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Isolation and engineering of a Listeria grayi bacteriophage.

ABSTRACT: The lack of bacteriophages capable of infecting the Listeria species, Listeria grayi, is academically intriguing and presents an obstacle to the development of bacteriophage-based technologies for Listeria. We describe the isolation and engineering of a novel L. grayi bacteriophage, LPJP1, isolated from farm silage. With a genome over 200,000 base pairs, LPJP1 is the first and only reported jumbo bacteriophage infecting the Listeria genus. Similar to other Gram-positive jumbo phages, LPJP1 appeared to contain modified base pairs, which complicated initial attempts to obtain genomic sequence using standard methods. Following successful sequencing with a modified approach, a recombinant of LPJP1 encoding the NanoLuc luciferase was engineered using homologous recombination. This luciferase reporter bacteriophage successfully detected 100 stationary phase colony forming units of both subspecies of L. grayi in four hours. A single log phase colony forming unit was also sufficient for positive detection in the same time period. The recombinant demonstrated complete specificity for this particular Listeria species and did not infect 150 non-L. grayi Listeria strains nor any other bacterial genus. LPJP1 is believed to be the first reported lytic bacteriophage of L. grayi as well as the only jumbo bacteriophage to be successfully engineered into a luciferase reporter.

SUBMITTER: Erickson S

PROVIDER: S-EPMC8460666 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIxodes scapularis is the most common tick species in North America and a vector of important pathogens that cause diseases in humans and animals including Lyme disease, anaplasmosis and babesiosis. Tick defensins have been identified as a new source of antimicrobial agents with putative medical applications due to their wide-ranging antimicrobial activities. Two multigene families of defensins were previously reported in I. scapularis. The objective of the present study was to characterise the potential antimicrobial activity of two defensins from I. scapularis with emphasis on human pathogenic bacterial strains and important phytopathogenic fungi.MethodsScapularisin-3 and Scapularisin-6 mature peptides were chemically synthesised. In vitro antimicrobial assays were performed to test the activity of these two defensins against species of different bacterial genera including Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Listeria spp. as well as Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa along with two plant-pathogenic fungi from the genus Fusarium. In addition, the tissue-specific expression patterns of Scapularisin-3 and Scapularisin-6 in I. scapularis midgut, salivary glands and embryo-derived cell lines were determined using PCR. Finally, tertiary structures of the two defensins were predicted and structural analyses were conducted.ResultsScapularisin-6 efficiently killed L. grayi, and both Scapularisin-3 and Scapularisin-6 caused strong inhibition (IC50 value: ~1 μM) of the germination of plant-pathogenic fungi Fusarium culmorum and Fusarium graminearum. Scapularisin-6 gene expression was observed in I. scapularis salivary glands and midgut. However, Scapularisin-3 gene expression was only detected in the salivary glands. Transcripts from the two defensins were not found in the I. scapularis tick cell lines ISE6 and ISE18.ConclusionOur results have two main implications. Firstly, the anti-Listeria and antifungal activities of Scapularisin-3 and Scapularisin-6 suggest that these peptides may be useful for (i) treatment of antibiotic-resistant L. grayi in humans and (ii) plant protection. Secondly, the antimicrobial properties of the two defensins described in this study may pave the way for further studies regarding pathogen invasion and innate immunity in I. scapularis.

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Isolation and engineering of a Listeria grayi bacteriophage.

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