Tn
            <i>6350</i>
            , a Novel Transposon Carrying Pyocin S8 Genes Encoding a Bacteriocin with Activity against Carbapenemase-Producing Pseudomonas aeruginosa

Turano, Helena; Gomes, Francisco Guilhien; Barros-Carvalho, Gesiele Almeida; Lopes, Ralf; Cerdeira, Louise; Netto, L.E.S.; Gales, Ana Cristina; Lincopán, Nilton

doi:10.1128/aac.00100-17

Cited by 10 publications

(14 citation statements)

References 20 publications

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“…Following exposure to UV light, LlpA BW11M1 expression is significantly enhanced. Similar observations have been made for several other ( Pseudomonas ) bacteriocins as well ( Ghazaryan et al, 2014 ; Godino et al, 2015 ; Hockett and Baltrus, 2017 ; Turano et al, 2017 ), and such DNA-damaging conditions (e.g., via mitomycin C treatments) constitute a common strategy to induce bacteriocin overproduction. Screening of a P. mosselii BW11M1 transposon mutant library revealed that LlpA expression is reduced in a recA and spoT mutant background, confirming the UV-light-induced expression of LlpA ( de los Santos et al, 2005 ).…”

Section: Stress-triggered Retaliation?supporting

confidence: 78%

Lectin-Like Bacteriocins

2018

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Bacteria produce a diverse array of antagonistic compounds to restrict growth of microbial rivals. Contributing to this warfare are bacteriocins: secreted antibacterial peptides, proteins and multi-protein complexes. These compounds typically eliminate competitors closely related to the producer. Lectin-like bacteriocins (LlpAs) constitute a distinct class of such proteins, produced by Pseudomonas as well as some other proteobacterial genera. LlpAs share a common architecture consisting of two B-lectin domains, followed by a short carboxy-terminal extension. Two surface-exposed moieties on susceptible Pseudomonas cells are targeted by the respective lectin modules. The carboxy-terminal domain binds D-rhamnose residues present in the lipopolysaccharide layer, whereas the amino-terminal domain interacts with a polymorphic external loop of the outer-membrane protein insertase BamA, hence determining selectivity. The absence of a toxin-immunity module as found in modular bacteriocins and other polymorphic toxin systems, hints toward a novel mode of killing initiated at the cellular surface, not requiring bacteriocin import. Despite significant progress in understanding the function of LlpAs, outstanding questions include the secretion machinery recruited by lectin-like bacteriocins for their release, as well as a better understanding of the environmental signals initiating their expression.

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Section: Stress-triggered Retaliation?supporting

confidence: 78%

Lectin-Like Bacteriocins

2018

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“…In contrast, the ET02 strain was completely resistant against pyocin S8, even at the highest concentrations tested ( Figure 2B, center panel). This finding was expected, as the ET02 strain contains a gene encoding ImS8 (33), which can bind to the cytotoxic domain and thereby neutralizes its endonuclease activity.…”

Section: Killing Spectrum Of Pyocin S8mentioning

confidence: 74%

“…We have previously shown that pyocin S8 displays a wide spectrum of killing activity against distinct MDR P. aeruginosa strains (33). Pyocin S8 is synthesized as a binary protein complex composed of a large killing subunit that contains 772 amino acids and its cognate smaller immunity protein with 85 amino acids.…”

Section: Amino Acid Sequence Identities and Domain Organization Of Pymentioning

confidence: 99%

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Structural and functional analysis of pyocin S8 fromPseudomonas aeruginosa: requirement of a glutamate in the H-N-H motif for the DNase activity

Turano

Fomes

Domingos

et al. 2020

Preprint

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Multi-drug resistance (MDR) is a serious threat to global 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 (Im) protein killed the strain PAO1. Furthermore, mutation of a highly conserved glutamic acid to alanine (Glu100Ala) completely inhibited this antimicrobial activity. Probably the integrity of the H-N-H motif is essential in the killing activity of S8, as Glu100 is a highly conserved component of this structure. 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 Ni 2+ strongly induced this DNase activity, whereas Mn 2+ and Mg 2+ exhibited moderate effects and Zn 2+ was inhibitory. Additionally, S8 bound Zn 2+ with a higher affinity than Ni 2+ 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 pyocin-S8DNase-Im complex at 1.38 Å, which gave us new insights into the endonuclease activity of S8. Our results reinforce the possible use of S8 as an alternative antibiotic for MDR Pseudomonas aeruginosa strains, while leaving commensal human microbiota intact.

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“…We have isolated a novel S-type pyocin displaying potent antibacterial activity against MDR P. aeruginosa isolates, demonstrating its potential as a protein antibiotic (33). This uncharacterized pyocin, previously designated S8 by in silico analysis (8), has a cytotoxic domain with metal-dependent DNase activity based on an H-N-H motif.…”

mentioning

confidence: 99%

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

et al. 2020

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Multi-drug 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. Probably the integrity of the H-N-H motif is 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 S8DNase-ImS8 complex at 1.38 Å, 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 50′s, 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 PAO1 strain. 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 towards a narrow-spectrum of P. aeruginosa strains make this protein an attractive antimicrobial alternative to combat MDR strains, while leaving commensal human microbiota intact.

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Tn 6350 , a Novel Transposon Carrying Pyocin S8 Genes Encoding a Bacteriocin with Activity against Carbapenemase-Producing Pseudomonas aeruginosa

Cited by 10 publications

References 20 publications

Lectin-Like Bacteriocins

Lectin-Like Bacteriocins

Structural and functional analysis of pyocin S8 fromPseudomonas aeruginosa: requirement of a glutamate in the H-N-H motif for the DNase activity

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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