Pseudomonas aeruginosa is a versatile Gram-negative pathogen that can infect a diversity of immunocompromised patients. Interest in alternatives to traditional antibiotics has inspired our investigation of R- and F-type pyocins as novel therapeutics. These phage tail-like bacteriocins are produced by P. aeruginosa to kill competing strains via pore formation in target cells. We aimed to characterize the diversity of pyocins and bacteriophages generated by diverse P. aeruginosa strains so as to identify pyocins of therapeutic value. Strategies to delineate pyocin and phage activities included physical methods, the modulation of pyocin regulation, and antibody-based detection of tail-like pyocins. We have identified the dominance of R- and F-type pyocins in impacting P. aeruginosa populations and revealed a small number of strains producing particularly potent pyocins. In addition, the co-regulation of phages and pyocins, the dependence of pyocins on pili for activity, and the striking diversity of pyocin susceptibility have all been recognized.

Pseudomonas aeruginosa is a versatile Gram-negative pathogen that can infect a diversity of immunocompromised patients. Interest in alternatives to traditional antibiotics has inspired our investigation of R- and F-type pyocins as novel therapeutics. These phage tail-like bacteriocins are produced by P. aeruginosa to kill competing strains via pore formation in target cells. We aimed to characterize the diversity of pyocins and bacteriophages generated by diverse P. aeruginosa strains so as to identify pyocins of therapeutic value. Strategies to delineate pyocin and phage activities included physical methods, the modulation of pyocin regulation, and antibody-based detection of tail-like pyocins. We have identified the dominance of R- and F-type pyocins in impacting P. aeruginosa populations and revealed a small number of strains producing particularly potent pyocins. In addition, the co-regulation of phages and pyocins, the dependence of pyocins on pili for activity, and the striking diversity of pyocin susceptibility have all been recognized.

Pseudomonas aeruginosa é um patógeno oportunista que ocasiona diferentes infecções em humanos. O surgimento de linhagens multirresistentes (MRs), contra os antibióticos comercialmente disponíveis, tem causado elevados níveis de morbidade e mortalidade. O objetivo deste estudo foi identificar uma molécula de piocina que apresente potente atividade bactericida contra cepas de P. aeruginosa MRs. Uma piocina de baixo peso molecular, produzida pela cepa ET02, apresentou potente atividade bactericida contra treze linhagens de P. aeruginosa produtoras de -lactamases (SPM-1, GIM-1, VIM-1, IMP-1, KPC-2, OXA-18 e GES-5). Essa piocina foi purificada e identificada como S8 através de espectrometria de massas e sequenciamento de DNA. Os genes codificadores da piocina S8 estão presentes no interior de um transposon localizado no cromossomo bacteriano da cepa ET02. Estes resultados demonstram que S8 possui potente atividade bactericida contra linhagens de P. aeruginosa MRs, podendo vir a ser utilizada como um composto antimicrobiano no tratamento de infecções hospitalares. / Pseudomonas aeruginosa is an opportunist pathogen that causes human infections. The emergence of multidrug resistant strains (MRs) against antibiotics commercially available has been causing elevated mortality and morbidity levels. The purpose of this study was to identify a molecule of pyocin with a potent bactericidal activity against P. aeruginosa MRs strains. A low molecular weight pyocin, produced by ET02 strain, has presented wide bactericidal activity against thirteen lineages of P. aeruginosa, producers of -lactamases (SPM-1, GIM-1, VIM-1, IMP-1, KPC-2, OXA-18 e GES-5). This pyocin was purified and identified as S8 through mass spectrometry and DNA sequencing. The pyocin S8 encoding genes are present in the interior of a transposon located in the bacterial chromosome of the ET02 strain. These results demonstrate that S8 have potent bactericidal activity against P. aeruginosa MRs lineages, and may well be used as an antimicrobial compound on treatment of hospital infections.

Multidrug resistant bacteria (MDR) have the potential to push back society to the pre-antibiotic era. Although discovered before penicillin, the inexorable rise in antibiotic resistance has revitalised interest in bacteriocins as treatments for bacterial infections. Bacteriocins are protein antibiotics principal to competition amongst pathogens and commensals, but the mechanisms by which they translocate across the Gram-negative cell envelope are poorly understood. The work presented in this thesis demonstrates how the endonuclease bacteriocin pyocin S2 (pyoS2) exploits the iron transporter FpvAI to translocate across the outer membrane (OM) of Pseudomonas aeruginosa. FpvAI is a 22-strand β-barrel and virulence factor in P. aeruginosa that transports iron into the cell in the form of a small siderophore, ferripyoverdine (Fe-Pvd). Uptake of Fe-Pvd requires the proton motive force (PMF), which is transduced to the ligand-bound receptor by TonB1 and its partner proteins ExbB-ExbD in the inner membrane (IM). The crystal structure of the high affinity complex (Kd = 240 pM) formed between the N-terminal domain of pyoS2 (pyoS2^NTD) and FpvAI is presented, which shows pyoS2^NTD mimics Fe-Pvd, and induces the same conformational changes in the receptor. Fluorescently-labelled pyoS2^NTD was actively imported into P. aeruginosa PAO1 cells and this import was dependent on the PMF, TonB1 and a TonB1-box motif at the N-terminus of pyoS2^NTD. Finally, photo-activated crosslinking of stalled translocation intermediates demonstrated pyoS2^NTD translocates through the FpvAI β-barrel lumen by a process analogous to that of Fe-Pvd. Following binding to FpvAI, translocation begins by the unfolding of a force-labile portion of the plug domain, opening a narrow channel through FpvAI. This enables pyoS2 to deliver its own TonB1-box to the periplasm where contact with TonB1 activates its import through the same channel, most likely as an unfolded polypeptide. Hence, this study demonstrates that bacteria possess a rudimentary protein import system that exploits energised nutrient transporters in the OM.