Targeted Killing of Pseudomonas aeruginosa by Pyocin G Occurs via the Hemin Transporter Hur

Iva Atanaskovic,Khedidja Mosbahi,Connor Sharp,Nicholas G Housden,Renata Kaminska,Daniel Walker,Colin Kleanthous

doi:10.1016/j.jmb.2020.04.020

Iva Atanaskovic, Khedidja Mosbahi + Show 5 more

Open Access

https://doi.org/10.1016/j.jmb.2020.04.020

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Abstract

Pseudomonas aeruginosa is a priority pathogen for the development of new antibiotics, particularly because 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 potent and diverse protein antibiotics that are deployed during bacterial competition. Pyocins are produced by more than 90% of P. aeruginosa strains and may have utility as last resort antibiotics against this bacterium. In this study, we explore the antimicrobial activity of a newly discovered pyocin called pyocin G (PyoG). We demonstrate that PyoG has broad killing activity against a collection of clinical P. aeruginosa isolates and is active in a Galleria mellonella infection model. We go on to identify cell envelope proteins that are necessary for the import of PyoG and its killing activity. PyoG recognizes bacterial cells by binding to Hur, an outer-membrane TonB-dependent transporter. Both pyocin and Hur interact with TonB1, which in complex with ExbB–ExbD links the proton motive force generated across the inner membrane with energy-dependent pyocin translocation across the outer membrane. Inner-membrane translocation of PyoG is dependent on the conserved inner-membrane AAA+ ATPase/protease, FtsH. We also report a functional exploration of the PyoG receptor. We demonstrate that Hur can bind to hemin in vitro and that this interaction is blocked by PyoG, confirming the role of Hur in hemin acquisition.

Highlights

Antibiotic-resistant Pseudomonas aeruginosa is a common cause of lung and wound infections in immunocompromised patients
Bacteriocins of P. aeruginosa, are potent and diverse protein antibiotics that are deployed during bacterial competition
pyocin G (PyoG) recognizes bacterial cells by binding to Hur, an outer-membrane TonBdependent transporter. Both pyocin and Hur interact with TonB1, which in complex with ExbB–ExbD links the proton motive force generated across the inner membrane with energy-dependent pyocin translocation across the outer membrane

Summary

Introduction

Antibiotic-resistant Pseudomonas aeruginosa is a common cause of lung and wound infections in immunocompromised patients. Nuclease pyocins translocate to the cytoplasm to degrade nucleic acids To do so, they first bind an outermembrane receptor, which can be common polysaccharide antigen [4] or an outer-membrane protein [5,6]. An important requirement for the potential use of pyocins in the clinic is to pair up different pyocin groups with their receptors If these pairs are known, it can be predicted from genomic data which strains of P. aeruginosa are likely to be sensitive to which pyocins. Resistance can occur through spontaneous mutations affecting proteins required for pyocin translocation These problems can, be overcome through the use of cocktails comprising toxins that have different cell envelope targets and different immunity proteins. We further show that Hur has a role in hemin acquisition and may have a role in P. aeruginosa virulence

Results and Discussion

Materials and Methods

Declaration of Competing Interests