MAPPATURA E CARATTERIZZAZIONE DEI G-QUADRUPLEX IN PROMOTORI DI MYCOBACTERIUM TUBERCULOSIS

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. The development of strains resistant to antitubercular drugs, makes it necessary to find new compounds against this pathogen in order to treat such disease. G-quadruplexes (G-4s), secondary structures that may form in G-rich regions, were found in promoter regions of several human and viral genes where they influence processes such as transcription and replication of DNA. G-4s are considered as promising targets for anticancer and antiviral chemotherapy thanks to the discovery of small molecules that bind and stabilize their structure, decreasing gene expression levels (G-4 ligands). Recently, bioinformatics analysis identified in M. tuberculosis 10,000 motifs with the potential to fold into G-4 structure, 45 of them are localized upstream or overlapping the transcription start site of genes distributed among different functional categories. Analysis in vitro (circular dichroism, UV spectroscopy and Taq polymerase stop assay) confirmed the formation of G4 structures in four of these genes: mosR, zwf1, clpX and ndhA. To test the possibility to use G-quadruplex structures as therapeutic targets, we evaluated the effect of two G-4 ligands (BRACO-19 e c-exNDI 2) on M. tuberculosis. Results showed that these compounds can inhibit the mycobacterial growth with a minimal inhibitory concentration (MIC) in the micromolar range and make the bacteria more sensitive to oxidative stress. We also evaluated the interactions between BRACO-19 and two well characterized antitubercular drugs (ethambutol and isoniazid), while interaction with isoniazid was shown to be addictive, that with ethambutol was indifferent. To study the influence of G4 structures on gene transcription, the zwf1, mosR, clpX, ndhA expression was evaluated in presence of BRACO-19. In particular the G4 sequence in zwf1 promoter was better characterized. Single strand oligonucleotides containing the sequence of the putative G-4 localized at the promoter region of zwf1 with different mutations were analyzed by circular dichroism, allowing us to identify a single point mutation able to disrupt the G-4 structure. This mutation was introduced in the zwf1 promoter and the final construct was cloned upstream a promoterless eGFP gene in a reporter plasmid. eGFP expression from this plasmid was compared to that of a similar plasmid in which the reporter gene was transcribed from the wild-type zwf1 promoter in physiologic conditions or after treatment of bacterium with BRACO-19 and H2O2.The results suggested that the G-quadruplex structure upstream of zwf1, at least in the observed conditions, hasn’t a preeminent role in gene regulation

Mapping and characterization of G-quadruplexes in Mycobacterium tuberculosis gene promoter regions

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), one of the top 10 causes of death worldwide in 2015. The recent emergence of strains resistant to all current drugs urges the development of compounds with new mechanisms of action. G-quadruplexes are nucleic acids secondary structures that may form in G-rich regions to epigenetically regulate cellular functions. Here we implemented a computational tool to scan the presence of putative G-quadruplex forming sequences in the genome of Mycobacterium tuberculosis and analyse their association to transcription start sites. We found that the most stable G-quadruplexes were in the promoter region of genes belonging to definite functional categories. Actual G-quadruplex folding of four selected sequences was assessed by biophysical and biomolecular techniques: all molecules formed stable G-quadruplexes, which were further stabilized by two G-quadruplex ligands. These compounds inhibited Mycobacterium tuberculosis growth with minimal inhibitory concentrations in the low micromolar range. These data support formation of Mycobacterium tuberculosis G-quadruplexes in vivo and their potential regulation of gene transcription, and prompt the use of G4 ligands to develop original antitubercular agents

MAPPATURA E CARATTERIZZAZIONE DEI G-QUADRUPLEX IN PROMOTORI DI MYCOBACTERIUM TUBERCULOSIS

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. The development of strains resistant to antitubercular drugs, makes it necessary to find new compounds against this pathogen in order to treat such disease. G-quadruplexes (G-4s), secondary structures that may form in G-rich regions, were found in promoter regions of several human and viral genes where they influence processes such as transcription and replication of DNA. G-4s are considered as promising targets for anticancer and antiviral chemotherapy thanks to the discovery of small molecules that bind and stabilize their structure, decreasing gene expression levels (G-4 ligands). Recently, bioinformatics analysis identified in M. tuberculosis 10,000 motifs with the potential to fold into G-4 structure, 45 of them are localized upstream or overlapping the transcription start site of genes distributed among different functional categories. Analysis in vitro (circular dichroism, UV spectroscopy and Taq polymerase stop assay) confirmed the formation of G4 structures in four of these genes: mosR, zwf1, clpX and ndhA. To test the possibility to use G-quadruplex structures as therapeutic targets, we evaluated the effect of two G-4 ligands (BRACO-19 e c-exNDI 2) on M. tuberculosis. Results showed that these compounds can inhibit the mycobacterial growth with a minimal inhibitory concentration (MIC) in the micromolar range and make the bacteria more sensitive to oxidative stress. We also evaluated the interactions between BRACO-19 and two well characterized antitubercular drugs (ethambutol and isoniazid), while interaction with isoniazid was shown to be addictive, that with ethambutol was indifferent. To study the influence of G4 structures on gene transcription, the zwf1, mosR, clpX, ndhA expression was evaluated in presence of BRACO-19. In particular the G4 sequence in zwf1 promoter was better characterized. Single strand oligonucleotides containing the sequence of the putative G-4 localized at the promoter region of zwf1 with different mutations were analyzed by circular dichroism, allowing us to identify a single point mutation able to disrupt the G-4 structure. This mutation was introduced in the zwf1 promoter and the final construct was cloned upstream a promoterless eGFP gene in a reporter plasmid. eGFP expression from this plasmid was compared to that of a similar plasmid in which the reporter gene was transcribed from the wild-type zwf1 promoter in physiologic conditions or after treatment of bacterium with BRACO-19 and H2O2.The results suggested that the G-quadruplex structure upstream of zwf1, at least in the observed conditions, hasn’t a preeminent role in gene regulation.La tubercolosi è una malattia infettiva causata da Mycobacterium tuberculosis. Lo sviluppo di ceppi resistenti ai farmaci antitubercolari normalmente utilizzati rende necessaria la ricerca di nuovi composti per la cura di tale malattia. I G-quadruplex (G4), strutture secondarie che possono formarsi in regioni del DNA ricche in guanina, sono state trovate in promotori di geni umani e virali dove influenzano processi biologici importanti come la trascrizione e la replicazione del DNA. I G4 sono considerati target promettenti per il trattamento di malattie tumorali e infezioni virali grazie alla scoperta di piccole molecole che legano e stabilizzano la loro struttura, inibendo il processo di trascrizione. Recentemente sono stati identificati nel genoma di M. tuberculosis più di 10000 motivi ricchi in guanina che potrebbero formare strutture G-quadruplex. Quarantacinque di essi si trovano a monte o sovrapposti al sito di inizio della trascrizione di geni distribuiti tra diverse categorie funzionali. Analisi in vitro hanno confermato la formazione delle strutture G-quadruplex in quattro di questi geni (mosR, zwf1, clpX e ndhA). Per saggiare la possibilità che le strutture G4 possano essere utilizzate come bersagli terapeutici, è stato valutato l’effetto di due molecole, in grado di legare e stabilizzare la struttura G4 (BRACO-19 e c-exNDI 2), sulla crescita di M. tuberculosis. Abbiamo dimostrato che tali composti riescono ad inibire la crescita micobatterica con una minima concentrazione inibente nel range micromolare rendendo anche il batterio più sensibile allo stress ossidativo. Inoltre sono state studiate le interazioni tra BRACO-19 e alcuni farmaci antitubercolari, il legante del G4 ha un effetto additivo se usato assieme all’isoniazide, mentre in combinazione con l’etambutolo ha un effetto indifferente. Per studiare l’influenza delle strutture G4 nella trascrizione, è stata valutata l’espressione dei geni mosR, zwf1, clpX e ndhA in presenza di BRACO-19. Uno studio più approfondito è stato svolto sul promotore del gene zwf1. Oligonucleotidi a singolo filamento con la stessa sequenza della regione promotoriale di zwf1 interessata alla formazione della struttura G4 sono stati mutati e analizzati con dicroismo circolare: una singola mutazione era richiesta per distruggere la struttura G-quadruplex nel promotore di zwf1. Tale mutazione è stata utilizzata in M. tuberculosis per caratterizzare in vivo l’attività del promotore attraverso la misurazione dell’espressione della proteina reporter eGFP (posta sotto il controllo del promotore wild-type e mutato) dopo trattamento del batterio con H202 o BRACO-19. I dati ottenuti suggeriscono, che almeno nelle condizioni da noi utilizzate, la struttura G4 a monte di tale gene non ha un ruolo preminente nella regolazione genica

Evaluation of new antibiotic cocktails against contaminating bacteria found in allograft tissues

Contamination of retrieved tissues is a major problem for allograft safety. Consequently, tissue banks have implemented decontamination protocols to eliminate microorganisms from tissues. Despite the widespread adoption of these protocols, few comprehensive studies validating such methods have been published. In this manuscript we compare the bactericidal activity of different antibiotic cocktails at different temperatures against a panel of bacterial species frequently isolated in allograft tissues collected at the Treviso Tissue Bank Foundation, a reference organization of the Veneto Region in Italy that was instituted to select, recover, process, store and distribute human tissues. We were able to identify at least two different formulations capable of killing most of the bacteria during prolonged incubation at 4\ua0\ub0C