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]
| Item Type: |
Tesi di dottorato
|
| Lingua: |
English |
| Title: |
DRUG DISCOVERY AND SEARCH FOR NEW THERAPEUTIC TARGETS: STRATEGIES TO COUNTERACT HUMAN PATHOGENS USING CAENORHABDITIS ELEGANS AND COLD-ADAPTED
MICROORGANISMS |
| Creators: |
| Creators | Email |
|---|
| Tedesco, Pietro | p.tedesco@ibp.cnr.it |
|
| Date: |
22 April 2016 |
| Number of Pages: |
177 |
| Institution: |
Università degli Studi di Napoli Federico II |
| Department: |
Scienze Chimiche |
| Scuola di dottorato: |
Biotecnologie |
| Dottorato: |
Scienze biotecnologiche |
| Ciclo di dottorato: |
28 |
| Coordinatore del Corso di dottorato: |
| nome | email |
|---|
| Sannia, Giovanni | sannia@unina.it |
|
| Tutor: |
| nome | email |
|---|
| Sannia, Giovanni | UNSPECIFIED | | de Pascale, Donatella | UNSPECIFIED |
|
| Date: |
22 April 2016 |
| Number of Pages: |
177 |
| Uncontrolled Keywords: |
Drug discovery; pathogens; cold-adapted bacteria; host model; bioprospecting |
| Settori scientifico-disciplinari del MIUR: |
Area 05 - Scienze biologiche > BIO/10 - Biochimica
Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare |
[error in script]
[error in script]
| Date Deposited: |
13 Apr 2016 08:43 |
| Last Modified: |
31 Oct 2016 13:54 |
| URI: |
http://www.fedoa.unina.it/id/eprint/10769 |
Abstract
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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