Tedesco, Pietro (2016) DRUG DISCOVERY AND SEARCH FOR NEW THERAPEUTIC TARGETS: STRATEGIES TO COUNTERACT HUMAN PATHOGENS USING CAENORHABDITIS ELEGANS AND COLD-ADAPTED MICROORGANISMS. [Tesi di dottorato]

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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.

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