Identification and characterization of biogenic silencers of Pseudomonas aeruginosa quorum sensing

Nemogućnost kontrole pojave i širenja rezistencije na antibiotike usmerila je istraživanja u poslednje dve dekade ka iznalaženju novih terapeutskih opcija, sa ciljem tretmana infekcija izazvanih patogenim bakterijama rezistentnim na veći broj kliniĉki znaĉajnih antibiotika. Uzevši u obzir ĉinjenicu da je antibiotska rezistencija kompleksan, multifaktorijalni fenomen, rešenje ovog problema ukljuĉuje niz pristupa usmerenih na kontrolu faktora koji olakšavaju nastanak i širenje rezistencije. Jedan od tih pristupa sastoji se u razvoju novih terapeutika koji bi delovali mehanizmima razliĉitim od trenutno dostupnih antibiotika. U tom pogledu, antivirulentna terapija zamišljena je kao obećavajuća alternativa sa ciljem kontrole virulencije specifiĉne za odreĊene patogene, bez vršenja snažnog selektivnog pritiska na bakterijske ćelije. Imajući u vidu da je Pseudomonas aeruginosa jedan od vodećih uzroĉnika unutarbolniĉkih infekcija širom sveta, leĉenje infekcija izazvanih ovim patogenom predstavlja veliki terapeutski izazov. P. aeruginosa svoj patogeni potencijal ostvaruje zahvaljujući mnogobrojnim uroĊenim, steĉenim i adaptivnim mehanizmima rezistencije. Pored toga, posedovanje sistema meĊućelijske komunikacije (eng. quorum sensing, QS) ovom patogenu omogućava fleksibilnost u regulaciji ekspresije gena ukljuĉenih u virulenciju, formiranje biofilma, produkciju sekundarnih metabolita i faktora koji imaju ulogu u zaštiti od imunskog sistema domaćina. Stoga bi primena antivirulentne terapije zasnovane na utišavanju meĊućelijske komunikacije bakterija mogla poslužiti kao obećavajuće oruĊe u kontroli infekcija izazvanih predstavnicima P. aeruginosa za koje ne postoji adekvatna terapija trenutno dostupnim antibioticima. U skladu sa time, predmet prouĉavanja ove teze bila je potraga za novim utišivaĉima meĊućelijske komunikacije bakterija (eng. quorum quenching, QQ) produkovanih od strane kliniĉkih izolata koji tokom infekcija dele istu ekološku nišu sa P. aeruginosa. Analizirana je kolekcija Gramnegativnih kliniĉkih izolata Laboratorije za molekularnu mikrobiologiju, Instituta za molekularnu genetiku i genetiĉko inženjerstvo, Univerziteta u Beogradu sa ciljem pronalaženja sojeva koji produkuju QQ molekule. Nakon odabira sojeva nosioca QQ fenotipa, usledila je njihova identifikacija kao i funkcionalna karakterizacija na model sistemu kliniĉkog izolata P. aeruginosa MMA83. Upotrebom Chromobacterium violaceum CV026 biosenzora, selektovano je 19 sojeva (od ukupno 633 analiziranih izolata) koji su ispoljavali QQ fenotip, od kojih su dva izolata Delftia sp. 11304 i Burkholderia sp. BCC4135, na osnovu najznaĉajnije QQ aktivnosti izdvojena za detaljniju karakterizaciju. UtvrĊeno je da Delftia sp. 11304 soj pripada Delftia tsuruhatensis vrsti, dok Burkholderia sp. BCC4135 pripada Burkholderia cepacia vrsti sa novim sekvencnim tipom ST1485. In silico analizom genomskih sekvenci ustanovljeno je da oba odabrana soja poseduju izuzetan potencijal virulencije i rezistencije na antimikrobna jedinjenja. Priroda QQ molekula koje produkuju ovi sojevi bila je razliĉita; konstatovano je da soj 11304 produkuje male molekule neproteinske prirode (QS inhibitore, QSI), dok BCC4135 produkuje QQ enzime. Pored toga, kod BCC4135 izolata utvrĊeno je prisustvo i QS fenomena. Ukupni etil-acetatni ekstrakt supernatanta soja D. tsuruhatensis 11304 (QSI ekstrakt) ostvario je znaĉajan antivirulentni potencijal na model sistemu kliniĉkog izolata P. aeruginosa MMA83...finding novel therapeutic options to combat multidrug-resistant pathogenic bacteria during the last two decades. Given the fact that antimicrobial resistance is a complex, multifactorial phenomenon, the solution to this problem comprises a range of approaches focused on monitoring the factors that facilitate the emergence and spread of resistance. One of proposed strategies consists of developing novel therapeutics that operate under different principles to the currently available antibiotics. In this respect, antivirulence therapy has been conceived as a promising alternative to control virulence in a pathogen-specific manner, without exerting strong selective pressure on the bacterial cells. Having in mind that Pseudomonas aeruginosa has been considered as a leading cause of nosocomial infections worldwide, the treatment of infections caused by this pathogen represents a major therapeutic challenge. The pathogenic potential of P. aeruginosa has been accomplished due to a numerous innate, acquired, and adaptive resistance mechanisms. In addition, the presence of cell-to-cell communication system (quorum sensing, QS) allows this pathogen the flexibility in the regulation of virulence gene expression responsible for biofilm development, production of secondary metabolites, and immuneevasive factors. Therefore, the use of antivirulence therapy based on the silencing of bacterial communication could serve as a promising tool in the control of infections caused by P. aeruginosa for which there is no adequate therapy with currently available antibiotics. Accordingly, the subject of this thesis was discovery and characterization of novel quenchers of bacterial cell-to-cell communication produced by clinical isolates that share the same ecological niche with P. aeruginosa during infections. A collection of Gram-negative clinical isolates from the Laboratory for Molecular Microbiology Institute of Molecular Genetics and Genetic Engineering University of Belgrade was analyzed in order to select the strains that produce quorum quenching (QQ) molecules. After the selection of strains carrying QQ phenotype, the identification of QQ molecules as well as their functional characterization on the model system of P. aeruginosa MMA83 clinical isolate was performed. Using Chromobacterium violaceum CV026 biosensor, 19 strains (out of a total of 633 analyzed isolates) with QQ phenotype were discovered, among which two clinical isolates Delftia sp. 11304 and Burkholderia sp. BCC4135 have been selected as strongest producers of QQ molecules for detailed characterization. According to the genomic sequence analysis, Delftia sp. 11304 was identified as a Delftia tsuruhatensis species while Burkholderia sp. BCC4135 was unveiled as Burkholderia cepacia with novel sequence type ST1485. In silico analysis of genomic sequences indicated that both selected strains possess significant antimicrobial resistance and virulence potential. The nature of the QQ molecules produced by these strains was determined as different; strain 11304 was found to produce small molecules of non-proteinaceous nature (QS inhibitors, QSI), while BCC4135 produced QQ enzymes. Besides, the presence of the QS phenomenon was determined in BCC4135 isolate. The total ethyl acetate extract of the D. tsuruhatensis 11304 culture supernatant (QSI extract) showed significant antivirulence potential on the model system of clinical isolate P. aeruginosa MMA83..