Use of High-Affinity Cell Wall-Binding Domains of Bacteriophage Endolysins for Immobilization and Separation of Bacterial Cells

Kretzer, Jan W.; Lehmann, Rainer P.; Schmelcher, Mathias; Banz, Manuel; Kim, Kwang-Pyo; Korn, Corinna; Loessner, Martin J.

doi:10.1128/aem.02402-06

Cited by 164 publications

(137 citation statements)

References 35 publications

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“…Following magnetic separation and washing, the resuspended LTF-MBs were surface plated on xylose lysine deoxycholate (XLD) agar in appropriate dilutions, and the plates incubated at 37°C overnight before colony enumeration. As previously found for bead-based Listeria separation (38), immobilization on beads did not affect the number of CFUs; i.e., the bead-bound bacteria remained viable and retained the same plating ability and growth pattern as freely suspended bacteria. This is explained by the fact that the number of beads always exceeded the number of cells to be recovered by more than 2 log.…”

supporting

confidence: 53%

“…A simplified alternative to applying whole phages for bacterial detection is to utilize phage-encoded host interaction proteins as affinity molecules. Cell wall binding domains (CBDs) of phage endolysins (peptidoglycan hydrolases) have proven effective for detecting and enriching Gram-positive bacteria, such as Listeria (36,37), Bacillus cereus, and Clostridium perfringens (38). Alternatively, phage receptor binding proteins (RBPs), which exist as long tail fibers (LTFs) or short tail spikes attached to the baseplate of tailed phages, have been applied for bacterial detection.…”

mentioning

confidence: 99%

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Modified Bacteriophage S16 Long Tail Fiber Proteins for Rapid and Specific Immobilization and Detection of Salmonella Cells

Denyes

Dunne

Steiner

et al. 2017

Appl Environ Microbiol

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Bacteriophage-based assays and biosensors rival traditional antibodybased immunoassays for detection of low-level Salmonella contaminations. In this study, we harnessed the binding specificity of the long tail fiber (LTF) from bacteriophage S16 as an affinity molecule for the immobilization, enrichment, and detection of Salmonella. We demonstrate that paramagnetic beads (MBs) coated with recombinant gp37-gp38 LTF complexes (LTF-MBs) are highly effective tools for rapid affinity magnetic separation and enrichment of Salmonella. Within 45 min, the LTF-MBs consistently captured over 95% of Salmonella enterica serovar Typhimurium cells from suspensions containing from 10 to 10 5 CFU · ml Ϫ1 , and they yielded equivalent recovery rates (93% Ϯ 5%, n ϭ 10) for other Salmonella strains tested. LTF-MBs also captured Salmonella cells from various food sample preenrichments, allowing the detection of initial contaminations of 1 to 10 CFU per 25 g or ml. While plating of bead-captured cells allowed ultrasensitive but time-consuming detection, the integration of LTF-based enrichment into a sandwich assay with horseradish peroxidaseconjugated LTF (HRP-LTF) as a detection probe produced a rapid and easy-to-use Salmonella detection assay. The novel enzyme-linked LTF assay (ELLTA) uses HRP-LTF to label bead-captured Salmonella cells for subsequent identification by HRP-catalyzed conversion of chromogenic 3,3=,5,5=-tetramethylbenzidine substrate. The color development was proportional for Salmonella concentrations between 10 2 and 10 7 CFU · ml Ϫ1 as determined by spectrophotometric quantification. The ELLTA assay took 2 h to complete and detected as few as 10 2 CFU · ml Ϫ1 S. Typhimurium cells. It positively identified 21 different Salmonella strains, with no cross-reactivity for other bacteria. In conclusion, the phage-based ELLTA represents a rapid, sensitive, and specific diagnostic assay that appears to be superior to other currently available tests. IMPORTANCEThe incidence of foodborne diseases has increased over the years, resulting in major global public health issues. Conventional methods for pathogen detection can be laborious and expensive, and they require lengthy preenrichment steps. Rapid enrichment-based diagnostic assays, such as immunomagnetic separation, can reduce detection times while also remaining sensitive and specific. A critical component in these tests is implementing affinity molecules that retain the ability to specifically capture target pathogens over a wide range of in situ applications. The protein complex that forms the distal tip of the bacteriophage S16 long tail fiber is shown here to represent a highly sensitive affinity molecule for the specific enrichment and detection of Salmonella. Phage-encoded long tail fibers have huge potential for development as novel affinity molecules for robust and specific diagnostics of a vast spectrum of bacteria.

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supporting

confidence: 53%

mentioning

confidence: 99%

Modified Bacteriophage S16 Long Tail Fiber Proteins for Rapid and Specific Immobilization and Detection of Salmonella Cells

Denyes

Dunne

Steiner

et al. 2017

Appl Environ Microbiol

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“…32 Cell wall binding of the endolysin is often necessary for bacteriolytic activity, 29,32,33 and endolysins often exhibit near species-specificity. 34,35 In rare cases endolysins can be serovar-specific, such as the L. monocytogenes phage endolysins Ply118 and Ply500 each unique to different serovars. 36,37 Endolysins with an intact CBD can also have a broad host range, such as with the streptococcal phage endolysin PlyC, which lyses several streptococcal species.…”

Section: Endolysins -Peptidoglycan Degrading Enzymesmentioning

confidence: 99%

Antimicrobial bacteriophage-derived proteins and therapeutic applications

2015

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“…Other approaches include use of an ice nucleation reporter phage (Wolber and Green 1990); concentration by IMS followed by phage mediated release of adenylate kinase (AK) (Blasco et al 1998;Wu et al 2001); fluorescently labelled phage (Jiang et al 2009); and an IMS-bacteriophage plaque formation assay requiring the addition of a virucide to inactivate free phage particles (Fravrin et al 2001). The usefulness of phagebased cell wall recognition proteins for magnetic capture has also been recently described utilizing cell-wall-binding domains (CBDs) highly specific for recognition and binding to target cells surfaces (Kretzer et al 2007;Korndoerfer et al 2006;Loessner et al 2002). Paramagnetic beads coated with CBD molecules were shown to outperform commercially available antibody-based magnetic beads with respect to sensitivity and percent recovery (Kretzer et al 2007).…”

Section: Phage Based Detection Methodsmentioning

confidence: 99%

“…The usefulness of phagebased cell wall recognition proteins for magnetic capture has also been recently described utilizing cell-wall-binding domains (CBDs) highly specific for recognition and binding to target cells surfaces (Kretzer et al 2007;Korndoerfer et al 2006;Loessner et al 2002). Paramagnetic beads coated with CBD molecules were shown to outperform commercially available antibody-based magnetic beads with respect to sensitivity and percent recovery (Kretzer et al 2007). An extension to this approach has been the use of phage-tail-associated recognition proteins for the immobilization of gram-negative cells (Galikowska et al 2011).…”

Section: Phage Based Detection Methodsmentioning

confidence: 99%