Dataset Information

Characterization of the Key Determinants of Phd Antitoxin Mediated Doc Toxin Inactivation in Salmonella.

ABSTRACT: In the search for novel antimicrobial therapeutics, toxin-antitoxin (TA) modules are promising yet underexplored targets for overcoming antibiotic failure. The bacterial toxin Doc has been associated with the persistence of Salmonella in macrophages, enabling its survival upon antibiotic exposure. After developing a novel method to produce the recombinant toxin, we have used antitoxin-mimicking peptides to thoroughly investigate the mechanism by which its cognate antitoxin Phd neutralizes the activity of Doc. We reveal insights into the molecular detail of the Phd-Doc relationship and discriminate antitoxin residues that stabilize the TA complex from those essential for inhibiting the activity of the toxin. Coexpression of Doc and antitoxin peptides in Salmonella was able to counteract the activity of the toxin, confirming our in vitro results with equivalent sequences. Our findings provide key principles for the development of chemical tools to study and therapeutically interrogate this important class of protein-protein interactions.

SUBMITTER: de Castro GV

PROVIDER: S-EPMC9207808 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Publications

Characterization of the Key Determinants of Phd Antitoxin Mediated Doc Toxin Inactivation in <i>Salmonella</i>.

de Castro Guilherme V GV Worm Dennis J DJ Grabe Grzegorz J GJ Rowan Fiona C FC Haggerty Lucy L de la Lastra Ana L AL Popescu Oana O Helaine Sophie S Barnard Anna A

ACS chemical biology 20220601 6

In the search for novel antimicrobial therapeutics, toxin-antitoxin (TA) modules are promising yet underexplored targets for overcoming antibiotic failure. The bacterial toxin Doc has been associated with the persistence of <i>Salmonella</i> in macrophages, enabling its survival upon antibiotic exposure. After developing a novel method to produce the recombinant toxin, we have used antitoxin-mimicking peptides to thoroughly investigate the mechanism by which its cognate antitoxin Phd neutralizes ...[more]

PMID: 35647667

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Project description:Toxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea, where they play a pivotal role in the establishment and maintenance of dormancy. Under normal growth conditions, the antitoxin neutralizes the toxin. However, under conditions of stress, such as nutrient starvation or antibiotic treatment, cellular proteases degrade the antitoxin, and the toxin functions to arrest bacterial growth. We characterized the specificity determinants of the interactions between VapB antitoxins and VapC toxins from nontypeable Haemophilus influenzae (NTHi) in an effort to gain a better understanding of how antitoxins control toxin activity and bacterial persistence. We studied truncated and full-length antitoxins with single amino acid mutations in the toxin-binding domain. Coexpressing the toxin and antitoxin in Escherichia coli and measuring bacterial growth by dilution plating assayed the ability of the mutant antitoxins to neutralize the toxin. Our results identified two single amino acid residues (W48 and F52) in the C-terminal region of the VapB2 antitoxin necessary for its ability to neutralize its cognate VapC2 toxin. Additionally, we attempted to alter the specificity of VapB1 by making a mutation that would allow it to neutralize its noncognate toxin. A mutation in VapB1 to contain the tryptophan residue identified herein as important in the VapB2-VapC2 interaction resulted in a VapB1 mutant (the T47W mutant) that binds to and neutralizes both its cognate VapC1 and noncognate VapC2 toxins. This represents the first example of a single mutation causing relaxed specificity in a type II antitoxin.ImportanceToxin-antitoxin systems are of particular concern in pathogenic organisms, such as nontypeable Haemophilus influenzae, as they can elicit dormancy and persistence, leading to chronic infections and failure of antibiotic treatment. Despite the importance of the TA interaction, the specificity determinants for VapB-VapC complex formation remain uncharacterized. Thus, our understanding of how antitoxins control toxin-induced dormancy and bacterial persistence requires thorough investigation of antitoxin specificity for its cognate toxin. This study characterizes the crucial residues of the VapB2 antitoxin from NTHi necessary for its interaction with VapC2 and provides the first example of a single amino acid change altering the toxin specificity of an antitoxin.

Dataset Information

Characterization of the Key Determinants of Phd Antitoxin Mediated Doc Toxin Inactivation in Salmonella.

Publications

Characterization of the Key Determinants of Phd Antitoxin Mediated Doc Toxin Inactivation in <i>Salmonella</i>.

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Dataset Information

Characterization of the Key Determinants of Phd Antitoxin Mediated Doc Toxin Inactivation in Salmonella.

Publications

Characterization of the Key Determinants of Phd Antitoxin Mediated Doc Toxin Inactivation in &lt;i&gt;Salmonella&lt;/i&gt;.

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OmicsDI is part of the ELIXIR infrastructure

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Characterization of the Key Determinants of Phd Antitoxin Mediated Doc Toxin Inactivation in <i>Salmonella</i>.