The opportunistic pathogen Pseudomonas aeruginosa is a common agent of infectious disease in immunocompromised individuals, and a dominant pathogen in late-stage of cystic fibrosis disease. P. aeruginosa represents a prototype of multidrug resistant “superbug” due to intrinsic and acquired resistance to antimicrobials agents, for which effective therapeutic options are very limited. In order to overcome the current resistance mechanisms, the identification and characterization of new cellular functions that are essential for P. aeruginosa viability could drive the development of new antibacterial compounds with novel mechanisms of action. The present PhD thesis is focused on the functional and structural characterization of the Transglutaminase protein A, TgpA, an inner membrane protein predicted to belong to the Transglutaminase-like family which contains a functional TG domain (TG180–544), localized in the periplasmic side, suggested to take part in an essential function involved in envelope structure. The protein was recently described as essential for the viability of P. aeruginosa, and a promising candidate for the design of new specific antimicrobial compounds. In Part I, the state of the art and main results are presented. First, the in vivo evaluation of the modulation of TgpA expression levels on the P. aeruginosa growth showed that, the increase of TgpA cellular levels dramatically affects the P. aeruginosa growth. On the other hand, the partial suppression of the chromosomal copy of the tgpA gene showed to compromised the envelope organization of the cells. The subsequent structural characterization of the functional TG domain allowed confirming that TgpA belongs to the poorly characterized Transglutaminase-like family, in which many of the prokaryotic members are proteases. Also, the presence of a carbohydrate-binding domain at the N-terminal portion of the protein, suggests that the binding to polysaccharides present in the cell wall might constitute a mechanism of regulation of the enzymatic activity of TgpA. Moreover, the active site of the protein shares homology with cysteine proteases and endopeptidases with described action in the maintenance/biosinthesys of the bacterial peptidoglycan, suggesting that TgpA might be involved in the cell wall metabolism. The transglutaminase and proteolytic activity of the TG domain were evaluated in vitro, showing residual activity over generic substrates. A manuscript in preparation, presented in Part II, describes in detail the characteristics found in the structure of the TG domain that are the foundations for the TgpA enzymatic activity. Finally, in the search for possible inhibitory molecules of the activity of TgpA, the results of a preliminary in silico docking analysis with in vivo results are presented in Part III.
UNRAVELLING THE ESSENTIAL ROLE OF TGPA IN THE VIABILITY OF PSEUDOMONAS AERUGINOSA: A PUTATIVE TARGET FOR NOVEL ANTIMICROBIAL AGENTS / M.m. Uruburu Gomez ; co-tutor: M. Milani, E. Mastrangelo ; scientific tutor: G. Bertoni. - Milano : Università degli studi di Milano. DIPARTIMENTO DI BIOSCIENZE, 2018 Apr 11. ((29. ciclo, Anno Accademico 2016.
|Titolo:||UNRAVELLING THE ESSENTIAL ROLE OF TGPA IN THE VIABILITY OF PSEUDOMONAS AERUGINOSA: A PUTATIVE TARGET FOR NOVEL ANTIMICROBIAL AGENTS|
|Data di pubblicazione:||11-apr-2018|
|Settore Scientifico Disciplinare:||Settore BIO/11 - Biologia Molecolare|
|Citazione:||UNRAVELLING THE ESSENTIAL ROLE OF TGPA IN THE VIABILITY OF PSEUDOMONAS AERUGINOSA: A PUTATIVE TARGET FOR NOVEL ANTIMICROBIAL AGENTS / M.m. Uruburu Gomez ; co-tutor: M. Milani, E. Mastrangelo ; scientific tutor: G. Bertoni. - Milano : Università degli studi di Milano. DIPARTIMENTO DI BIOSCIENZE, 2018 Apr 11. ((29. ciclo, Anno Accademico 2016.|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.13130/uruburu-gomez-monica-maria_phd2018-04-11|
|Appare nelle tipologie:||Tesi di dottorato|