Pseudomonas aeruginosa is a highly adaptable bacterium that thrives in a broad range of ecological niches. In addition, it can infect hosts as diverse as plants, nematodes, and mammals. In humans, it is an important opportunistic pathogen in compromised individuals, such as patients with cystic fibrosis, severe burns, or impaired immunity. P. aeruginosa is difficult to control because of its ability to develop resistance, often multiple, to all current classes of clinical antibiotics. The discovery of novel essential genes or pathways that have not yet been targeted by clinical antibiotics can underlie the development of alternative effective antibacterials to overcome existing mechanisms of resistance. The main focus of my PhD thesis was the discovery of novel essential genes of P. aeruginosa by shotgun antisense screening. Our antisense screenings identified 33 growth-impairing single-locus genomic inserts that allowed us to generate a list of 27 “essential-for-growth” genes: among them 15 were “novel” essential genes with no homologs reported to have an essential role in other bacterial species. Interestingly, the essential genes in our panel were suggested to take part in a broader range of cellular functions than those currently targeted by extant antibiotics. Our study also identified 43 growth-impairing inserts carrying multiple loci targeting 105 genes. Taken together, our results show the feasibility of antisense technology in P. aeruginosa for identifying novel essential genes and we expect that our methodology could be well suited for antisense-mediated searches of essential genes in other Gram-negative bacterial species. These results were presented in publication 1 (PART II). Then, we focused on two loci, PA2873 and P0580, found in our antisense screenings in PAO1. The PA2873 gene product was annotated as a hypothetical membrane protein endowed with a periplasmic region harboring a structural domain belonging to the transglutaminase-like superfamily, a group of archaeal, bacterial, and eukaryotic proteins homologous to animal transglutaminases. We show that the periplasmic portion of the PA2873 protein, which we named TgpA, does possess transglutaminase activity in vitro. This is the first report of transglutaminase activity in P. aeruginosa. In addition, we have provided strong evidence that TgpA plays a critical role in the viability of P. aeruginosa. The results on TgpA were presented in publication 2 (PART II). The PA0580 locus encodes the protein Gcp belonging to the Kae1/Qri7/YgjD family. Combining several genetic approaches, we showed that Gcp is essential for P. aeruginosa. In addition, we focused on the product of PA3685 locus, the YeaZ protein, a putative partner of Gcp. We obtained evidence that YeaZ also plays a critical role in P. aeruginosa viability. A manuscript describing the results on Gcp is in preparation and presented in PART III.
IDENTIFICATION AND CHARACTERIZATION OF NEW ESSENTIAL FUNCTIONS IN THE OPPORTUNISTIC PATHOGEN PSEUDOMONAS AERUGINOSA / R. Rusmini ; scientific tutor: G. Bertoni. - Milano : Università degli studi di Milano. DIPARTIMENTO DI BIOSCIENZE, 2014 Jun 24. ((26. ciclo, Anno Accademico 2013.
|Titolo:||IDENTIFICATION AND CHARACTERIZATION OF NEW ESSENTIAL FUNCTIONS IN THE OPPORTUNISTIC PATHOGEN PSEUDOMONAS AERUGINOSA|
|Data di pubblicazione:||24-giu-2014|
|Settore Scientifico Disciplinare:||Settore BIO/18 - Genetica|
Settore BIO/19 - Microbiologia Generale
|Citazione:||IDENTIFICATION AND CHARACTERIZATION OF NEW ESSENTIAL FUNCTIONS IN THE OPPORTUNISTIC PATHOGEN PSEUDOMONAS AERUGINOSA / R. Rusmini ; scientific tutor: G. Bertoni. - Milano : Università degli studi di Milano. DIPARTIMENTO DI BIOSCIENZE, 2014 Jun 24. ((26. ciclo, Anno Accademico 2013.|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.13130/rusmini-ruggero_phd2014-06-24|
|Appare nelle tipologie:||Tesi di dottorato|