Effects of P. aeruginosa quorum sensing molecules on Drosophila genome: a new tool to identify candidate genes involved in host-pathogen crosstalk

Efectele moleculelor de quorum sensing produse de P. aeruginosa asupra genomului de Drosophila: o noua cale de identificare a genelor candidat implicate in comunicarea gazda-agent infectios

Pseudomonas aeruginosa, un microorganism multirezistent, responsabil de rate ridicate de  morbiditate și mortalitate datorate infecțiilor oportuniste și nosocomiale, mai ales în tarile in curs de dezvoltare, comunică intraspecific si cu gazdele eucariote prin mecanisme de semnalizare intercelulara, mediate de mecanisme de quorum sensing care se bazeaza pe producerea de molecule cu greutate moleculară mică implicate in reglarea expresiei coordonate a factorilor de virulență. Moleculele de semnalizare produse de P. aeruginosa (MSPA) pot exercita  efecte pleiotropice asupra celulelor eucariote în cultura. În prezentul proiect ne propunem un model original, integrativ de abordare in vivo pentru investigarea modului în care astfel de molecule de semnalizare pot modula genomul de Drosophila melanogaster, în scopul identificarii genelor implicate in imunitate si a ortologilor  lor umani. În acest scop, vor fi evaluate efectele fenotipice ale MSPA și ale celulelor viabile de P. aeruginosa (tulpini virulente versus defective pentru sistemul de quorum sensing) asupra liniilor de D. melanogaster de tip sălbatic și mutante, in scopul evidențierii genelor cu niveluri de expresie modulate semnificativ, urmate de identificarea prin mijloace bioinformatice a genelor ortoloage umane, evaluarea efectelor MSPA si ale celulelor viabile de P. aeruginosa asupra  profilurilor de expresie genica in celule eucariote umane și de formularea unor concluzii privind contribuția rezultatelor obtinute la dezvoltarea de noi strategii terapeutice utile in managementul bolilor infecțioase și al celor cu substrat imunologic.

Pseudomonas aeruginosa, an emerging multi-drug resistant organism, one of the major contributors to morbidity and mortality in opportunistic and nosocomial infections, communicates intra-species and with the eukaryotic hosts by cross-kingdom inter-cellular signaling mechanisms such as quorum sensing (QS), relying on low-molecular weight excreted molecules, to control the production of virulence factors. The P. aeruginosa QS signaling molecules (QSSMs) have in vitro pleiotropic effects on eukaryotic cells. In the present project we propose an original, integrative in vivo approach for the investigation of the way in which such signaling molecules modulate the Drosophila melanogaster genome, in order to identify the most sensitive eukaryotic genes targeted by the QSSMs and the specific orthologous genes in the Homo sapiens genome. In order to accomplish our purpose, we will evaluate the phenotypic effects of QSSMs and viable P. aeruginosa cells (virulent versus quorum sensing defective strains) on D. melanogaster wild type and mutant strains, highlight the genes expressing a significant up- or down-regulation, identify by bioinformatics the orthologous human genes, assess the effects of P. aeruginosa QSSMs and viable P. aeruginosa cells on the respective genes profiles in the human eukaryotic cells and finally formulate the clinical contribution of our findings to future development of novel therapeutics for a multitude of infectious and immunological diseases.

(Engleza)

In the present project, we propose an original, integrative in vivo approach for the investigation of the way in which P. aeruginosa QSSMs modulate the genome of D. melanogaster, in order to further identify significantly up- or down-regulated genes. Our in vivo approach concerning gene expression modulation by QSSMs molecules is intended to reveal genes involved in the immune response, but also genes involved in other stress adaptive response pathways, such as apoptosis. This integrative model will allow us to identify specific orthologous genes in the genome of Homo sapiens and to investigate how they are modulated by QSSMs . Since the concentration of the synthetic QS molecules is controllable, we will be able to simulate different scenarios of infections with various bacterial charges and to monitor accordingly the modulation of the transcription rate of GOIs (genes of interest). In order to accomplish our purpose, the following objectives will be followed:
Ob.1: Evaluation of the phenotypic effects of QSSM and viable P. aeruginosa cells (virulent versus quorum sensing defective strains) on D. melanogaster wild type and mutant strains.
Ob.2: Highlighting the gene expression profile of inoculated/infected fruit flies versus untreated organisms.
Ob. 3: Bioinformatic analysis of the functions of the genes expressing a significant up- or down-regulation and identifying orthologous human genes.
Ob. 4: Assessment of the effects of P. aeruginosa QSSMs and viable P. aeruginosa cells (virulent versus quorum sensing defective strains) on the identified orthologous human genes expression and subsequent phenotypic traits using eukaryotic cells in culture.
Ob.5: Formulation of the clinical significance of the findings, based on the evidenced bioactivities of the chemically defined QSSMs.
Ob.6: Project management, dissemination of results and human resource development.