Import mechanisms of pyocins G and AP41

Thesis

Pseudomonas aeruginosa is a priority pathogen for antibiotic development. Multi-drug-resistant strains of this bacterium cause serious nosocomial infections and are the leading cause of death in cystic fibrosis patients. Pyocins, bacteriocins of P. aeruginosa, are protein antibiotics deployed during intraspecies bacterial competition. Pyocins are produced by more than 90 % of P. aeruginosa strains. Due to their diversity and potency, pyocins may have utility as last resort antibiotics against P. aeruginosa. In this thesis, the import mechanism of two poorly-understood nuclease pyocins, G and AP41, was explored using microbiological, biochemical, biophysical and cell-based approaches. Pyocin G (PyoG) was previously identified by the Kleanthous lab through bioinformatics analysis of P. aeruginosa genomes. I demonstrate that PyoG binds Hur (Hemin uptake receptor), an outer membrane TonB1 dependent transporter. Hur binds hemin in vitro, and this interaction is blocked by PyoG. Both PyoG and Hur interact with TonB1, which links proton motive force generation across the inner membrane with energy-dependent pyocin translocation across the outer membrane, most likely through Hur. Inner membrane translocation of PyoG requires the conserved inner-membrane AAA+ATPase/protease, FtsH, similar to the situation for E. coli-specific colicins. Surprisingly, however, this translocation step also requires TonB1 as well as the unstructured N-terminus of PyoG that binds TonB1. Moreover, inner membrane translocation is dependent on the so-called Pyocin S domain, which is conserved amongst nuclease bacteriocins. Pyocin AP41, like PyoG, requires TonB1 and FtsH to kill P. aeruginosa and the unstructured N-terminus of AP41 is similarly involved in binding TonB1. The outer membrane receptor of AP41 remains unknown, but there are indications that FhaC, a putative hemagglutinin transporter, may be the outer membrane receptor and/or translocator.

Authors: Atanaskovic, I

• DOI: 10.5287/ora-qyogayg1r
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: bacteriocin; protein import; antibiotic; Pseudomonas aeruginosa; pyocin
• Subjects: Molecular microbiology; Protein biochemistry