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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Author(s):
Turano, Helena [1] ; Gomes, Fernando [1] ; Domingos, Renato M. [2] ; Degenhardt, Maximilia F. S. [3] ; Oliveira, Cristiano L. P. [3] ; Garratt, Richard C. [4] ; Lincopan, Nilton [5] ; Netto, Luis E. S. [1]
Total Authors: 8
Affiliation:
[1] Univ Sao Paulo, Inst Biociencias, Dept Genet & Biol Evolut, Sao Paulo - Brazil
[2] Univ Nova Lisboa, Inst Tecnol Quim & Biol, Oeiras - Portugal
[3] Univ Sao Paulo, Inst Fis, Dept Fis Expt, Sao Paulo - Brazil
[4] Univ Sao Paulo, Inst Fis Sao Carlos, Dept Fis & Ciencia Interdisciplinar, Sao Carlos - Brazil
[5] Univ Sao Paulo, Inst Ciencias Biomed, Dept Microbiol, Sao Paulo - Brazil
Total Affiliations: 5
Document type: Journal article
Source: Journal of Bacteriology; v. 202, n. 21 NOV 2020.
Web of Science Citations: 0
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 angstrom, 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. IMPORTANCE Pyocins are proteins produced by Pseudomonas aeruginosa strains that participate in intraspecific competition and host-pathogen interactions. They were first described in the 1950s and since then have gained attention as possible new antibiotics. However, there is still only scarce information about the molecular mechanisms by which these molecules induce cell death. Here, we show that the metal-dependent endonuclease activity of pyocin S8 is involved with its antimicrobial action against strain PAO1. We also describe that this killing activity is dependent on a conserved Glu residue within the H-N-H motif. The potency and selectivity of pyocin S8 toward a narrow spectrum of P. aeruginosa strains make this protein an attractive antimicrobial alternative for combatting MDR strains, while leaving commensal human microbiota intact. (AU)

FAPESP's process: 13/07937-8 - Redoxome - Redox Processes in Biomedicine
Grantee:Ohara Augusto
Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC