Dataset Information

Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI.

ABSTRACT: The microcin PDI inhibits a diverse group of pathogenic Escherichia coli strains. Coculture of a single-gene knockout library (BW25113; n=3,985 mutants) against a microcin PDI-producing strain (E. coli 25) identified six mutants that were not susceptible (?atpA, ?atpF, ?dsbA, ?dsbB, ?ompF, and ?ompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts in E. coli O157:H7 Sakai. Heterologous expression of E. coli ompF conferred susceptibility to Salmonella enterica and Yersinia enterocolitica strains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49 region within the first extracellular loop of E. coli OmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator for ompF, and consequently loss of susceptibility by the ?ompR strain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. In trans expression of ompF in the ?dsbA and ?dsbB strains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

SUBMITTER: Zhao Z

PROVIDER: S-EPMC4579430 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI.

Zhao Zhe Z Eberhart Lauren J LJ Orfe Lisa H LH Lu Shao-Yeh SY Besser Thomas E TE Call Douglas R DR

Applied and environmental microbiology 20150724 20

The microcin PDI inhibits a diverse group of pathogenic Escherichia coli strains. Coculture of a single-gene knockout library (BW25113; n=3,985 mutants) against a microcin PDI-producing strain (E. coli 25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts in E. coli O157:H7 Sakai. Heterologous expressio ...[more]

PMID: 26209678

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Project description:Microcin PDI (MccPDI), a class IIa microcin that is produced by Escherichia coli strains 25 and 284, is known to inhibit foodborne pathogenic enterohemorrhagic E. coli serotypes O157:H7 and O26. Here we demonstrate that MccPDI can inhibit Shigella strains and E. coli isolates that are multidrug resistant, the latter including strains known to cause urinary tract infections in people and companion animals. Two exceptions out of 17 strains were identified. One of the two resistant E. coli isolates (AR0349) has a mutation in a critical amino acid residue that was identified in previous work as a requisite for the MccPDI precursor protein (McpM) to interact with outer membrane porin F (OmpF) on susceptible cells. The second resistant E. coli strain (MAD 96) had no mutations in ompF, but it was PCR positive for two antimicrobial peptides, of which colicin Ia/Ib likely inhibits the MccPDI-producing strain during coculture. Recombinant McpM was still effective against strain MAD 96. In an assessment of how MccPDI affects susceptible strains, results from both an extracellular ATP assay and a nucleic acid staining assay were consistent with membrane damage, while the addition of 200- to 600-Da polyethylene glycol (PEG) to cocultures protected against MccPDI (>600-Da PEG did not provide protection). Further studies using a paraformaldehyde cross-linking experiment and a bacterial two-hybrid assay demonstrated that MccPDI immunity protein (McpI) forms a multimeric complex with itself and presumably protects the producer strain from within the periplasm through an unknown mechanism.IMPORTANCE Microcins represent potential alternatives to conventional antibiotics for human and veterinary medicine. For them to be applied in this manner, however, we need to better understand their spectrum of activity, how these proteins interact with susceptible cells, and how producer cells are protected against the antimicrobial properties of the microcins. For microcin PDI (MccPDI), we report that the spectrum of activity likely includes most E. coli strains due to a conserved binding motif found on an outer membrane protein. Shigella has this motif as well and is susceptible to MccPDI killing via damage to the bacterial membrane. Receptor specificity suggests that these proteins could be used without causing large-scale disruptions to a microbiota, but this also increases the likelihood that resistance can evolve via random mutations. As with conventional antibiotics, good stewardship will be needed to preserve the efficacy of microcins should they be deployed for clinical use.

Dataset Information

Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI.

Publications

Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI.

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