Establishment of a transparent soil system to study Bacillus subtilis chemical ecology

Bacterial secondary metabolites are structurally diverse molecules that drive microbial interaction by altering growth, cell differentiation, and signaling. Bacillus subtilis, a Gram-positive soil-dwelling bacterium, produces a wealth of secondary metabolites, among them, lipopeptides have been vastly studied by their antimicrobial, antitumor, and surfactant activities. However, the natural functions of secondary metabolites in the lifestyles of the producing organism remain less explored under natural conditions, i.e. in soil. Here, we describe a hydrogel-based transparent soil system to investigate B. subtilis chemical ecology under controllable soil-like conditions. The transparent soil matrix allows the growth of B. subtilis and other isolates gnotobiotically and under nutrient-controlled conditions. Additionally, we show that transparent soil allows the detection of lipopeptides production and dynamics by HPLC-MS and MALDI-MS imaging, along with fluorescence imaging of 3-dimensional bacterial assemblages. We anticipate that this affordable and highly controllable system will promote bacterial chemical ecology research and help to elucidate microbial interactions driven by secondary metabolites.

MALDI mass spectrometry imaging of fresh and processed food: constituents, ingredients, contaminants and additives

Mass Spectrometry imaging (MS imaging) provides spatial information for a wide range of compound classes in different sample matrices. We used MS imaging to investigate the distribution of components in fresh and processed food, including meat, dairy and bakery products. The MS imaging workflow was optimized to cater to the specific properties and challenges of the individual samples. We successfully detected highly nonpolar and polar constituents such as beta-carotene and anthocyanins, respectively. For the first time, the distribution of a contaminant and a food additive was visualized in processed food. We detected acrylamide in German gingerbread and investigated the penetration of the preservative natamycin into cheese. For this purpose, a new data analysis tool was developed to study the penetration of analytes from uneven surfaces. Our results show that MS imaging has great potential in food analysis to provide relevant information about components' distributions, particularly those underlying official regulations.

Towards the production of tikitericin: A novel peptide from Thermogemmatispora strain T81

<p>The utilisation of natural products for treatment of human ailments has been rooted in various cultures for centuries. Extraction of natural products has been essential for the discovery of new drugs and inspiration for synthetic analogues. Since the success of penicillin, microbial natural products have been of interest. Genome mining of Thermogemmatisporastrain T81, a thermophile from the Taupo Volcanic Zone, found the potential for the production of novel ribosomally synthesised and post-translationally modified peptides (RiPPs). Previous work showed that T81 exhibited antimicrobial activity against a wide variety of extremophillic bacteria. Although the three thiopeptides encoded forin the genome of T81 have not been found, the lanthipeptide tikitericin has recently been isolated and described. Unfortunately tikitericin is produced in low quantities by T81 andbioactivity data has not yet been obtained. Because of its potential antimicrobial activity, different routes to produce it are of interest. The aim of this project wasto synthesisetikitericin by solid phase peptide synthesis. MS imaging was also utilised to search for the presence of tikitericin as an antimicrobial agent in situ.</p>

Towards the production of tikitericin: A novel peptide from Thermogemmatispora strain T81

<p>The utilisation of natural products for treatment of human ailments has been rooted in various cultures for centuries. Extraction of natural products has been essential for the discovery of new drugs and inspiration for synthetic analogues. Since the success of penicillin, microbial natural products have been of interest. Genome mining of Thermogemmatisporastrain T81, a thermophile from the Taupo Volcanic Zone, found the potential for the production of novel ribosomally synthesised and post-translationally modified peptides (RiPPs). Previous work showed that T81 exhibited antimicrobial activity against a wide variety of extremophillic bacteria. Although the three thiopeptides encoded forin the genome of T81 have not been found, the lanthipeptide tikitericin has recently been isolated and described. Unfortunately tikitericin is produced in low quantities by T81 andbioactivity data has not yet been obtained. Because of its potential antimicrobial activity, different routes to produce it are of interest. The aim of this project wasto synthesisetikitericin by solid phase peptide synthesis. MS imaging was also utilised to search for the presence of tikitericin as an antimicrobial agent in situ.</p>

Interleukin-13 overexpressing mice represent an advanced pre-clinical model for detecting the distribution of anti-mycobacterial drugs within centrally necrotizing granulomas

The Mycobacterium tuberculosis (Mtb)-harboring granuloma with a necrotic center surrounded by a fibrous capsule is the hallmark of tuberculosis (TB). For a successful treatment, antibiotics need to penetrate these complex structures to reach their bacterial targets. Hence, animal models reflecting the pulmonary pathology of TB patients are of particular importance to improve the pre-clinical validation of novel drug candidates. Mtb-infected interleukin-13 overexpressing (IL-13 tg ) mice develop a TB pathology very similar to patients and, in contrast to other mouse models, also share pathogenetic mechanisms. Accordingly, IL-13 tg animals represent an ideal model for analyzing the penetration of novel anti-TB drugs into various compartments of necrotic granulomas by matrix-assisted-laser-desorption/ionization-mass spectrometry imaging (MALDI MS imaging). In the present study, we evaluated the suitability of BALB/c IL-13 tg mice for determining the antibiotic distribution within necrotizing lesions. To this end, we established a workflow based on the inactivation of Mtb by gamma irradiation while preserving lung tissue integrity and drug distribution, which is essential for correlating drug penetration with lesion pathology. MALDI MS imaging analysis of clofazimine, pyrazinamide and rifampicin revealed a drug-specific distribution within different lesion types including cellular granulomas, developing in BALB/c wild-type mice, and necrotic granulomas of BALB/c IL-13 tg animals, emphasizing the necessity of pre-clinical models reflecting human pathology. Most importantly, our study demonstrates that BALB/c IL-13 tg mice recapitulate the penetration of antibiotics into human lesions. Therefore, our workflow in combination with the IL-13 tg mouse model provides an improved and accelerated evaluation of novel anti-TB drugs and new regimens in the pre-clinical stage.

On-tissue spatial proteomics integrating MALDI-MS imaging with shotgun proteomics reveals soy consumption-induced biomarkers in a fragile X syndrome mouse model

Soy-based diets are associated with increased seizures and autism. Thus, there is an acute need for unbiased protein biomarker identification in Fragile X syndrome (FXS) in response to soy consumption. Herein, we present a spatial proteomics approach integrating mass spectrometry imaging (MSI) with label-free proteomics in a mouse model of FXS to map the spatial distribution and quantify the levels of proteins in the hippocampus and hypothalamus brain regions. In total, 1,004 unique peptides were spatially resolved, demonstrating the diverse array of peptidomes present in the tissue slices and the broad coverage of the strategy. A group of proteins that are known to be involved in the GABAergic system, synaptic transmission, and co-expression network analysis indicated that protein in soy group was significantly associated with metabolism and synapse modules in the Fmr1KO brain. Ultimately, this spatial proteomics work laid the ground for identifying novel therapeutic targets and biomarkers for FXS.

Structural library and visualization of endogenously oxidized phosphatidylcholines using mass spectrometry-based techniques

Although oxidized phosphatidylcholines (oxPCs) play critical roles in numerous pathological events, the type and production sites of endogenous oxPCs remain unknown because of the lack of structural information and dedicated analytical methods. Herein, a library of 465 oxPCs is constructed using high-resolution mass spectrometry-based non-targeted analytical methods and employed to detect 70 oxPCs in mice with acetaminophen-induced acute liver failure. We show that doubly oxygenated polyunsaturated fatty acid (PUFA)-PCs (PC PUFA;O2), containing epoxy and hydroxide groups, are generated in the early phase of liver injury. Hybridization with in-vivo 18O labeling and matrix-assisted laser desorption/ionization-tandem MS imaging reveals that PC PUFA;O2 are accumulated in cytochrome P450 2E1-expressing and glutathione-depleted hepatocytes, which are the major sites of liver injury. The developed library and visualization methodology should facilitate the characterization of specific lipid peroxidation events and enhance our understanding of their physiological and pathological significance in lipid peroxidation-related diseases.