Genome and Proteome of Campylobacter jejuni Bacteriophage NCTC 12673

ABSTRACTCampylobacter jejunicontinues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome ofC. jejunibacteriophage NCTC 12673 and the exploitation of its receptor-binding protein for specific bacterial detection. Remarkably, the 135-kbMyoviridaegenome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (includingC. jejuniphages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putativetrans-splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the receptor-binding protein (Gp48) revealed features common to theSalmonella entericaP22 phage tailspike protein, including the ability to specifically recognize a host organism. Bacteriophage receptor-binding proteins may offer promising alternatives for use in pathogen detection platforms.

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Specific detection of Campylobacter jejuni using the bacteriophage NCTC 12673 receptor binding protein as a probe

The Analyst ◽

10.1039/c1an15547d ◽

2011 ◽

Vol 136 (22) ◽

pp. 4780 ◽

Cited By ~ 62

Author(s):

Amit Singh ◽

Denis Arutyunov ◽

Mark T. McDermott ◽

Christine M. Szymanski ◽

Stephane Evoy

Keyword(s):

Campylobacter Jejuni ◽

Receptor Binding ◽

Binding Protein ◽

Specific Detection ◽

Receptor Binding Protein

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Developing Innolysins Against Campylobacter jejuni Using a Novel Prophage Receptor-Binding Protein

Frontiers in Microbiology ◽

10.3389/fmicb.2021.619028 ◽

2021 ◽

Vol 12 ◽

Author(s):

Athina Zampara ◽

Martine C. Holst Sørensen ◽

Yilmaz Emre Gencay ◽

Dennis Grimon ◽

Sebastian Hougaard Kristiansen ◽

...

Keyword(s):

Campylobacter Jejuni ◽

Receptor Binding ◽

Capsular Polysaccharide ◽

Binding Protein ◽

Log Reduction ◽

Cell Counts ◽

Phage Receptor ◽

Receptor Binding Protein

Campylobacter contaminated poultry remains the major cause of foodborne gastroenteritis worldwide, calling for novel antibacterials. We previously developed the concept of Innolysin composed of an endolysin fused to a phage receptor binding protein (RBP) and provided the proof-of-concept that Innolysins exert bactericidal activity against Escherichia coli. Here, we have expanded the Innolysin concept to target Campylobacter jejuni. As no C. jejuni phage RBP had been identified so far, we first showed that the H-fiber originating from a CJIE1-like prophage of C. jejuni CAMSA2147 functions as a novel RBP. By fusing this H-fiber to phage T5 endolysin, we constructed Innolysins targeting C. jejuni (Innolysins Cj). Innolysin Cj1 exerts antibacterial activity against diverse C. jejuni strains after in vitro exposure for 45 min at 20°C, reaching up to 1.30 ± 0.21 log reduction in CAMSA2147 cell counts. Screening of a library of Innolysins Cj composed of distinct endolysins for growth inhibition, allowed us to select Innolysin Cj5 as an additional promising antibacterial candidate. Application of either Innolysin Cj1 or Innolysin Cj5 on chicken skin refrigerated to 5°C and contaminated with C. jejuni CAMSA2147 led to 1.63 ± 0.46 and 1.18 ± 0.10 log reduction of cells, respectively, confirming that Innolysins Cj can kill C. jejuni in situ. The receptor of Innolysins Cj remains to be identified, however, the RBP component (H-fiber) recognizes a novel receptor compared to lytic phages binding to capsular polysaccharide or flagella. Identification of other unexplored Campylobacter phage RBPs may further increase the repertoire of new Innolysins Cj targeting distinct receptors and working as antibacterials against Campylobacter.

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Bacteriophage Receptor Binding Protein Based Assays for the Simultaneous Detection of Campylobacter jejuni and Campylobacter coli

PLoS ONE ◽

10.1371/journal.pone.0069770 ◽

2013 ◽

Vol 8 (7) ◽

pp. e69770 ◽

Cited By ~ 27

Author(s):

Muhammad A. Javed ◽

Somayyeh Poshtiban ◽

Denis Arutyunov ◽

Stephane Evoy ◽

Christine M. Szymanski

Keyword(s):

Campylobacter Jejuni ◽

Receptor Binding ◽

Binding Protein ◽

Simultaneous Detection ◽

Campylobacter Coli ◽

Receptor Binding Protein

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Enzyme-Linked Phage Receptor Binding Protein Assays (ELPRA) Enable Identification of Bacillus anthracis Colonies

Viruses ◽

10.3390/v13081462 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1462

Author(s):

Peter Braun ◽

Nadja Rupprich ◽

Diana Neif ◽

Gregor Grass

Keyword(s):

Bacillus Anthracis ◽

Receptor Binding ◽

Pathogen Detection ◽

Fluorescent Proteins ◽

Binding Protein ◽

Host Cells ◽

Close Relationship ◽

Phage Receptor ◽

Protein Assays ◽

Receptor Binding Protein

Bacteriophage receptor binding proteins (RBPs) are employed by viruses to recognize specific surface structures on bacterial host cells. Recombinant RBPs have been utilized for detection of several pathogens, typically as fusions with reporter enzymes or fluorescent proteins. Identification of Bacillus anthracis, the etiological agent of anthrax, can be difficult because of the bacterium’s close relationship with other species of the Bacillus cereussensu lato group. Here, we facilitated the identification of B. anthracis using two implementations of enzyme-linked phage receptor binding protein assays (ELPRA). We developed a single-tube centrifugation assay simplifying the rapid analysis of suspect colonies. A second assay enables identification of suspect colonies from mixed overgrown solid (agar) media derived from the complex matrix soil. Thus, these tests identified vegetative cells of B. anthracis with little processing time and may support or confirm pathogen detection by molecular methods such as polymerase chain reaction.

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A Smooth-Type, Phage-ResistantKlebsiella pneumoniaeMutant Strain Reveals that OmpC Is Indispensable for Infection by Phage GH-K3

Applied and Environmental Microbiology ◽

10.1128/aem.01585-18 ◽

2018 ◽

Vol 84 (21) ◽

Cited By ~ 7

Author(s):

Ruopeng Cai ◽

Mei Wu ◽

Hao Zhang ◽

Yufeng Zhang ◽

Mengjun Cheng ◽

...

Keyword(s):

Receptor Binding ◽

Binding Protein ◽

Multidrug Resistant ◽

Proteinase K ◽

Adsorption Efficiency ◽

Content Type ◽

Therapeutic Applications ◽

Rapid Emergence ◽

Receptor Binding Protein ◽

Phage Treatment

ABSTRACTBacteriophage can be used as an alternative or complementary therapy to antibiotics for treating multidrug-resistant bacterial infections. However, the rapid emergence of resistant host variants during phage treatment has limited its therapeutic applications. In this study, a potential phage-resistant mechanism ofKlebsiella pneumoniaewas revealed. After phage GH-K3 treatment, a smooth-type colony, named K7RB, was obtained from theK. pneumoniaeK7 culture. Treatment with IO4−and/or proteinase K indicated that polysaccharides of K7 played an important role in phage recruitment, and protein receptors on K7 were essential for effective infection by GH-K3. Differences in protein expression between K7 and K7RBwere quantitatively analyzed by liquid chromatography-tandem mass spectrometry. Among differentially expressed proteins, OmpC, OmpN, KPN_02430, and OmpF were downregulated significantly in K7RB.trans-Complementation of OmpC in K7RBconferred rapid adsorption and sensitivity to GH-K3. In contrast, a single-base deletion mutation ofompCin K7, which resulted in OmpC silencing, led to lower adsorption efficiency and resistance to GH-K3. These assays proved that OmpC is the key receptor-binding protein for GH-K3. In addition, the nativeK. pneumoniaestrains KPP14, KPP27, and KPP36 showed low or no sensitivity to GH-K3. However, these strains became more sensitive to GH-K3 after their native receptors were replaced by OmpC of K7, suggesting that OmpCK7was the most suitable receptor for GH-K3. This study revealed that K7RBbecame resistant to GH-K3 due to gene mutation ofompCand that OmpC of K7 is essential for effective infection by GH-K3.IMPORTANCEWith increased incidence of multidrug-resistant (MDR) bacterial strains, phages have regained attention as promising potential antibacterial agents. However, the rapid emergence of resistant variants during phage treatment has limited the therapeutic applications of phage. According to ourtrans-complementation,ompCmutation, and phage adsorption efficiency assays, we identified OmpC as the key receptor-binding protein (RBP) for phage GH-K3, which is essential for effective infection. This study revealed that the phage secondary receptor ofK. pneumoniae, OmpC, is the essential RBP not only for phage infecting Gram-negative bacteria, such asEscherichia coliandSalmonella, but also forK. pneumoniae.

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