as a new method to test up to thousands of compounds to classify them into groups based on ultra-structural changes that correlate to their modes of action. First experiments in E. coli (gram-negative) have demonstrated that using conventional and experimental antimicrobials a classification of compounds according to their mode of action was possible. Results were backed up by transmission electron microscopy. Further work showed that also gram-positive bacteria (Staphylococcus aureus) can be used and the effects of novel antimicrobial peptides on both types of bacteria were studied. Preliminary experiments also show that BioSAXS can be used to classify antifungal drugs, demonstrated on Candida albicans. In summary, BioSAXS can accelerate and enrich the discovery of antimicrobial compounds from screening projects with a novel mode of action and hence de-risk the development of urgently needed antimicrobial drugs.

Spatial Distribution of Intracellular Ion Concentrations in Aggregate-Forming HeLa Cells Analyzed by µ-XRF Imaging.

Protein aggregation is a hallmark of several severe neurodegenerative disorders such as Huntington's, Parkinson's, or Alzheimer's disease. Metal ions play a profound role in protein aggregation and altered metal-ion homeostasis is associated with disease progression. Here we utilize µ-X-ray fluorescence imaging in combination with rapid freezing to resolve the elemental distribution of phosphorus, sulfur, potassium, and zinc in huntingtin exon-1-mYFP expressing HeLa cells. Using quantitative XRF analysis, we find a threefold increase in zinc and a 10-fold enrichment of potassium that can be attributed to cellular stress response. While the averaged intracellular ion areal masses are significantly different in aggregate-containing cells, a local intracellular analysis shows no different ion content at the location of intracellular inclusion bodies. The results are compared to corresponding experiments on HeLa cells forming pseudoisocyanine chloride aggregates. As those show similar results, changes in ion concentrations are not exclusively linked to huntingtin exon-1 amyloid formation.

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.

Effect of ozone stress on the intracellular metabolites from Cobetia marina.

A GCxGC-MS system was employed with a non-polar × mid-polar column set for the metabolic non-target analysis of Cobetia marina, the model bacteria for marine biofouling. C. marina was treated with ozone to investigate the intracellular metabolic state change under oxidative stress. A minimal inhibitory concentration test was involved to guarantee that the applied ozone dosages were not lethal for the cells. In this study, non-target analyses were performed to identify the metabolites according to the NIST database. As a result, over 170 signals were detected under normal living conditions including 35 potential metabolites. By the comparison of ozone-treated and non-treated samples, five compounds were selected to describe observed trends of signals in the contour plots. Oleic acid exhibited a slight growth by increasing ozone dosage. In contrast, other metabolites such as the amino acid L-proline showed less abundance after ozone treatment, which was more evident once ozone dosage was raised. Thus, this work could provide a hint for searching for up/downregulating factors in such environmental stress conditions for C. marina. Graphical abstract.

Soft X-ray diffraction patterns measured by a LiF detector with sub-micrometre resolution and an ultimate dynamic range.

The unique diagnostic possibilities of X-ray diffraction, small X-ray scattering and phase-contrast imaging techniques applied with high-intensity coherent X-ray synchrotron and X-ray free-electron laser radiation can only be fully realized if a sufficient dynamic range and/or spatial resolution of the detector is available. In this work, it is demonstrated that the use of lithium fluoride (LiF) as a photoluminescence (PL) imaging detector allows measuring of an X-ray diffraction image with a dynamic range of ∼107 within the sub-micrometre spatial resolution. At the PETRA III facility, the diffraction pattern created behind a circular aperture with a diameter of 5 µm irradiated by a beam with a photon energy of 500 eV was recorded on a LiF crystal. In the diffraction pattern, the accumulated dose was varied from 1.7 × 105 J cm-3 in the central maximum to 2 × 10-2 J cm-3 in the 16th maximum of diffraction fringes. The period of the last fringe was measured with 0.8 µm width. The PL response of the LiF crystal being used as a detector on the irradiation dose of 500 eV photons was evaluated. For the particular model of laser-scanning confocal microscope Carl Zeiss LSM700, used for the readout of the PL signal, the calibration dependencies on the intensity of photopumping (excitation) radiation (λ = 488 nm) and the gain have been obtained.

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 Gram-negative bacteria Escherichia coli and the Gram-positive bacteria methicillin-resistant 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 Gram-positive 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.

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 1.7·106. 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 9.5·104 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.

A Multiperspective Approach to Solvent Regulation of Enzymatic Activity: HMG-CoA Reductase.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was investigated in different organic cosolvents by means of kinetic and calorimetric measurements, molecular dynamics simulations, and small-angle X-ray scattering. The combined experimental and theoretical techniques were essential to complement each other's limitations in the investigation of the complex interaction pattern between the enzyme, different solvent types, and concentrations. In this way, the underlying mechanisms for the loss of enzyme activity in different water-miscible solvents could be elucidated. These include direct inhibitory effects onto the active center and structural distortions.