Molecular structure and functional analysis of pyocin s8 from pseudomonas aeruginosa reveals the essential requirement of a glutamate residue in the H-N-H motif for DNase activity

Helena Turano, Fernando Gomes, Renato M. Domingos, Maximilia F.S. Degenhardt, Cristiano L.P. Oliveira, Richard C. Garratt, Nilton Lincopan, Luis E.S. Netto

Research output: Contribution to journalArticlepeer-review

Abstract

Multidrug resistance (MDR) is a serious threat to public health, making the development of new antimicrobials an urgent necessity. Pyocins are protein antibiotics produced by Pseudomonas aeruginosa strains to kill closely related cells during intraspecific competition. Here, we report an in-depth biochemical, microbicidal, and structural characterization of a new S-type pyocin, named S8. Initially, we described the domain organization and secondary structure of S8. Subsequently, we observed that a recombinant S8 composed of the killing subunit in complex with the immunity (ImS8) protein killed the strain PAO1. Furthermore, mutation of a highly conserved glutamic acid to alanine (Glu100Ala) completely inhibited this antimicrobial activity. The integrity of the H-N-H motif is probably essential in the killing activity of S8, as Glu100 is a highly conserved residue of this motif. Next, we observed that S8 is a metal-dependent endonuclease, as EDTA treatment abolished its ability to cleave supercoiled pUC18 plasmid. Supplementation of apo S8 with Ni2_ strongly induced this DNase activity, whereas Mn2_ and Mg2_ exhibited moderate effects and Zn2_ was inhibitory. Additionally, S8 bound Zn2_ with a higher affinity than Ni2_ and the Glu100Ala mutation decreased the affinity of S8 for these metals, as shown by isothermal titration calorimetry (ITC). Finally, we describe the crystal structure of the Glu100Ala S8 DNase-ImS8 complex at 1.38 A, which gave us new insights into the endonuclease activity of S8. Our results reinforce the possibility of using pyocin S8 as an alternative therapy for infections caused by MDR strains, while leaving commensal human microbiota intact.

Original languageEnglish
Article numbere00346-20
JournalJournal of Bacteriology
Volume202
Issue number21
DOIs
Publication statusPublished - 8 Oct 2020

Keywords

  • H-N-H motif
  • MDR
  • Metals
  • Multidrug resistance
  • Protein structure
  • Protein structure-function
  • Pseudomonas aeruginosa
  • Pyocin

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