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Characterization and prediction of the mechanism of action of antibiotics through NMR metabolomics
Background: The emergence of antibiotic resistant pathogenic bacteria has reduced our ability to combat infectious diseases. At the same time the numbers of new antibiotics reaching the market have decreased. This situation has created an urgent need to discover novel antibiotic scaffolds. Recently, the application of pattern recognition techniques to identify molecular fingerprints in ‘omics’ studies, has emerged as an important tool in biomedical research and laboratory medicine to identify pathogens, to monitor therapeutic treatments or to develop drugs with improved metabolic stability, toxicological profile and efficacy. Here, we hypothesize that a combination of metabolic intracellular fingerprints and extracellular footprints would provide a more comprehensive picture about the mechanism of action of novel antibiotics in drug discovery programs. Results: In an attempt to integrate the metabolomics approach as a classification tool in the drug discovery processes, we have used quantitative 1H NMR spectroscopy to study the metabolic response of Escherichia coli cultures to different antibiotics. Within the frame of our study the effects of five different and well-known antibiotic classes on the bacterial metabolome were investigated both by intracellular fingerprint and extracellular footprint analysis. The metabolic fingerprints and footprints of bacterial cultures were affected in a distinct manner and provided complementary information regarding intracellular and extracellular targets such as protein synthesis, DNA and cell wall. While cell cultures affected by antibiotics that act on intracellular targets showed class-specific fingerprints, the metabolic footprints differed significantly only when antibiotics that target the cell wall were applied. In addition, using a training set of E. coli fingerprints extracted after treatment with different antibiotic classes, the mode of action of streptomycin, tetracycline and carbenicillin could be correctly predicted. Conclusion: The metabolic profiles of E. coli treated with antibiotics with intracellular and extracellular targets could be separated in fingerprint and footprint analysis, respectively and provided complementary information. Based on the specific fingerprints obtained for different classes of antibiotics, the mode of action of several antibiotics could be predicted. The same classification approach should be applicable to studies of other pathogenic bacteria.
Structural insights into heme binding to IL-36α proinflammatory cytokine
Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins.
Bacterial community and PHB-accumulating bacteria associated with the wall and specialized niches of the hindgut of the forest cockchafer (Melolontha hippocastani)
A characterization of the bacterial community of the hindgut wall of two larval and the adult stages of the forest cockchafer (Melolontha hippocastani) was carried out using amplicon sequencing of the 16S rRNA gene fragment. We found that, in second-instar larvae, Caulobacteraceae and Pseudomonadaceae showed the highest relative abundances, while in third-instar larvae, the dominant families were Porphyromonadaceae and Bacteroidales-related. In adults, an increase of the relative abundance of Bacteroidetes, Proteobacteria (γ- and δ- classes) and the family Enterococcaceae (Firmicutes) was observed. This suggests that the composition of the hindgut wall community may depend on the insect’s life stage. Additionally, specialized bacterial niches hitherto very poorly described in the literature were spotted at both sides of the distal part of the hindgut chamber. We named these structures “pockets.” Amplicon sequencing of the 16S rRNA gene fragment revealed that the pockets contained a different bacterial community than the surrounding hindgut wall, dominated by Alcaligenaceae and Micrococcaceae-related families. Poly-β-hydroxybutyrate (PHB) accumulation in the pocket was suggested in isolated Achromobacter sp. by Nile Blue staining, and confirmed by gas chromatography–mass spectrometry analysis (GC-MS) on cultured bacterial mass and whole pocket tissue. Raman micro-spectroscopy allowed to visualize the spatial distribution of PHB accumulating bacteria within the pocket tissue. The presence of this polymer might play a role in the colonization of these specialized niches.
Light-responsive paper strips as CO-releasing material with a colourimetric response
Carbon monoxide (CO) is known for its multifaceted role in human physiology, and molecules that release CO in a controlled way have been proposed as therapeutic drugs. In this work, a light-responsive CO-releasing molecule (CORM-Dabsyl) showed a strong colourimetric response upon photochemical CO-release, owing to the tight conjugation of a Mn(i) tricarbonyl centre to a dabsyl chromophoric ligand (L). Whereas the complex was very stable in the dark in nitrogen-purged aqueous media, CO-release was effectively triggered using 405 nm irradiation. CORM-Dabsyl, L and the inactive product iCORM-Dabsyl have been investigated by DFT and TD-DFT calculations. Only mild toxicity of CORM-Dabsyl was observed against LX-2 and HepaRG® human cell lines (IC50 ∼ 30 μM). Finally, to develop a CO storage and release material that is readily applicable to therapeutic situations, CORM-Dabsyl was loaded on low-cost and easily disposable paper strips, from which the light triggered CO-release was conveniently visible with the naked eye.
New perspectives for viability studies with high-content analysis Raman spectroscopy (HCA-RS)
Raman spectroscopy has been widely used in clinical and molecular biological studies, providing high chemical specificity without the necessity of labels and with little-to-no sample preparation. However, currently performed Raman-based studies of eukaryotic cells are still very laborious and time-consuming, resulting in a low number of sampled cells and questionable statistical validations. Furthermore, the approach requires a trained specialist to perform and analyze the experiments, rendering the method less attractive for most laboratories. In this work, we present a new high-content analysis Raman spectroscopy (HCA-RS) platform that overcomes the current challenges of conventional Raman spectroscopy implementations. HCA-RS allows sampling of a large number of cells under different physiological conditions without any user interaction. The performance of the approach is successfully demonstrated by the development of a Raman-based cell viability assay, i.e., the effect of doxorubicin concentration on monocytic THP-1 cells. A statistical model, principal component analysis combined with support vector machine (PCA-SVM), was found to successfully predict the percentage of viable cells in a mixed population and is in good agreement to results obtained by a standard cell viability assay. This study demonstrates the potential of Raman spectroscopy as a standard high-throughput tool for clinical and biological applications.
Raman spectroscopy follows time-dependent changes in T lymphocytes isolated from spleen of endotoxemic mice
T lymphocytes (T cells) are highly specialized members of the adaptive immune system and hold the key to the understanding the hosts’ response toward invading pathogen or pathogen-associated molecular patterns such as LPS. In this study, noninvasive Raman spectroscopy is presented as a label-free method to follow LPS-induced changes in splenic T cells during acute and postacute inflammatory phases (1, 4, 10, and 30 d) with a special focus on CD4+ and CD8+ T cells of endotoxemic C57BL/6 mice. Raman spectral analysis reveals highest chemical differences between CD4+ and CD8+ T cells originating from the control and LPS-treated mice during acute inflammation, and the differences are visible up to 10 d after the LPS insult. In the postacute phase, CD4+ and CD8+ T cells from treated and untreated mice could not be differentiated anymore, suggesting that T cells largely regained their original status. In sum, the biological information obtained from Raman spectra agrees with immunological readouts demonstrating that Raman spectroscopy is a well-suited, label-free method for following splenic T cell activation in systemic inflammation from acute to postacute phases. The method can also be applied to directly study tissue sections as is demonstrated for spleen tissue one day after LPS insult.T lymphocytes (T cells) are highly specialized members of the adaptive immune system and hold the key to the understanding the hosts’ response toward invading pathogen or pathogen-associated molecular patterns such as LPS. In this study, noninvasive Raman spectroscopy is presented as a label-free method to follow LPS-induced changes in splenic T cells during acute and postacute inflammatory phases (1, 4, 10, and 30 d) with a special focus on CD4+ and CD8+ T cells of endotoxemic C57BL/6 mice. Raman spectral analysis reveals highest chemical differences between CD4+ and CD8+ T cells originating from the control and LPS-treated mice during acute inflammation, and the differences are visible up to 10 d after the LPS insult. In the postacute phase, CD4+ and CD8+ T cells from treated and untreated mice could not be differentiated anymore, suggesting that T cells largely regained their original status. In sum, the biological information obtained from Raman spectra agrees with immunological readouts demonstrating that Raman spectroscopy is a well-suited, label-free method for following splenic T cell activation in systemic inflammation from acute to postacute phases. The method can also be applied to directly study tissue sections as is demonstrated for spleen tissue one day after LPS insult.
Microfluidic Cultivation and Laser Tweezers Raman Spectroscopy of E. coli under Antibiotic Stress
Analyzing the cells in various body fluids can greatly deepen the understanding of the mechanisms governing the cellular physiology. Due to the variability of physiological and metabolic states, it is important to be able to perform such studies on individual cells. Therefore, we developed an optofluidic system in which we precisely manipulated and monitored individual cells of Escherichia coli. We tested optical micromanipulation in a microfluidic chamber chip by transferring individual bacteria into the chambers. We then subjected the cells in the chambers to antibiotic cefotaxime and we observed the changes by using time-lapse microscopy. Separately, we used laser tweezers Raman spectroscopy (LTRS) in a different micro-chamber chip to manipulate and analyze individual cefotaxime-treated E. coli cells. Additionally, we performed conventional Raman micro-spectroscopic measurements of E. coli cells in a micro-chamber. We found observable changes in the cellular morphology (cell elongation) and in Raman spectra, which were consistent with other recently published observations. The principal component analysis (PCA) of Raman data distinguished between the cefotaxime treated cells and control. We tested the capabilities of the optofluidic system and found it to be a reliable and versatile solution for this class of microbiological experiments.
Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells
Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.
A Computational Pipeline for Sepsis Patients’ Stratification and Diagnosis
Sepsis is still a little acknowledged public health issue, despite its increasing incidence and the growing mortality rate. In addition, a clear diagnosis can be lengthy and complicated, due to highly variable symptoms and non-specific criteria, causing the disease to be diagnosed and treated too late. This paper presents the HemoSpec platform, a decision support system which, by collecting and automatically processing data from several acquisition devices, can help in the early diagnosis of sepsis.