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Identification of host receptor and receptor-binding module of a newly sequenced T5-like phage EPS7.

ABSTRACT: The virulent bacteriophage EPS7 active against a number of Salmonella serovar and Escherichia coli strains, isolated from the local sewage in Korea, belongs to the family Siphoviridae. The ESP7 genome constitutes a linear double-stranded DNA of 111 382 bp. DNA sequencing and genomic analysis of EPS7 showed that it belongs to the phage T5 family. We identified the EPS7 genes involved in DNA repair, replication, viral structure and bacterial lysis by comparing the EPS7 genome with that of T5. In contrast, the tail genes encoding for putative host receptor-binding protein and the putative receptor-blocking lipoprotein precursor of EPS7 exhibit high homologies with the corresponding gene products of BF23, another member of the T5-family. BF23 binds to BtuB, a surface receptor in the host and involved in vitamin B12 uptake, but its infection is independent of TonB. By constructing a series of deletion mutants in Salmonella and in E. coli and studying phage infection in the mutant hosts, we showed that BtuB is also the host receptor of the phage EPS7. Whether EPS7 infection depends on TonB needs to be further studied.

SUBMITTER: Hong J

PROVIDER: S-EPMC9021807 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Coeliac disease (CD) is a common and complex disorder of the small intestine caused by intolerance to wheat gluten and related edible cereals like barley and rye. Peptides originating from incomplete gliadin digestion activate the lamina propria infiltrating T cells to release proinflammatory cytokines, which in turn cause profound tissue remodelling of the small intestinal wall. There is no cure for CD except refraining from consuming gluten-containing products.Phage from a random oligomer display library were enriched by repeated pannings against immobilised gliadin proteins. Phage from the final panning round were plated, individual plaques picked, incubated with host bacteria, amplified to a population size of 1011 to 1012 and purified. DNA was isolated from 1000 purified phage populations and the region covering the 36 bp oligonucleotide insert from which the displayed peptides were translated, was sequenced. Altogether more than 150 different peptide-encoding sequences were identified, many of which were repeatedly isolated under various experimental conditions. Amplified phage populations, each expressing a single peptide, were tested first in pools and then one by one for their ability to inhibit binding of human anti-gliadin antibodies in ELISA assays. These experiments showed that several of the different peptide-expressing phage tested inhibited the interaction between gliadin and anti-gliadin antibodies. Finally, four different peptide-encoding sequences were selected for further analysis, and the corresponding 12-mer peptides were synthesised in vitro. By ELISA assays it was demonstrated that several of the peptides inhibited the interaction between gliadin molecules and serum anti-gliadin antibodies. Moreover, ELISA competition experiments as well as dot-blot and western blot revealed that the different peptides interacted with different molecular sites of gliadin.We believe that several of the isolated and characterised gliadin-binding peptides described here could provide valuable tools for researchers in the field of CD by facilitating studies on localisation and uptake of various gliadin peptides in the small intestine. In future work, the potential of these peptides to detoxify gluten will be investigated.

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Identification of host receptor and receptor-binding module of a newly sequenced T5-like phage EPS7.

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