DRUG DISCOVERY AND SEARCH FOR NEW THERAPEUTIC TARGETS: STRATEGIES TO COUNTERACT HUMAN PATHOGENS USING CAENORHABDITIS ELEGANS AND COLD-ADAPTED MICROORGANISMS

The alarming diffusion of multidrug-resistant pathogens represents a serious threat to human health and economy. To counteract this phenomenon two main strategies has been pursued: the research of novel therapeutic targets and the identification of novel drugs exploiting natural products. The first strategy is focused principally on the identification of genes involved into bacterial virulence mechanism, to “disarm” pathogens. Dissecting and validating the pathogenicity determinants of human pathogens have been facilitated by the use of non-vertebrate host models such us Caenorhabditis elegans. The second strategy aims at the utilization of the huge potential of secondary metabolites produced by microorganims focusing on bacteria living in extreme environments such as the oceans, the poles and the deserts. In this research project we applied these two approaches with special emphasis on pathogens belonging to the Burkholderia cepacia complex Bcc and parasite nematodes. To this aim we exploited the versatility of C. elegans as versatile model system and the potential of psychrophilic microorganisms as source of novel bioactive compounds. The first part of the project was focused on establishing an infection model between a selected panel of strains belonging to Bcc and the nematode C. elegans. With this aim, two different toxicity tests were performed to monitos host mortality by accumulation in the intestine or by toxins production. A Virulence Ranking scheme was defined based on the percentage of surviving worms. Our results suggested that only the cystic fibrosis isolated strains possessed profound nematode killing ability to accumulate in worms’ intestines. We also, investigated the role of host transporer during the infection. For this analysis a complete set of isogenic nematode single Multidrug Resistance associated Protein efflux mutants and a number of efflux inhibitors were interrogated in the host toxicity assays. We demonstrated that disabling host transporters genetically (C. elegans knock out mutants) or chemically (efflux inhibitors) enhanced nematodes mortality, suggesting a role in toxin-substrate recognition for some of the tested transporters. The work performed has provided useful information on Bcc pathogenicity and it achieved the development of a suitable platform for dissecting Bcc virulence factors and for drug discovery and validation of anti-Bcc molecules. The second part of the project was focused on the identification of new bioactive compounds targeting Bcc strains and parasite nematodes. To achieve this goal a biodiscovery pipeline was developed starting with isolation of cold-adapted bacteria from sediments collected from Antarctica and Tibet. The isolates were first evaluated for their antimicrobial and anthelmintic activity with cell-based assays. Antimicrobial capability was evaluated using the cross-streaking experiments targeting human pathogens, while for the anthelmintic activity, we assayed isolates ability to survive and kill the nematodes C. elegans that was used as model helmint. Positive isolates to primary screening were grown in liquid cultures to produce crude extracts in order to perform secondary assays. Positive extracts were then fractionated using Solid Phase Extraction, and HPLC, and pure bioactive compounds were identified with LC-MS and NMR. With this strategy, we achieved the isolation of 3 Rhamnolipids, two of which were new, embedded with high (MIC < 1 μg/mL) antimicrobial activity against Bcc strains. We found also one positive extract able to kill the nematodes and a second one that completely inhibit the growth of Francisella tularensis an opportunistic human pathogen, at the concentration of 25 μg/mL. LC-MS analysis of this fraction revealed the presence of 16§-hydroxycrambescidin, a known alkaloid with unreported antimicrobial activity

Enhanced production of biobased, biodegradable, Poly(3-hydroxybutyrate) using an unexplored marine bacterium Pseudohalocynthiibacter aestuariivivens, isolated from highly polluted coastal environment.

The production and disposal of plastics from limited fossil reserves, has prompted research for greener and sustainable alternatives. Polyhydroxyalkanoates (PHAs) are biocompatible, biodegradable, and thermoprocessable polyester produced by microbes. PHAs found several applications but their use is limited due to high production cost and low yields. Herein, for the first time, the isolation and characterization of Pseudohalocynthiibacter aestuariivivens P96, a marine bacterium able to produce surprising amount of PHAs is reported. In the best growth condition P96 was able to reach a maximum production of 4.73 g/L, corresponding to the 87 % of total cell dry-weight. Using scanning and transmission microscopy, lab-scale fermentation, spectroscopic techniques, and genome analysis, the production of thermoprocessable polymer Polyhydroxybutyrate P(3HB), a PHAs class, endowed with mechanical and thermal properties comparable to that of petroleum-based plastics was confirmed. This study represents a milestone toward the use of this unexplored marine bacterium for P(3HB) production. [Abstract copyright: Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Isolation and characterization of strain exiguobacterium sp. Krl4, a producer of bioactive secondary metabolites from a Tibetan glacier

Extremophilic microorganisms represent a unique source of novel natural products. Among them, cold adapted bacteria and particularly alpine microorganisms are still underexplored. Here, we describe the isolation and characterization of a novel Gram-positive, aerobic rod-shaped alpine bacterium (KRL4), isolated from sediments from the Karuola glacier in Tibet, China. Complete phenotypic analysis was performed revealing the great adaptability of the strain to a wide range of temperatures (5–40 ◦C), pHs (5.5–8.5), and salinities (0–15% w/v NaCl). Genome sequencing identified KRL4 as a member of the placeholder genus Exiguobacterium_A and annotation revealed that only half of the protein-encoding genes (1522 of 3079) could be assigned a putative function. An analysis of the secondary metabolite clusters revealed the presence of two uncharacterized phytoene synthase containing pathways and a novel siderophore pathway. Biological assays confirmed that the strain produces molecules with antioxidant and siderophore activities. Furthermore, intracellular extracts showed nematocidal activity towards C. elegans, suggesting that strain KRL4 is a source of anthelmintic compounds

Antibiotics from Deep-Sea Microorganisms : Current Discoveries and Perspectives

Funding: This research was funded by the MarPipe project: Improving the flow in the pipeline of the next generation of marine biodiscovery scientists, funded through the European Commission H2020-MSCA-ITN-ETN scheme, GA721421.Peer reviewedPublisher PD

Cryo-protective effect of an ice-binding protein derived from Antarctic bacteria

Cold environments are populated by organisms able to contravene deleterious effects of low temperature by diverse adaptive strategies, including the production of ice binding proteins (IBPs) that inhibit the growth of ice crystals inside and outside cells. We describe the properties of such a protein (EfcIBP) identified in the metagenome of an Antarctic biological consortium composed of the ciliate Euplotes focardii and psychrophilic non-cultured bacteria. Recombinant EfcIBP can resist freezing without any conformational damage and is moderately heat stable, with a midpoint temperature of 66.4 °C. Tested for its effects on ice, EfcIBP shows an unusual combination of properties not reported in other bacterial IBPs. First, it is one of the best-performing IBPs described to date in the inhibition of ice recrystallization, with effective concentrations in the nanomolar range. Moreover, EfcIBP has thermal hysteresis activity (0.53 °C at 50 μm) and it can stop a crystal from growing when held at a constant temperature within the thermal hysteresis gap. EfcIBP protects purified proteins and bacterial cells from freezing damage when exposed to challenging temperatures. EfcIBP also possesses a potential N-terminal signal sequence for protein transport and a DUF3494 domain that is common to secreted IBPs. These features lead us to hypothesize that the protein is either anchored at the outer cell surface or concentrated around cells to provide survival advantage to the whole cell consortium

Identification of a Sorbicillinoid-Producing Aspergillus Strain with Antimicrobial Activity Against Staphylococcus aureus: a New Polyextremophilic Marine Fungus from Barents Sea

The exploration of poorly studied areas of Earth can highly increase the possibility to discover novel bioactive compounds. In this study, the cultivable fraction of fungi and bacteria from Barents Sea sediments has been studied to mine new bioactive molecules with antibacterial activity against a panel of human pathogens. We isolated diverse strains of psychrophilic and halophilic bacteria and fungi from a collection of nine samples from sea sediment. Following a full bioassay-guided approach, we isolated a new promising polyextremophilic marine fungus strain 8Na, identified as Aspergillusprotuberus MUT 3638, possessing the potential to produce antimicrobial agents. This fungus, isolated from cold seawater, was able to grow in a wide range of salinity, pH and temperatures. The growth conditions were optimised and scaled to fermentation, and its produced extract was subjected to chemical analysis. The active component was identified as bisvertinolone, a member of sorbicillonoid family that was found to display significant activity against Staphylococcus aureus with a minimum inhibitory concentration (MIC) of 30 μg/mL. © 2018, Springer Science+Business Media, LLC, part of Springer Nature

CL316,243, a β3-adrenergic receptor agonist, induces muscle hypertrophy and increased strength.

Studies in vitro have demonstrated that β3-adrenergic receptors (β3-ARs) regulate protein metabolism in skeletal muscle by promoting protein synthesis and inhibiting protein degradation. In this study, we evaluated whether activation of β3-ARs by the selective agonist CL316,243 modifies the functional and structural properties of skeletal muscles of healthy mice. Daily injections of CL316,243 for 15 days resulted in a significant improvement in muscle force production, assessed by grip strength and weight tests, and an increased myofiber cross-sectional area, indicative of muscle hypertrophy. In addition, atomic force microscopy revealed a significant effect of CL316,243 on the transversal stiffness of isolated muscle fibers. Interestingly, the expression level of mammalian target of rapamycin (mTOR) downstream targets and neuronal nitric oxide synthase (NOS) was also found to be enhanced in tibialis anterior and soleus muscles of CL316,243 treated mice, in accordance with previous data linking β3-ARs to mTOR and NOS signaling pathways. In conclusion, our data suggest that CL316,243 systemic administration might be a novel therapeutic strategy worthy of further investigations in conditions of muscle wasting and weakness associated with aging and muscular diseases

Antimicrobial Activity of Monoramnholipids Produced by Bacterial Strains Isolated from the Ross Sea (Antarctica)

Microorganisms living in extreme environments represent a huge reservoir of novel antimicrobial compounds and possibly of novel chemical families. Antarctica is one of the most extraordinary places on Earth and exhibits many distinctive features. Antarctic microorganisms are well known producers of valuable secondary metabolites. Specifically, several Antarctic strains have been reported to inhibit opportunistic human pathogens strains belonging to Burkholderia cepacia complex (Bcc). Herein, we applied a biodiscovery pipeline for the identification of anti-Bcc compounds. Antarctic sub-sea sediments were collected from the Ross Sea, and used to isolate 25 microorganisms, which were phylogenetically affiliated to three bacterial genera (Psychrobacter, Arthrobacter, and Pseudomonas) via sequencing and analysis of 16S rRNA genes. They were then subjected to a primary cell-based screening to determine their bioactivity against Bcc strains. Positive isolates were used to produce crude extracts from microbial spent culture media, to perform the secondary screening. Strain Pseudomonas BNT1 was then selected for bioassay-guided purification employing SPE and HPLC. Finally, LC-MS and NMR structurally resolved the purified bioactive compounds. With this strategy, we achieved the isolation of three rhamnolipids, two of which were new, endowed with high (MIC < 1 μg/mL) and unreported antimicrobial activity against Bcc strains

Bisdemethoxycurcumin and Its Cyclized Pyrazole Analogue Differentially Disrupt Lipopolysaccharide Signalling in Human Monocyte-Derived Macrophages

Several studies suggest that curcumin and related compounds possess antioxidant and anti-inflammatory properties including modulation of lipopolysaccharide- (LPS-) mediated signalling in macrophage cell models. We here investigated the effects of curcumin and the two structurally unrelated analogues GG6 and GG9 in primary human blood-derived macrophages as well as the signalling pathways involved. Macrophages differentiated from peripheral blood monocytes for 7 days were activated with LPS or selective Toll-like receptor agonists for 24 h. The effects of test compounds on cytokine production and immunophenotypes evaluated as CD80+/CCR2+ and CD206+/CD163+ subsets were examined by ELISA and flow cytometry. Signalling pathways were probed by Western blot. Curcumin (2.5–10 μM) failed to suppress LPS-induced inflammatory responses. While GG6 reduced LPS-induced IκB-α degradation and showed a trend towards reduced interleukin-1β release, GG9 prevented the increase in proinflammatory CD80+ macrophage subset, downregulation of the anti-inflammatory CD206+/CD163+ subset, increase in p38 phosphorylation, and increase in cell-bound and secreted interleukin-1β stimulated by LPS, at least in part through signalling pathways not involving Toll-like receptor 4 and nuclear factor-κB. Thus, the curcumin analogue GG9 attenuated the LPS-induced inflammatory response in human blood-derived macrophages and may therefore represent an attractive chemical template for macrophage pharmacological targeting