Novel Colicin F
            <sub>Y</sub>
            of Yersinia frederiksenii Inhibits Pathogenic Yersinia Strains via YiuR-Mediated Reception, TonB Import, and Cell Membrane Pore Formation

Bosák, Juraj; Laiblová, Petra; Šmarda, Jan; Dědičová, Daniela; Šmajs, David

doi:10.1128/jb.05885-11

Cited by 15 publications

(22 citation statements)

References 79 publications

(64 reference statements)

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“…Our strategy was based on first determining if the pore-forming domain of PyoS5, if imported, could kill E. coli cells and then engineering the minimal requirements into E. coli in order for PyoS5 to be recognized and transported. A similar strategy was reported by Bosák et al, where E. coli was engineered to be susceptible to a bacteriocin specific for Yersinia kristensenii (28). In the present work, we first showed that a chimera of the PyoS5 pore-forming domain fused to the C terminus of the colicin B translocation and receptor-binding region (replacing colicin B's own pore-forming domain) was cytotoxic against E. coli BL21(DE3) cells.…”

Section: Resultssupporting

confidence: 74%

Pyocin S5 Import into Pseudomonas aeruginosa Reveals a Generic Mode of Bacteriocin Transport

Behrens

Lowe

Gault

et al. 2020

mBio

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Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria. IMPORTANCE Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets Pseudomonas aeruginosa. In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of Escherichia coli cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting Gram-negative bacteria.

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Section: Resultssupporting

confidence: 74%

Pyocin S5 Import into Pseudomonas aeruginosa Reveals a Generic Mode of Bacteriocin Transport

Behrens

Lowe

Gault

et al. 2020

mBio

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“…The genus Yersinia has previously been shown to contain a number of bactericidal toxins only two of which have been characterized as bacteriocins. Pesticins from Yersinia pestis degrade peptidoglycan using murimidase activity and colicin F Y is a pore forming bacteriocin [ 68 , 69 ]. We identified 18 NB sequence clusters at 90% sequence identity in 8 Yersinia species and identified both DNase and rRNase cytotoxic motifs.…”

Section: Resultsmentioning

confidence: 99%

Diversity and distribution of nuclease bacteriocins in bacterial genomes revealed using Hidden Markov Models

et al. 2017

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Bacteria exploit an arsenal of antimicrobial peptides and proteins to compete with each other. Three main competition systems have been described: type six secretion systems (T6SS); contact dependent inhibition (CDI); and bacteriocins. Unlike T6SS and CDI systems, bacteriocins do not require contact between bacteria but are diffusible toxins released into the environment. Identified almost a century ago, our understanding of bacteriocin distribution and prevalence in bacterial populations remains poor. In the case of protein bacteriocins, this is because of high levels of sequence diversity and difficulties in distinguishing their killing domains from those of other competition systems. Here, we develop a robust bioinformatics pipeline exploiting Hidden Markov Models for the identification of nuclease bacteriocins (NBs) in bacteria of which, to-date, only a handful are known. NBs are large (>60 kDa) toxins that target nucleic acids (DNA, tRNA or rRNA) in the cytoplasm of susceptible bacteria, usually closely related to the producing organism. We identified >3000 NB genes located on plasmids or on the chromosome from 53 bacterial species distributed across different ecological niches, including human, animals, plants, and the environment. A newly identified NB predicted to be specific for Pseudomonas aeruginosa (pyocin Sn) was produced and shown to kill P. aeruginosa thereby validating our pipeline. Intriguingly, while the genes encoding the machinery needed for NB translocation across the cell envelope are widespread in Gram-negative bacteria, NBs are found exclusively in γ-proteobacteria. Similarity network analysis demonstrated that NBs fall into eight groups each with a distinct arrangement of protein domains involved in import. The only structural feature conserved across all groups was a sequence motif critical for cell-killing that is generally not found in bacteriocins targeting the periplasm, implying a specific role in translocating the nuclease to the cytoplasm. Finally, we demonstrate a significant association between nuclease colicins, NBs specific for Escherichia coli, and virulence factors, suggesting NBs play a role in infection processes, most likely by enabling pathogens to outcompete commensal bacteria.

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“…Our strategy was based on first determining if the pore-forming domain of PyoS5, if imported, could kill E. coli cells and then engineering the minimal requirements into E. coli in order for PyoS5 to be recognised and transported. A similar strategy was reported by Bosak et al (2012) where E. coli was engineered to be susceptible to a bacteriocin specific for Yersinia kristensenii (Bosák et al , 2012). In the present work, we first showed that a chimera of the PyoS5 pore-forming domain fused to the C-terminus of the colicin B translocation and receptor-binding region (replacing colicin B’s own pore-forming domain) was cytotoxic against E. coli BL21 (DE3) cells.…”

Section: Resultsmentioning

confidence: 92%

Pyocin S5 import intoPseudomonas aeruginosareveals a generic mode of bacteriocin transport

Behrens¹,

Lowe²,

Gault³

et al. 2019

Preprint

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25Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen P. 26 aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, 27 using crystallography, biophysical and biochemical analysis and live-cell imaging, we define 28 the entry process of PyoS5 and reveal links to the transport mechanisms of other 29 bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises 30 two novel tandemly repeated kinked three helix bundle domains that structure-based 31 alignments identify as key import domains in other pyocins. The central domain binds the 32 lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell 33 surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its 34 associated disordered region binds the inner membrane protein TonB1, which together drive 35 import of the bacteriocin across the outer membrane. Finally, we identify the minimal 36 requirements for sensitizing Escherichia coli towards PyoS5, as well as other pyocins, and 37 suggest that a generic pathway likely underpins the import of all TonB-dependent 38 bacteriocins across the outer membrane of Gram-negative bacteria. 39 40 Word count, 170 41 42 48only the release of bacteriocins does not rely on physical contact between bacterial cells. 49Bacteriocin production generally occurs following a stress signal, such as DNA damage, 50 inducing expression and release of the bacteriocin from auto-lysed cells (Kleanthous, 2010). 51The bacteriocin then diffuses through the medium to kill a neighbouring cell. Bacteriocins 52 range in size, from small peptides to large proteins with both types currently being 53 evaluated/developed as antimicrobials against multidrug resistant bacteria (Rios et al, 2016; 54 Behrens et al, 2017). In many instances, however, developments are hindered by a lack of 55 understanding as to how these molecules work. In the case of protein bacteriocins, 56 extensive sequence diversification and homologous recombination further hamper efforts to 57 find generic mechanisms of uptake. Here, we focus on the uptake mechanism of PyoS5, a 58 protein bacteriocin that specifically targets the opportunistic human pathogen P. aeruginosa 59 and shown recently in animal models to be more effective at clearing lung infections than 60 tobramycin, the antibiotic generally used to treat P. aeruginosa in cystic fibrosis patients 61 (McCaughey et al, 2016b). Through a structure-led approach, we deconstruct the energised 62 uptake pathway of PyoS5 and show that its transport across the outer membrane likely 63 represents the default pathway for all TonB-dependent bacteriocins. 64There is a pressing need for new antibiotics against Gram-negative bacteria but in 65 particular P. aeruginosa which has been designated a priority pathogen (WHO, 2017). The 66 intrinsic low permeability of its outer membrane renders P. aeruginosa insensitive to many 67 classes of antibiotics. Many strains also express mult...

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Novel Colicin F _Y of Yersinia frederiksenii Inhibits Pathogenic Yersinia Strains via YiuR-Mediated Reception, TonB Import, and Cell Membrane Pore Formation

Cited by 15 publications

References 79 publications

Pyocin S5 Import into Pseudomonas aeruginosa Reveals a Generic Mode of Bacteriocin Transport

Pyocin S5 Import into Pseudomonas aeruginosa Reveals a Generic Mode of Bacteriocin Transport

Diversity and distribution of nuclease bacteriocins in bacterial genomes revealed using Hidden Markov Models

Pyocin S5 import intoPseudomonas aeruginosareveals a generic mode of bacteriocin transport

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Novel Colicin F Y of Yersinia frederiksenii Inhibits Pathogenic Yersinia Strains via YiuR-Mediated Reception, TonB Import, and Cell Membrane Pore Formation

Cited by 15 publications

References 79 publications

Pyocin S5 Import into Pseudomonas aeruginosa Reveals a Generic Mode of Bacteriocin Transport

Pyocin S5 Import into Pseudomonas aeruginosa Reveals a Generic Mode of Bacteriocin Transport

Diversity and distribution of nuclease bacteriocins in bacterial genomes revealed using Hidden Markov Models

Pyocin S5 import intoPseudomonas aeruginosareveals a generic mode of bacteriocin transport

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Product

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About

Novel Colicin F _Y of Yersinia frederiksenii Inhibits Pathogenic Yersinia Strains via YiuR-Mediated Reception, TonB Import, and Cell Membrane Pore Formation